The chromosome-scale genome and the genetic resistance machinery against insect herbivores of the Mexican toloache, Datura stramonium.

Plant resistance refers to the heritable ability of plants to reduce damage caused by natural enemies, such as herbivores and pathogens, either through constitutive or induced traits like chemical compounds or trichomes. However, the genetic architecture-the number and genome location of genes that affect plant defense and the magnitude of their effects-of plant resistance to arthropod herbivores in natural populations remains poorly understood. In this study, we aimed to unveil the genetic architecture of plant resistance to insect herbivores in the annual herb Datura stramonium (Solanaceae) through quantitative trait loci mapping. We achieved this by assembling the species' genome and constructing a linkage map using an F2 progeny transplanted into natural habitats. Furthermore, we conducted differential gene expression analysis between undamaged and damaged plants caused by the primary folivore, Lema daturaphila larvae. Our genome assembly resulted in 6,109 scaffolds distributed across 12 haploid chromosomes. A single quantitative trait loci region on chromosome 3 was associated with plant resistance, spanning 0 to 5.17 cM. The explained variance by the quantitative trait loci was 8.44%. Our findings imply that the resistance mechanisms of D. stramonium are shaped by the complex interplay of multiple genes with minor effects. Protein-protein interaction networks involving genes within the quantitative trait loci region and overexpressed genes uncovered the key role of receptor-like cytoplasmic kinases in signaling and regulating tropane alkaloids and terpenoids, which serve as powerful chemical defenses against D. stramonium herbivores. The data generated in our study constitute important resources for delving into the evolution and ecology of secondary compounds mediating plant-insect interactions.

Optimization and physicochemical characterization of bacterial cellulose by Komagataeibacter nataicola and Komagataeibacter maltaceti strains isolated from grape, thorn apple and apple vinegars.

Bacterial cellulose (BC) is a valuable biopolymer that is increasingly used in medical, pharmaceutical and food industries with its excellent physicochemical properties as high water-holding capacity, nanofibrillar structure, large surface area, porosity, mechanical strength and biocompatibility. Accordingly, the isolation, identification and characterization of potent BC producers from grape, thorn apple and apple vinegars were performed in this study. The strains isolated from grape and apple vinegars were identified as Komagataeibacter maltaceti and the strain isolated from thorn apple vinegar was identified as Komagataeibacter nataicola with 16S rRNA analysis. Optimized conditions were found as 8% dextrin, 1.5% (peptone + yeast extract) and 10% inoculation amount at pH 6.0 with a productivity rate of 1.15 g/d/L, a yield of 8.06% and a dry weight of 6.45 g/L for K. maltaceti, and 10% maltose, 1% (peptone + yeast extract) and 10% inoculation amount at pH 6.0 with a productivity rate of 0.96 g/L/d, a yield of 5.35% and a dry weight of 5.35 g/L for K. nataicola. Obtained BC from K. maltaceti and K. nataicola strains was more than 2.56- and 1.86-fold when compared with BC obtained from HS media and exhibited 95.1% and 92.5% WHC, respectively. Based on the characterization results, BC pellicles show characteristic FT-IR bands and have ultrafine 3D structures with high thermal stability. By means of having ability to assimilate monosaccharides, disaccharides and polysaccharide used in this study, it is predicted that both isolated Komagataeibacter species can be used in the production of biopolymers from wastes containing complex carbon sources in the future.

One Hundred Years of Gene Balance: How Stoichiometric Issues Affect Gene Expression, Genome Evolution, and Quantitative Traits.

A century ago experiments with the flowering plant Datura stramonium and the fruit fly Drosophila melanogaster revealed that adding an extra chromosome to a karyotype was much more detrimental than adding a whole set of chromosomes. This phenomenon was referred to as gene balance and has been recapitulated across eukaryotic species. Here, we retrace some developments in this field. Molecular studies suggest that the basis of balance involves stoichiometric relationships of multi-component interactions. This concept has implication for the mechanisms controlling gene expression, genome evolution, sex chromosome evolution/dosage compensation, speciation mechanisms, and the underlying genetics of quantitative traits.

Genomic signatures of the evolution of defence against its natural enemies in the poisonous and medicinal plant Datura stramonium (Solanaceae).

Tropane alkaloids and terpenoids are widely used in the medicine and pharmaceutic industry and evolved as chemical defenses against herbivores and pathogens in the annual herb Datura stramonium (Solanaceae). Here, we present the first draft genomes of two plants from contrasting environments of D. stramonium. Using these de novo assemblies, along with other previously published genomes from 11 Solanaceae species, we carried out comparative genomic analyses to provide insights on the genome evolution of D. stramonium within the Solanaceae family, and to elucidate adaptive genomic signatures to biotic and abiotic stresses in this plant. We also studied, in detail, the evolution of four genes of D. stramonium-Putrescine N-methyltransferase, Tropinone reductase I, Tropinone reductase II and Hyoscyamine-6S-dioxygenase-involved in the tropane alkaloid biosynthesis. Our analyses revealed that the genomes of D. stramonium show signatures of expansion, physicochemical divergence and/or positive selection on proteins related to the production of tropane alkaloids, terpenoids, and glycoalkaloids as well as on R defensive genes and other important proteins related with biotic and abiotic pressures such as defense against natural enemies and drought.

Pharmacognostic standardization and DNA fingerprinting of leaves of Datura stramonium, growing naturally in Asir region of Saudi Arabia.

Evaluation of the various pharmacognostic quality parameters and DNA fingerprint of Saudi Arabian medicinal plant namely Datura stramonium growing in Asir region was the objective of the study. The pharmacognostical parameters were done in terms of macroscopic characters, microscopic details, physico-chemical evaluations, phytochemical analysis, fluorescence analysis and DNA fingerprint by using standard techniques and random polymorphic DNA primer. The detailed microscopy of the leaf revealed the presence of pre-medullary phloem, xylem, endodermis, parenchymatous pericycle, lower epidermis and calcium oxalate crystals. There are various amounts of foreign material, ash values, moisture content and extractive values, found after estimations. Preliminary phytochemical screening exhibited the presence of alkaloids, flavonoids, glycosides, tannins and sterols in variable amounts. Random amplified polymorphic DNA (RAPD) showed there are a prominent scorbale DNA bands. These evaluations provided referential information for correct authentication and quality standardization of the important medicinal plant material. These information will also be supportive to differentiate Datura stramonium from the closely related other species.

Genomic and chemical evidence for local adaptation in resistance to different herbivores in Datura stramonium.

Because most species are collections of genetically variable populations distributed to habitats differing in their abiotic/biotic environmental factors and community composition, the pattern and strength of natural selection imposed by species on each other's traits are also expected to be highly spatially variable. Here, we used genomic and quantitative genetic approaches to understand how spatially variable selection operates on the genetic basis of plant defenses to herbivores. To this end, an F2 progeny was generated by crossing Datura stramonium (Solanaceae) parents from two populations differing in their level of chemical defense. This F2 progeny was reciprocally transplanted into the parental plants' habitats and by measuring the identity by descent (IBD) relationship of each F2 plant to each parent, we were able to elucidate how spatially variable selection imposed by herbivores operated on the genetic background (IBD) of resistance to herbivory, promoting local adaptation. The results highlight that plants possessing the highest total alkaloid concentrations (sum of all alkaloid classes) were not the most well-defended or fit. Instead, specific alkaloids and their linked loci/alleles were favored by selection imposed by different herbivores. This has led to population differentiation in plant defenses and thus, to local adaptation driven by plant-herbivore interactions.

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.

Evaluation of Two New Methods for DNA Extraction of "Legal High" Plant Species.

A quick, simple, and high-yield nucleic acid isolation process is crucial for high-quality DNA analysis. The ability of the MicroGEM PDQeX phytoGEM system and Omega Bio-tek E.Z.N.A.® Plant DS Mini kit to extract PCR-ready DNA was evaluated by extracting the forensically relevant "legal high" plant species: Ipomoea purpurea, Artemisia absinthium, Mitragyna speciosa, Datura stramonium, and Papaver somniferum. The plant material was pulverized, processed using the manufacturer's plant protocol for the PDQeX Nucleic Acid Extraction or the manufacturer's protocol for the Omega extraction, quantified using the Invitrogen Qubit 2.0 Fluorometer, and analyzed for amplifiability by PCR using a Qiagen Rotor-Gene Q instrument and published assays. The DNA amplicons for the legal high species produced high-resolution melt curves concordant with the melts observed when DNA was isolated using the Qiagen DNeasy Plant Mini Kit in previous studies.

Methods for Purifying Datura stramonium Agglutinin and Producing Recombinant Agglutinin Protein in a Heterologous Plant Host.

Datura stramonium seeds contain at least three chitin-binding isolectins as homo- or heterodimers of A and B subunits. This lectin has been used for the detection and isolation of sugar chains with N-acetyllactosaminyl structures on highly branched N-glycans. In terms of future diagnostic use, the development of a recombinant lectin will be the most effective approach for producing homogeneous lectin preparations. This chapter presents details of the procedure used for lectin purification and also describes a method that can be used for producing active recombinant homodimeric BB-isolectin in Arabidopsis plants.

Development of a PCR High-Resolution Melt Assay for Artemisia absinthium (Wormwood) and a Triplex Assay with Two Additional "Unregulated Legal High" Species Datura stramonium (Jimson Weed) and Merremia tuberosa (Hawaiian Woodrose).

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an "unregulated legal high" in higher concentrations. The European Union limits thujone from Artemisia species to 35 mg/kg while the U.S. Food and Drug Administration requires less than 10 ppm to be "thujone-free." However, individuals can smoke or ingest A. absinthium in different forms. This study developed a polymerase chain reaction (PCR) high-resolution melt (HRM) assay to detect and identify A. absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three "unregulated legal high" species: Datura stramonium, Merremia tuberosa, and A. absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42 ± 0.20°C, 83.88 ± 0.22°C, and 87.77 ± 0.15°C, respectively. The primer set developed distinguished A. absinthium from a variety of plant species and was successfully triplexed.

Changes in floral biology and inbreeding depression in native and invaded regions of Datura stramonium.

Plant populations invading new environments might compromise their fitness contribution to the next generation, because of the lack of native specialist pollinators and/or potential mates. Thus, changes in plant mating system and traits linked to it are expected in populations colonising new environments where selection would favour selfing and floral traits that maximise reproductive output. To test this, we studied native (Mexico) and non-native (Spain) populations of the obligate sexual reproducing annual weed Datura stramonium. Flower size, herkogamy, total number of seeds per plant, number of visits by and type of pollinators, and inbreeding depression were assessed in native and non-native populations. Finally, we measured phenotypic selection on corolla size and herkogamy in each population. Flower size and herkogamy showed wide and similar variation in both ranges. However, the largest average flower size was found in one non-native population whereas the highest average positive herkogamy was detected in one native population. On average, flowers in the native range received more visits by pollinators. Hawkmoths were the main visitors in the native populations while only bees were observed visiting flowers in Spain's populations. Only in the native range was inbreeding depression detected. Selection to reduce herkogamy was found only in one native population. Absence of both inbreeding depression and selection on floral traits suggest a change in mating system of D. stramonium in a new range where generalist pollinators may be promoting high reproductive success. Selection against deleterious alleles might explain the reduction of inbreeding depression, promoting the evolution of selfing.

Simultaneous Identification of Four "Legal High" Plant Species in a Multiplex PCR High-Resolution Melt Assay.

The international prevalence of "legal high" drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real-time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high-resolution melt using LCGreen Plus® . The PCR assay was evaluated based on the following: (i) specificity and selectivity-primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity-serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability-sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging "legal high" species.

Datura stramonium agglutinin: cloning, molecular characterization and recombinant production in Arabidopsis thaliana.

Datura stramonium seeds contain at least three chitin-binding isolectins [termed Datura stramonium agglutinin (DSA)] as homo- or heterodimers of A and B subunits. We isolated a cDNA encoding isolectin B (DSA-B) from an immature fruit cDNA library; this contained an open reading frame encoding 279 deduced amino acids, which was confirmed by partial sequencing of the native DSA-B peptide. The sequence consisted of: (i) a cysteine (Cys)-rich carbohydrate-binding domain composed of four conserved chitin-binding domains and (ii) an extensin-like domain of 37 residues containing four SerPro4-6 motifs that was inserted between the second and third chitin-binding domains (CBDs). Although each chitin-binding domain contained eight conserved Cys residues, only the second chitin-binding domain contained an extra Cys residue, which may participate in dimerization through inter-disulfide bridge formation. Using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, the molecular mass of homodimeric lectin composed of two B-subunits was determined as 68,821 Da. The molecular mass of the S-pyridilethylated B-subunit were found to be 37,748 Da and that of the de-glycosylated form was 26,491 Da, which correlated with the molecular weight estimated from the deduced sequence. Transgenic Arabidopsis plants overexpressing the dsa-b demonstrated hemagglutinating activity. Recombinant DSA-B was produced as a homodimeric glycoprotein with a similar molecular mass to that of the native form. Moreover, the N-terminus of the purified recombinant DSA-B protein was identical to that of the native DSA-B, confirming that the cloned cDNA encoded DSA-B.

Complete chloroplast genome sequence of poisonous and medicinal plant Datura stramonium: organizations and implications for genetic engineering.

Datura stramonium is a widely used poisonous plant with great medicinal and economic value. Its chloroplast (cp) genome is 155,871 bp in length with a typical quadripartite structure of the large (LSC, 86,302 bp) and small (SSC, 18,367 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,601 bp). The genome contains 113 unique genes, including 80 protein-coding genes, 29 tRNAs and four rRNAs. A total of 11 forward, 9 palindromic and 13 tandem repeats were detected in the D. stramonium cp genome. Most simple sequence repeats (SSR) are AT-rich and are less abundant in coding regions than in non-coding regions. Both SSRs and GC content were unevenly distributed in the entire cp genome. All preferred synonymous codons were found to use A/T ending codons. The difference in GC contents of entire genomes and of the three-codon positions suggests that the D. stramonium cp genome might possess different genomic organization, in part due to different mutational pressures. The five most divergent coding regions and four non-coding regions (trnH-psbA, rps4-trnS, ndhD-ccsA, and ndhI-ndhG) were identified using whole plastome alignment, which can be used to develop molecular markers for phylogenetics and barcoding studies within the Solanaceae. Phylogenetic analysis based on 68 protein-coding genes supported Datura as a sister to Solanum. This study provides valuable information for phylogenetic and cp genetic engineering studies of this poisonous and medicinal plant.

Assessing the tobacco-rattle-virus-based vectors system as an efficient gene silencing technique in Datura stramonium (Solanaceae).

Datura stramonium is a well-known medicinal plant, which is important for its alkaloids. There are intrinsic limitations for the natural production of alkaloids in plants; metabolic engineering methods can be effectively used to conquer these limitations. In order for this the genes involved in corresponding pathways need to be studied. Virus-Induced Gene Silencing is known as a functional genomics technique to knock-down expression of endogenous genes. In this study, we silenced phytoene desaturase as a marker gene in D. stramonium in a heterologous and homologous manner by tobacco-rattle-virus-based VIGS vectors. Recombinant TRV vector containing pds gene from D. stramonium (pTRV2-Dspds) was constructed and injected into seedlings. The plants injected with pTRV2-Dspds showed photobleaching 2 weeks after infiltration. Spectrophotometric analysis demonstrated that the amount of chlorophylls and carotenoids in leaves of the bleached plants decreased considerably compared to that of the control plants. Semi-Quantitative RT-PCR results also confirmed that the expression of pds gene in the silenced plants was significantly reduced in comparison with the control plants. The results showed that the viral vector was able to influence the levels of total alkaloid content in D. stramonium. Our results illustrated that TRV-based VIGS vectors are able to induce effective and reliable functional gene silencing in D. stramonium as an alternative tool for studying the genes of interest in this plant, such as the targeted genes in tropane alkaloid biosynthetic pathway. The present work is the first report of establishing VIGS as an efficient method for transient silencing of any gene of interest in D. stramonium.

The effect of inbreeding on defence against multiple enemies in Datura stramonium.

The ability of plants to respond to natural enemies might depend on the availability of genetic variation for the optimal phenotypic expression of defence. Selfing can affect the distribution of genetic variability of plant fitness, resistance and tolerance to herbivores and pathogens. The hypothesis of inbreeding depression influencing plant defence predicts that inbreeding would reduce resistance and tolerance to damage by natural enemies relative to outcrossing. In a field experiment entailing experimentally produced inbred and outcrossed progenies, we assessed the effects of one generation of selfing on Datura stramonium resistance and tolerance to three types of natural enemies, herbivores, weevils and a virus. We also examined the effect of damage on relative growth rate (RGR), flower, fruit, and seed production in inbred and outcrossed plants. Inbreeding significantly reduced plant defence to natural enemies with an increase of 4% in herbivore damage and 8% in viral infection. These results indicate inbreeding depression in total resistance. Herbivory increased 10% inbreeding depression in seed number, but viral damage caused inbred and outcrossed plants to have similar seed production. Inbreeding and outcrossing effects on fitness components were highly variable among families, implying that different types or numbers of recessive deleterious alleles segregate following inbreeding in D. stramonium. Although inbreeding did not equally alter all the interactions, our findings indicate that inbreeding reduced plant defence to herbivores and pathogens in D. stramonium.

[Identification of Daturae flos and its adulterants based on DNA barcoding technique].

To identify the original plant of Daturae Flos from its adulterants by DNA barcoding, the sequences of ITS2, psbA-trnH, matK, rbcL of four species including Datura metel, Darura innoxia, Darura stramonium and Brugmansia arborea were compared and analyzed. The PCR and sequencing success rate of the four regions (ITS2, psbA-trnH, matK, rbcL) was 100%, 90%, 100% and 85%, respectively. Sequences were assembled with CodonCode Aligner. K2P distances were calculated and NJ tree was performed by MEGA 4.1. Thirty SNPs were found among ITS2 sequences, and 33 insert/deletes were found among psbA-trnH intergenic regions. The interspecific K2P distance of ITS2 and psbA-trnH was obviously higher than that of the intraspecific one. As to matK and rbcL, there was no "Barcoding Gap" existing between inter- and intra-specific distances. The NJ trees of the four regions/combinations were built separately. Samples of Brugmansia arborea were clustered into one clade, and the other species of Datura L. formed another clade. The results showed that either ITS2 or psbA-trnH was useful to identify Daturae Flos from its adulterants.

Evolution of putrescine N-methyltransferase from spermidine synthase demanded alterations in substrate binding.

Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM)-dependent methylation of putrescine in tropane alkaloid biosynthesis. PMT presumably evolved from the ubiquitous spermidine synthase (SPDS). SPDS protein structure suggested that only few amino acid exchanges in the active site were necessary to achieve PMT activity. Protein modelling, mutagenesis, and chimeric protein construction were applied to trace back evolution of PMT activity from SPDS. Ten amino acid exchanges in Datura stramonium SPDS dismissed the hypothesis of facile generation of PMT activity in existing SPDS proteins. Chimeric PMT and SPDS enzymes were active and indicated the necessity for a different putrescine binding site when PMT developed.

Spermine facilitates recovery from drought but does not confer drought tolerance in transgenic rice plants expressing Datura stramonium S-adenosylmethionine decarboxylase.

Polyamines are known to play important roles in plant stress tolerance but it has been difficult to determine precise functions for each type of polyamine and their interrelationships. To dissect the roles of putrescine from the higher polyamines spermidine and spermine, we generated transgenic rice plants constitutively expressing a heterologous S-adenosylmethionine decarboxylase (SAMDC) gene from Datura stramonium so that spermidine and spermine levels could be investigated while maintaining a constant putrescine pool. Whereas transgenic plants expressing arginine decarboxylase (ADC) produced higher levels of putrescine, spermidine and spermine, and were protected from drought stress, transgenic plants expressing SAMDC produced normal levels of putrescine and showed drought symptoms typical of wild type plants under stress, but the transgenic plants showed a much more robust recovery on return to normal conditions (90% full recovery compared to 25% partial recovery for wild type plants). At the molecular level, both wild type and transgenic plants showed transient reductions in the levels of endogenous ADC1 and SAMDC mRNA, but only wild type plants showed a spike in putrescine levels under stress. In transgenic plants, there was no spike in putrescine but a smooth increase in spermine levels at the expense of spermidine. These results confirm and extend the threshold model for polyamine activity in drought stress, and attribute individual roles to putrescine, spermidine and spermine.

Flow cytometric investigations of diploid and tetraploid plants and in vitro cultures of Datura stramonium and Hyoscyamus niger.

Plant in vitro systems are valuable sources for the production of biological active substances. However, changed profiles of secondary metabolites, and low, variable yields possibly caused by genetic instabilities complicate their industrial implementation. DNA profiling of plant in vitro cultures may provide data for the selection of highly producing in vitro cultures. Diploid and tetraploid Datura stramonium and Hyoscyamus niger plant as well as calli, and hairy root lines derived from them were analyzed by flow cytometry. Plant in vitro cultures undergo several cycles of endoreduplication more than the explants from which they were obtained. The highest cycle values were observed in calli (e.g. 1.19 for diploid H. niger) possibly induced by the growth factors. However, hairy roots cultivated without growth factor exhibited significant degrees of endoreduplication (cycle value 0.88 for diploid H. niger). Sets of five hairy root lines from each plant and ploidy level showed consistent within-set ploidy patterns. The ploidy profiles of investigated plant in vitro and in vivo differ. For the first time we report that hairy roots of two Solanaceae species undergo endoreduplication. Theploidy profiles of in vitro cultures (hairy roots and calli) seem to be influenced by the genome size, the growth factors applied, and the type of in vitro culture. The transformation of several hairy root lines showed no differences in the ploidy patterns.