Brace roots in C 3 Poaceae: where have they gone?

Brace roots are common in large C 4 Poaceae species, such as maize and sorghum. However, in other species, these roots were either never reported, or the existence of the trait was neglected. Here we report the presence of brace roots in a high-performing Avena sativa L. (oat) line.

Leaf manganese and phenolics as proxies of soil acidification and phosphorus acquisition mechanisms in lentil cultivars on alkaline soil.

Leaf manganese (Mn) concentration has been used as a proxy for root exudation and phosphorus (P) uptake under controlled conditions, but there are limited field studies that confirm its validity. On an alkaline, P-poor soil, four lentil cultivars ('Samos', 'Thessaly', 'Flip', 'Algeria') received two P rates (0 and 26.2kgPha-1 ), for two growing seasons, to study whether aboveground assessments [leaf P, Mn, phenolic concentration (TPhe)] can approximate rhizosphere physiological traits related to P acquisition [soil acidification (ΔpH), arbuscular mycorrhizal fungi (AMF) colonisation, acid phosphatase activity (APase)]. Phosphorus addition had no effect on the determined traits. Cultivars differed in leaf P, Mn, TPhe and AMF, but there was no clear pattern relating aboveground traits to rhizosphere traits related to P acquisition, thus not confirming that leaf Mn can be a proxy of root exudation. Of three growth stages [V 7-8, R1 (first bloom), R4 (flat pod)], R1 seemed to be critical, showing the highest leaf P, ΔpH, AMF and TPhe. Precipitation and temperatures over the growing season were determinants of lentil responses affecting rhizosphere activity, soil P availability and finally leaf traits. In conclusion, in lentil on alkaline and P-limiting soils, high leaf Mn and phenolic concentration are not reliable indicators of rhizosphere P-acquiring mechanisms.

Comparative Studies on the Anti-Inflammatory and Apoptotic Activities of Four Greek Essential Oils: Involvement in the Regulation of NF-κΒ and Steroid Receptor Signaling.

Essential oils (EOs) are well-known for their anti-fungal, anti-microbial, anti-inflammatory and relaxing activities. Steroid hormones, especially glucocorticoids, are also well-known for their anti-inflammatory activities and control of the hypothalamus-pituitary-adrenal (HPA) axis and glucose homeostasis. The biological activities of glucocorticoids render them the most widely prescribed anti-inflammatory drugs, despite their adverse side effects. In this study, comparative studies of the anti-inflammatory activities and interference with glucocorticoids receptor (GR) and estrogen receptor (ER) signaling of EOs from Greek Oregano, Melissa officinalis, Lavender and from the Chios Mastic, produced from the Greek endemic mastic tree, were performed in Human Embryonic Kidney 293 (HEK-293) cells. Chios Mastic (Mastiha) and oregano EOs exhibited the highest anti-inflammatory activities. The former showed a reduction in both NF-κB activity and protein levels. Mastic essential oil also caused a reduction in GR protein levels that may compensate for its boosting effect on dexamethasone (DEX)-induced GR transcriptional activation, ending up in no induction of the gluconeogenic phoshoenolpyruvate carboxykinase (PEPCK) protein levels that constitute the GR target. Oregano, Melissa officinalis and lavender EOs caused the suppression of the transcriptional activation of GR. Furthermore, the most active EO, that taken from Melissa officinalis, showed a reduction in both GR and PEPCK protein levels. Thus, the anti-inflammatory and anti-gluconeogenic activities of the EOs were uncovered, possibly via the regulation of GR signaling. Moreover, cytotoxic actions of Melissa officinalis and lavender EOs via the induction of mitochondrial-dependent apoptosis were revealed. Our results highlight these essentials oils' anti-inflammatory and apoptotic actions in relation to their implication on the regulation of steroid hormones' actions, uncovering their potential use in steroid therapy, with many applications in pharmaceutical and health industries as anti-cancer, anti-hyperglycemic and anti-inflammatory supplements.

Multidisciplinary docking, kinetics and X-ray crystallography studies of baicalein acting as a glycogen phosphorylase inhibitor and determination of its' potential against glioblastoma in cellular models.

Glycogen phosphorylase (GP) is the rate-determining enzyme in the glycogenolysis pathway. Glioblastoma (GBM) is amongst the most aggressive cancers of the central nervous system. The role of GP and glycogen metabolism in the context of cancer cell metabolic reprogramming is recognised, so that GP inhibitors may have potential treatment benefits. Here, baicalein (5,6,7-trihydroxyflavone) is studied as a GP inhibitor, and for its effects on glycogenolysis and GBM at the cellular level. The compound is revealed as a potent GP inhibitor against human brain GPa (Ki = 32.54 µM), human liver GPa (Ki = 8.77 µM) and rabbit muscle GPb (Ki = 5.66 µM) isoforms. It is also an effective inhibitor of glycogenolysis (IC50 = 119.6 µM), measured in HepG2 cells. Most significantly, baicalein demonstrated anti-cancer potential through concentration- and time-dependent decrease in cell viability for three GBM cell-lines (U-251 MG, U-87 MG, T98-G) with IC50 values of ∼20-55 µM (48- and 72-h). Its effectiveness against T98-G suggests potential against GBM with resistance to temozolomide (the first-line therapy) due to a positive O6-methylguanine-DNA methyltransferase (MGMT) status. The solved X-ray structure of rabbit muscle GP-baicalein complex will facilitate structure-based design of GP inhibitors. Further exploration of baicalein and other GP inhibitors with different isoform specificities against GBM is suggested.

Regulation of Energy Metabolism and Anti-Inflammatory Activities of Mastiha Fractions from Pistacia lentiscus L. var. chia.

Pistacia lentiscus L. var. chia resin (Chios Mastiha), the first natural chewing gum, is widely used in Mediterranean cuisine and has been used in traditional medicine from ancient times. Regarding its chemical composition, Chios Mastiha is known to be rich in triterpenes. Triterpenes have a similar structure to glucocorticoids (GCs), the steroid hormones that exert strong anti-inflammatory activities and play crucial roles in the regulation of cellular metabolism. To simplify the characterization of the bioactive compounds of Mastiha resin, three different polarity fractions were isolated and were further analyzed regarding their main chemical composition and an assessment of their biological activities. The biological assessment focused on the evaluation of the potential anti-proliferative, anti-inflammatory, and apoptotic activities as well as the possible interference of the three different polarity Mastiha fractions with the glucocorticoid receptor signaling, with the aim of characterizing the biochemical mechanisms of the actions of the Mastiha fraction. Applying MTT cell viability assay, luciferase/ß-galactosidase assay, and Western blot analysis showed that Chios Mastiha apolar, medium-polar, and polar fractions reduced the HEK293 cell viability in a dose-dependent manner, possibly by mitochondrial-mediated induction of apoptosis. Medium-polar and polar Mastiha fractions also suppressed the GR and NF-κΒ transcriptional activation and the p65 protein levels. These activities were accompanied by the modulation of protein levels of regulatory molecules playing a crucial role in cellular energy homeostasis, such as GR, phosphoenolpyruvate carboxykinase (PEPCK), and/or peroxisome proliferator-activated receptor alpha (PPARα), and by the induction of phosphorylation and the activation of the AMP-activated protein kinase (AMPK). The medium-polar fraction was found to be enriched in triterpenes, such as lupeol, 24Z-masticadienonic acid methyl ester, and 24Z-isomasticadienonic acid methyl ester, and it was the most active one, so we propose that triterpenes in medium-polar fraction are possibly the bioactive compounds responsible for Mastiha's regulatory actions on energy metabolism and anti-inflammatory activities via interference with GR, NF-κΒ, and AMPK signaling. This highlights its potential applications in many fields of pharmaceutical, cosmetic, and nutraceutical interest.

Increased Expression of the Mitochondrial Glucocorticoid Receptor Enhances Tumor Aggressiveness in a Mouse Xenograft Model.

Mitochondria are important organelles for cellular physiology as they generate most of the energy requirements of the cell and orchestrate many biological functions. Dysregulation of mitochondrial function is associated with many pathological conditions, including cancer development. Mitochondrial glucocorticoid receptor (mtGR) is proposed as a crucial regulator of mitochondrial functions via its direct involvement in the regulation of mitochondrial transcription, oxidative phosphorylation (OXPHOS), enzymes biosynthesis, energy production, mitochondrial-dependent apoptosis, and regulation of oxidative stress. Moreover, recent observations revealed the interaction of mtGR with the pyruvate dehydrogenase (PDH), a key player in the metabolic switch observed in cancer, indicating direct involvement of mtGR in cancer development. In this study, by using a xenograft mouse model of mtGR-overexpressing hepatocarcinoma cells, we showed increased mtGR-associated tumor growth, which is accompanied by reduced OXPHOS biosynthesis, reduction in PDH activity, and alterations in the Krebs cycle and glucose metabolism, metabolic alterations similar to those observed in the Warburg effect. Moreover, autophagy activation is observed in mtGR-associated tumors, which further support tumor progression via increased precursors availability. Thus, we propose that increased mitochondrial localization of mtGR is associated with tumor progression possible via mtGR/PDH interaction, which could lead to suppression of PDH activity and modulation of mtGR-induced mitochondrial transcription that ends up in reduced OXPHOS biosynthesis and reduced oxidative phosphorylation versus glycolytic pathway energy production, in favor of cancer cells.

Apoptotic, Anti-Inflammatory Activities and Interference with the Glucocorticoid Receptor Signaling of Fractions from Pistacia lentiscus L. var. chia Leaves.

In this study acetonic extracts of leaves of Pistacia lentiscus L. var. chia (mastiha tree) grown in the south as well as in the north Chios Greek island were isolated and further fractionated to give three different polarity fractions: apolar, medium-polar, and polar. The isolated fractions were assessed as regards their main composition, cytotoxic, anti-inflammatory activities, and interference with the glucocorticoid receptor (GR) signaling, applying cytotoxic assay, luciferase assays, and Western blot analysis of apoptosis-, energy-, and inflammation-associated molecules. Differences in cell viability have been detected among different polarity leaf fractions as well as among fractions of different plant origin with polar fractions showing the highest cytotoxicity. Fractions-induced anti-inflammatory activities and suppressive effects on the dexamethasone (DEX)-induced GR transcriptional activation were unveiled. The partition protocol of leaves fractions applied uncovers the enhanced glucocorticoid-associated biological activities of the medium-polar fractions, which may be associated with their enrichment in the triterpenoids that showed structural similarity with the glucocorticoids. A reduction in GR protein levels is observed by the fraction which is shown to be associated with the medium polar-induced proteolytic degradation of the receptor. In addition, the enhanced cytotoxic, anti-inflammatory, and potential anti-glycemic activities of the fractions from the Southern P. lentiscus L. that exclusively produce the mastiha resin, is revealed, indicating that leaves fractions from mastiha tree, similarly to mastiha tree resin, may have the potential to be further analyzed for their potent applications in the pharmaceutical cosmetic and nutraceutical fields.

Phosphorus fertilisation may induce Zn deficiency in cotton (Gossypium hirsutum) on calcareous Mediterranean soils.

On a P-poor, calcareous soil, three upland cotton (Gossypium hirsutum L.) cultivars (ST 402, ST 405, Zeta 2) were tested for 2years under three P rates (0, 13.1, 26.2kgPha-1 ). Leaf traits (SPAD values; specific leaf area, SLA; carbon isotope discrimination, Δ; 15 N natural abundance, δ15 N) and elements (N, P, K, C, Na, Zn) along with arbuscular mycorrhizal (AM) colonisation were measured at first open flower, full bloom and first open boll stages. Phosphorus addition decreased yield, but had no effect on fibre quality, a response attributed to P-induced Zn deficiency, previously reported for cereals. The best-performing cv., ST 405, had high SPAD and SLA, but the lowest P, N and Zn concentrations, an indication of cultivar's high use efficiency for these nutrients. At full bloom, SPAD was lowest, while SLA was highest. AM increased gradually with growth stages, while N, P, K and Zn concentrations showed an opposite trend, possibly due to a dilution effect. On Mediterranean calcareous soils, P fertilisation should take into account soil Zn levels in order to avoid P-Zn antagonistic relationships, which could impact negatively on yield.

Anti-Apoptotic and Antioxidant Activities of the Mitochondrial Estrogen Receptor Beta in N2A Neuroblastoma Cells.

Estrogens are steroid hormones that play a crucial role in the regulation of the reproductive and non-reproductive system physiology. Among non-reproductive systems, the nervous system is mainly affected by estrogens due to their antioxidant, anti-apoptotic, and anti-inflammatory activities, which are mediated by membranous and nuclear estrogen receptors, and also by non-estrogen receptor-associated estrogen actions. Neuronal viability and functionality are also associated with the maintenance of mitochondrial functions. Recently, the localization of estrogen receptors, especially estrogen receptor beta, in the mitochondria of many types of neuronal cells is documented, indicating the direct involvement of the mitochondrial estrogen receptor beta (mtERß) in the maintenance of neuronal physiology. In this study, cell lines of N2A cells stably overexpressing a mitochondrial-targeted estrogen receptor beta were generated and further analyzed to study the direct involvement of mtERß in estrogen neuroprotective antioxidant and anti-apoptotic actions. Results from this study revealed that the presence of estrogen receptor beta in mitochondria render N2A cells more resistant to staurosporine- and H2O2-induced apoptotic stimuli, as indicated by the reduced activation of caspase-9 and -3, the increased cell viability, the increased ATP production, and the increased resistance to mitochondrial impairment in the presence or absence of 17-ß estradiol (E2). Thus, the direct involvement of mtERß in antioxidant and anti-apoptotic activities is documented, rendering mtERß a promising therapeutic target for mitochondrial dysfunction-associated degenerative diseases.

Boswellic acids and their derivatives as potent regulators of glucocorticoid receptor actions.

Glucocorticoid (GCs) hormones exert their actions via their cognate steroid receptors the Glucocorticoid Receptors (GR), by genomic or non-genomic mechanisms of actions. GCs regulate many cellular functions among them growth, metabolism, immune response and apoptosis. Due to their cell type specific induction of apoptosis GCs are used for the treatment of certain type of cancer. In addition, due to their anti-inflammatory actions, GCs are among the most highly prescribed drug to treat chronic inflammatory disorders, albeit to the many adverse side effects arising by their long term and high doses use. Thus, there is a high need for selective glucocorticoid receptor agonist - modulators (SEGRA- SGRMs) as effective as classic GCs, but with a reduced side effect profile. Boswellic acids (BAs) are triterpenes that show structural similarities with GCs and exhibit anti-inflammatory and anti-cancer activities. In this study we examined whether BA alpha and beta and certain BAs derivatives exert their actions, at least in part, through the regulation of GR activities. Applying docking analysis we found that BAs can bind stably into the deacylcortivazol (DAC) accommodation pocket of GR. Moreover we showed that certain boswellic acids derivatives induce glucocorticoid receptor nuclear translocation, no activation of GRE dependent luciferase gene expression, and suppression of the TNF-α induced NF-κB transcriptional activation in GR positive HeLa and HEK293 cells, but not in low GR level COS-7 cells. Furthermore, certain boswellic acids compounds exert antagonistic effect on the DEX-induced GR transcriptional activation and induce cell type specific mitochondrial dependent apoptosis. Our results indicate that certain BAs are potent selective glucocorticoid receptor regulators and could have great potential for therapeutic use.

Affinity Crystallography Reveals Binding of Pomegranate Juice Anthocyanins at the Inhibitor Site of Glycogen Phosphorylase: The Contribution of a Sugar Moiety to Potency and Its Implications to the Binding Mode.

Anthocyanins (ACNs) are dietary phytochemicals with an acknowledged therapeutic significance. Pomegranate juice (PJ) is a rich source of ACNs with potential applications in nutraceutical development. Glycogen phosphorylase (GP) catalyzes the first step of glycogenolysis and is a molecular target for the development of antihyperglycemics. The inhibitory potential of the ACN fraction of PJ is assessed through a combination of in vitro assays, ex vivo investigation in hepatic cells, and X-ray crystallography studies. The ACN extract potently inhibits muscle and liver isoforms of GP. Affinity crystallography reveals the structural basis of inhibition through the binding of pelargonidin-3-O-glucoside at the GP inhibitor site. The glucopyranose moiety is revealed as a major determinant of potency as it promotes a structural binding mode different from that observed for other flavonoids. This inhibitory effect of the ACN scaffold and its binding mode at the GP inhibitor binding site may have significant implications for future structure-based drug design endeavors.

Neurotoxic effects of aluminum are associated with its interference with estrogen receptors signaling.

Aluminum compounds have been observed in various brain regions, and their accumulation has been associated with many neurodegenerative disorders. Neurotoxic effects of aluminum are attributed to reactive oxygen species generation, induction of apoptosis and inflammatory reactions activation. Metalloestrogen activity of aluminum has also been linked to breast cancer progression and metastasis. In this study, taking into account the anti-apoptotic and anti-oxidant activities of estrogens in neuronal cells, which are mediated by estrogen receptors, the possible estrogenic activity of aluminum in SH-SY5Y neuroblastoma cells was studied. Our results showed that aluminum in the form of aluminum chlorohydrate (ACH) exhibited no effect on estrogen receptors transcriptional activation, and differential effect on estrogen receptor alpha (ERα) and estrogen receptor beta (ERß) protein levels. ACH caused reduction in ERß protein levels, and increase in its mitochondrial localization. ACH-induced reduction in ERß protein level may be linked, at least in part, to the ACH-induced increase in ERα protein level. This statement is based on our observations showing aluminum-induced reduction in the E2-induced increase in ERα S118 phosphorylation, in MCF-7 and SH-SH5Y cells. Phosphorylation at S118 residue is known to be associated with inhibition of the ubiquitin-induced proteolytic degradation of ERα, leading to its accumulation. Since it is known that ERα negatively regulate ERß expression, increase in ERα, may contribute to reduction in ERß levels and subsequent weakening of its anti-apoptotic and anti-oxidant activity, justified by the observed reduction in procaspase 9, mitochondrial cytochrome c, Bcl-2, Bcl-xL and mitochondrial thioredoxin protein level, as well as by the increase in proapoptotic BAX level, in ACH treated SH-SY5Y cells. In addition, increase in mitochondrial ERß localization may also trigger mitochondrial metabolism, suppress biosynthetic process of gluconeogenesis, as indicated by the observed reduction in the phosphoenolpyruvate carboxykinase protein level, and eventually lead to increase in reactive oxygen species (ROS) generation, known to be implicated in aluminum induced neurodegeneration. This statement was verified by the observed ACH-induced increase in ERß mitochondrial localization, induction of the mitochondrial membrane depolarization and increase in ROS production, in neuronal-like differentiated SH-SY5Y cells.

Diversity and nodulation effectiveness of rhizobia and mycorrhizal presence in climbing dry beans grown in Prespa lakes plain, Greece.

The Prespa lakes plain is an isolated area where about 1000 ha are seeded to Phaseolus vulgaris L. and Phaseolus coccineus L. Nodulation, arbuscular mycorrhizal fungal (AMF) presence and the genetic diversity of rhizobia were evaluated by 16S-ITS-23S-RFLP patterns and by sequencing. The bean rhizobial population in the region was diverse, despite its geographic isolation. No biogeographic relationships were detected, apart from a Rhizobium tropici-related strain that originated from an acidic soil. No clear pattern was detected in clustering with bean species and all isolates formed nodules with both bean species. Most strains were related to Rhizobium leguminosarum and a number of isolates were falling outside the already characterized species of genus Rhizobium. Application of heavy fertilization has resulted in high soil N and P levels, which most likely reduced nodulation and AMF spore presence. However, considerable AMF root length colonization was found in most of the fields.

Potential Dissociative Glucocorticoid Receptor Activity for Protopanaxadiol and Protopanaxatriol.

Glucocorticoids are steroid hormones that regulate inflammation, growth, metabolism, and apoptosis via their cognate receptor, the glucocorticoid receptor (GR). GR, acting mainly as a transcription factor, activates or represses the expression of a large number of target genes, among them, many genes of anti-inflammatory and pro-inflammatory molecules, respectively. Transrepression activity of glucocorticoids also accounts for their anti-inflammatory activity, rendering them the most widely prescribed drug in medicine. However, chronic and high-dose use of glucocorticoids is accompanied with many undesirable side effects, attributed predominantly to GR transactivation activity. Thus, there is a high need for selective GR agonist, capable of dissociating transrepression from transactivation activity. Protopanaxadiol and protopanaxatriol are triterpenoids that share structural and functional similarities with glucocorticoids. The molecular mechanism of their actions is unclear. In this study applying induced-fit docking analysis, luciferase assay, immunofluorescence, and Western blot analysis, we showed that protopanaxadiol and more effectively protopanaxatriol are capable of binding to GR to activate its nuclear translocation, and to suppress the nuclear factor-kappa beta activity in GR-positive HeLa and HEK293 cells, but not in GR-low level COS-7 cells. Interestingly, no transactivation activity was observed, whereas suppression of the dexamethasone-induced transactivation of GR and induction of apoptosis in HeLa and HepG2 cells were observed. Thus, our results indicate that protopanaxadiol and protopanaxatriol could be considered as potent and selective GR agonist.

Potential interference of aluminum chlorohydrate with estrogen receptor signaling in breast cancer cells.

Aluminum salts are widely used as the active antiperspirant in underarm cosmetic. Experimental observations indicate that its long term application may correlate with breast cancer development and progression. This action is proposed to be attributed, among others, to aluminum possible estrogen-like activities. In this study we showed that aluminum, in the form of aluminum chlorohydrate (ACH), caused increase in estrogen receptor alpha (ERα) protein levels, in ERα-positive MCF-7 cells. This effect was accompanied by moderate activation of Estrogen Response Elements (ERE)-driven reporter gene expression and 20%-50% increase in certain estrogen responsive, ERE-independent genes expression. Genes affected were ERα, p53, cyclin D1, and c-fos, crucial regulators of breast cancer development and progression. ACH-induced genes expression was eliminated in the presence of the estrogen antagonist: ICI 182780, in MCF-7 cells, whereas it was not observed in ERα-negative MDA-MB-231 breast cancer cells, indicating aluminum interference with estrogen signaling. Moreover, ACH caused increase in the perinuclear localization of estrogen receptor alpha in MCF-7 breast cancer cells and increase in the mitochondrial Bcl-2 protein, possibly affecting receptors-mediated mitochondrial actions and mitochondrial-dependent apoptosis. ACH-induced perinuclear localization of estrogen receptor beta was also observed in MDA-MB-231. Our findings indicate that aluminum actions on estrogen receptors protein level and subcellular localization possibly affect receptors-mediated actions and thus, aluminum interference with estrogen signaling.

Urate crystals directly activate the T-cell receptor complex and induce T-cell proliferation.

Uric acid is a known danger associated molecular pattern molecule able to induce inflammation following internalization of its crystals by cells of the innate immune system. By activating antigen-presenting cells, urate boosts adaptive immunity as well. Furthermore, urate crystals can induce proliferation of isolated T-cells, which are unable to phagocytose crystal particles. In light of the evidence that urate crystals can also activate dendritic cells and macrophages without prior internalization but through sequestration of lipid rafts (and consequently receptors clustering in a non specific manner), the authors evaluated whether such a mechanism is involved in the direct activation of T-cells by urate crystals. In the present study, isolated human T-cells were cultured with or without urate at a concentration above its crystallization level. The expression and phosphorylation state of the T-cell receptor (TCR) complex zeta chain and the expression of the master regulator of cell proliferation c-Myc were assessed by western blotting. T-cell proliferation was measured by bromodeoxyuridine assay. Collectively, the results indicated that urate increased zeta chain phosphorylation indicating that it induces activation of TCR complex directly, since zeta chain phosphorylation takes place at the cell membrane and is a very proximal event in TCR complex-mediated signal transduction. In parallel, urate increased the expression of the transcription factor c-Myc and induced T-cell proliferation. In conclusion, urate crystals directly activate the TCR complex and induce T-cell proliferation.

In the wild hybridization of annual Datura species as unveiled by morphological and molecular comparisons.

BACKGROUND: The present work aimed to verify whether intermediate variants were natural crosses between Datura species (D. stramonium forms and D. ferox). Their existence has been long ago insinuated but has not been studied using morphological features and molecular tools. The variants differed in stem coloring, upper bearing forks, and fruit characters. RESULTS: Principal Components Analysis of 11 morphological characteristics showed that D. ferox and D. stramonium (forms stramonium and tatula) were quite different and the putative hybrids were intermittent. The D. ferox × D. stramonium f. tatula was closer to the latter of its parents. Sequencing analysis revealed identical amplified trnL intron in all variants and a 100% homology with D. stramonium accession number EU580984.1 suggested that this plastid cannot discern Datura variants. However, genomic analysis with URP markers indicated that the hybrids had >60% genetic makeup similarity with both parents suggesting that the intermediate variants were putative inter-specific hybrids. Moreover, the dendrogram stemmed from cluster analysis of the fingerprint profile of variants placed D. stramonium and D. ferox in different branches indicating their genetic differentiation from each other as well as from their hybrids. CONCLUSIONS: The findings suggest that the natural hybridization of annual Datura species occurs. Extrapolating, this hybridization could be the first step for speciation. More possibly, it can alter population composition, its weediness and adaptability to local conditions.