Acceptor Plants Research Articles

Pyrrolizidine Alkaloids in Food on the Italian Market.

Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by over 6000 plant species worldwide. PAs enter the food chain through accidental co-harvesting of PA-containing weeds and through soil transfer from the living plant to surrounding acceptor plants. In animal studies, 1,2-unsaturated PAs have proven to be genotoxic carcinogens. According to the scientific opinion expressed by the 2017 EFSA, the foods with the highest levels of PA contamination were honey, tea, herbal infusions, and food supplements. Following the EFSA's recommendations, data on the presence of PAs in relevant food were monitored and collected. On 1 July 2022, the Commission Regulation (EU) 2020/2040 came into force, repealed by Commission Regulation (EU) 2023/915, setting maximum levels for the sum of pyrrolizidine alkaloids in certain food. A total of 602 food samples were collected from the Italian market between 2019 and 2022 and were classified as honey, pollen, dried tea, dried herbal infusions, dried herbs, and fresh borage leaves. The food samples were analyzed for their PA content via an in-house LC-MS/MS method that can detect PAs according to Regulation 2023/915. Overall, 42% of the analyzed samples were PA-contaminated, 14% exceeded the EU limits, and the items most frequently contaminated included dried herbs and tea. In conclusion, the number of food items containing considerable amounts of PAs may cause concern because they may contribute to human exposure, especially considering vulnerable populations-most importantly, children and pregnant women.

Novel Cognitions in Allelopathy: Implications from the "Horizontal Natural Product Transfer".

Whereas the translocation of allelochemicals between plants is well established, a related general transfer of genuine specialized metabolites has not been considered so far. The elucidation of the so-called "Horizontal Natural Product Transfer" revealed that alkaloids, such as nicotine and pyrrolizidine alkaloids, which are leached out from decomposing alkaloid-containing plants (donor plants), are indeed taken up by the roots of plants growing in the vicinity (acceptor plants). Further studies demonstrated that phenolic compounds, such as coumarins or stilbenes, are also taken up by acceptor plants. Contemporary analyses from co-cultivation experiments outlined that natural products are not exclusively transferred from dead and rotting donor plant materials, but also from vital plants. In analogy to xenobiotics, the imported specialized metabolites might also be modified within the acceptor plants. As known from the uptake of xenobiotics, the import of specialized metabolites is also generally due to a simple diffusion of the substances across the biomembranes and does not require a carrier. The uptake depends in stricto sensu on the physicochemical properties of the certain compound. This article presents a current overview of the phenomenon of "Horizontal Natural Product Transfer" and discusses its relevance for our understanding of allelopathic interactions. The knowledge that specialized metabolites might in general be readily translocated from one plant into others should significantly contribute to our understanding of plant-plant interactions and-in particular-to the evolution of typical allelopathic effects, such as inhibition of growth and germination of potential competitors.

ROS Metabolism Perturbation as an Element of Mode of Action of Allelochemicals.

The allelopathic interaction between plants is one of the elements that influences plant communities. It has been commonly studied by applying tissue extracts onto the acceptors or by treating them with isolated allelotoxins. Despite descriptive observations useful for agricultural practice, data describing the molecular mode of action of allelotoxins cannot be found. Due to the development of -omic techniques, we have an opportunity to investigate specific reactive oxygen species (ROS)-dependent changes in proteome or transcriptome that are induced by allelochemicals. The aim of our review is to summarize data on the ROS-induced modification in acceptor plants in response to allelopathic plants or isolated allelochemicals. We present the idea of how ROS are involved in the hormesis and plant autotoxicity phenomena. As an example of an -omic approach in studies of the mode of action of allelopatic compounds, we describe the influence of meta-tyrosine, an allelochemical exudated from roots of fescues, on nitration—one of nitro-oxidative posttranslational protein modification in the roots of tomato plants. We conclude that ROS overproduction and an induction of oxidative stress are general plants’ responses to various allelochemicals, thus modification in ROS metabolisms is regarded as an indirect mode of action of allelochemicals.

Effects of Ozone (O3) on leaf secretory cell characteristics related to allelopathy of woody plants: Modelling allelopathic Interactions

We studied the effects of ozone (O3) on the allelopathic medicinal trees/shrubs that influenced other species growing under their canopy in National Park Sochi, Russia (Caucasus region). Using model reactions, the action of rain leachates from these allelopathic trees/shrubs on the plants growing under their canopy was determined. Depending on the duration and intensity of exposure, the O3 treatment of test woody plants changed their (i) characteristics of leaves and their water extracts, (ii) colour and (iii) autofluorescence of secretory cells. Water extracts from the allelopathic woody donor plants served as model of rain leachates, and their effects were tested on the seeds and pollens germination of acceptor plants (Lavatera trimestris L. var.Rubin and Hippeastrum (Herb.) hybridum ). The effects of water extracts from the ozone treated plants differed from the untreated plants. The allelochemicals in donor plants stimulated, inhibited or had no effects on the recipient plants compared with control. The test-reactions in tropospheric ozone stress should be used in Modelling System of tree/shrub-herbaceous plants interactions, to find the allelopathic interactions in urban conditions.

Improving the phytochemical profile and bioactivity of Chinese cabbage sprouts by interspecific transfer of metabolites

The high demand for healthy food in recent years has led to an increasing need for highly bioactive plant materials. One such plant, Chinese cabbage, possesses flavonoids with antioxidant and antidiabetic properties, but they appear in low quantities. The interspecific transfer of metabolites is a promising technique that could contribute to the increase of the beneficial properties of food. The objective of the study was to determine how an interspecific source-sink phytochemical transfer from donor extracts to the Chinese cabbage affects its phenolic and vitamin C profile and intestinal bioaccessibility, hypoglycemic potential and antioxidant capacity. In addition, sprouts treated with Rosa sp. and Hypericum perforatum extracts showed better antiproliferative effect towards human breast adenocarcinoma cells than untreated sprouts. The results suggest that treatment of plants with donor extracts is a promising approach to improve the nutritional and phytochemical profile and bioactive properties of acceptor plants.

The uptake of alkaloids by plants from the soil is determined by rhizosphere pH

A tremendous high number of herbal products such as spices, herbal teas, or phytopharmaceuticals, contain significant amounts of toxic alkaloids. These widespread contaminations are mainly due to the uptake of alkaloids from the soil. Due to their alkalinity, it is obvious that the import of alkaloids is strongly affected by the pH of the rhizosphere. This so far unconsidered issue was analyzed in a model study by applying various alkaloids to hydroponically grown barley seedlings, adjusted to several pH values, i.e., 5, 6, 7, and 8. In most cases, the resulting concentration of the alkaloids imported into the barley leaves ranged from about 0.8 to 7.7 μmol/g d.w. However, caffeine was imported to a higher extent, resulting in concentration up to 19 μmol/g d.w. In contrast, the uptake of monocrotaline (0.04–0.13 μmol/g d.w.) and atropine (just traces) was quite lower. For sparteine, no uptake was recorded. Apart from caffeine, the uptake of alkaloids strongly depends on the pH in the medium. Accordingly, these experiments demonstrated the significance of the rhizosphere pH for the alkaloid uptake. Both parameters, the overall concentration of alkaloids as well as the share of the unprotonated alkaloids, are determined by the pH. Accordingly, for the first time, it became obvious that the alkaloidal contaminations of plant derived commodities significantly depend on the pH of the rhizosphere during the cultivation of the crop plants. Moreover, the extent of alkaloid accumulation in acceptor plants is also influenced by the rate of transpiration. In consequence, the uptake of the various alkaloids and its pH-dependency might be quite different and no general prediction on the extent of alkaloid uptake can be made.

Pilot study on the uptake and modification of harmaline in acceptor plants: An innovative approach to visualize the interspecific transfer of natural products

Substances which have been leached out from decomposing plant parts or exuded from vital plants (donor plants), are taken up by acceptor plants and subsequently modified. This phenomenon was likewise established for harmala alkaloids. Employing hydroponically grown barley seedlings, it becomes evident that harmaline and harmine are taken up by the roots of the acceptor plants. Furthermore, based on HPLC and GC-MS analyses, it was demonstrated that these alkaloids also are present in Setaria viridis plants, which grew in the direct vicinity of the alkaloid containing Peganum harmala plants.Since harmaline exhibits a bright green fluorescence, this alkaloid was employed to visualize the uptake into the acceptor plants by feeding it to roots of barley seedlings. In the further course, the imported harmaline was converted in the leaves to yield harmine, which exhibits a dark blue fluorescence. This conversion was also verified by HPLC and GC-MS analyses. Based on the massive differences in the fluorescence properties, both processes, uptake and modification in the acceptor plants, could be monitored by macroscopical studies as well as by confocal laser scanning microscopical analyses. As result, for the first time, the phenomenon of ”Horizontal Natural Product Transfer” is visualized vividly.

ВЛИЯНИЕ ВОЗДУШНЫХ ЭКЗОМЕТАБОЛИТОВ РАСТЕНИЙ И ИХ ИНФОРМАЦИОННО-ЭНЕРГЕТИЧЕСКИХ ПОЛЕЙ НА БИОТЕСТЫ

The paper demonstrates that air exometabolites of plant natural and cut leaves and flowers are proved to effect the bio tests – seed germination. The degree and character of this influence quantity and character differ for various donor and acceptor plants. The biological action of products of plants transpiration remains under its accumulation in the activated carbon and frozen water. It is shown that the interaction of some plants on the others is caused by the interrelation of both aboveground organs and root systems. So,under beetroot seed generation the size of the seedlings roots in the controlled variant was 5.5 ± 0.9 cm, the stem size was 4.6 ± 06 cm. When growing sorrel and beetroot seeds together,the beetroot size of the roots was 3.1 ± 1.0 cm; the stem size was 3.1 ± 1.0 cm. It seems feasible to take into consideration this influence under combined planting of several crops.

Horizontal Natural Product Transfer: Intriguing Insights into a Newly Discovered Phenomenon.

Just recently, the "horizontal natural product transfer" was unveiled: alkaloids, which have been leached out from decomposing alkaloidal donor plants, are taken up by the roots of acceptor plants. In the same manner, many other natural products, such as coumarins or stilbenes, are also taken up from the soil. Recent research outlined that alkaloids are transferred also from a living donor plant to plants growing in their vicinity. In the acceptor plants, the imported natural products might be modified by hydroxylation and glucosylation. These insights will strongly impact our understanding of contamination of plant-derived commodities as well as plant-plant interactions.

Transfer of pyrrolizidine alkaloids between living plants: A disregarded source of contaminations

To elucidate the origin of the wide-spread contaminations of plant derived commodities with various alkaloids, we employed co-cultures of pyrrolizidine alkaloid (PA) containing Senecio jacobaea plants with various alkaloid free acceptor plants. Our analyses revealed that all plants grown in the vicinity of the Senecio donor plants indeed contain significant amounts of the PAs, which previously had been synthesized in the Senecio plants. These findings illustrate that typical secondary metabolites, such as pyrrolizidine alkaloids, are commonly transferred and exchanged between living plants. In contrast to the broad spectrum of alkaloids in Senecio, in the acceptor plants nearly exclusively jacobine is accumulated. This indicates that this alkaloid is exuded specifically by the Senecio roots. Although the path of alkaloid transfer from living donor plants is not yet fully elucidated, these novel insights will extend and change our understanding of plant-plant interactions and reveal a high relevance with respect to the widespread alkaloidal contaminations of plant-derived commodities. Moreover, they could be the basis for the understanding of various so far not fully understood phenomena in cultivation of various crops, e.g. the beneficial effects of crop rotations or the co-cultivation of certain vegetables.

Uptake and modification of umbelliferone by various seedlings

Inspired by the recently discovered phenomenon of “horizontal natural product transfer” we investigated the putative uptake of phenolic specialized metabolites. Umbelliferone was chosen for this case study, since this coumarin as well as its derivatives can easily be determined by HPLC analyses. Barley (Hordeum vulgare L.), radish (Raphanus sativus L.), pea (Pisum sativum L.), flax (Linum usitatissimum L.), and garden cress (Lepidium sativum L.) were cultivated in hydroponic media, to which the coumarin was applied. Uptake of umbelliferone was verified by corresponding HPLC analyses of extracts obtained from the aerial parts of the seedlings.In all cases, a tremendous uptake of umbelliferone was observed. In plants that genuinely contain coumarins, the umbelliferone taken up was modified: in garden cress, it was hydroxylated and glucosylated to yield esculin, while in barley seedlings, the imported umbelliferone was modified by methoxylation to yield scopoletin. Corresponding reactions are known from modifications of xenobiotics to be catalyzed by cytochrome P450 enzymes. Accordingly, in an additional approach, umbelliferone was applied together with naproxen, which is reported to reduce enzyme activity of P450 enzymes. As predicted, the conversion of umbelliferone to scopoletin in barley as well as the modification to esculin in garden cress was strongly reduced by the addition of naproxen.These data for the first time demonstrate that – in addition to alkaloids – also phenolic compounds are taken up by various acceptor plants. Apart from the leaching of rotting plants, coumarins are known to be exuded by many plants. Accordingly, these compounds are frequently present in soils and will be taken up. These coherences imply that the horizontal natural product transfer might represent a more general phenomenon in plant ecology. Moreover, this study outlines that – in analogy to the modification of xenobiotics – also natural products taken up are modified in the acceptor plants.

Uptake of nicotine from discarded cigarette butts – A so far unconsidered path of contamination of plant-derived commodities

This study aimed to elucidate the origin of the widespread nicotine contamination of plant-derived commodities, by conducting field experiments with various herbs and spice plants. By scattering tobacco and cigarette butts on the field and subsequent nicotine analyses of the acceptor plants, we verified that the alkaloid is leached out into the soil and is taken up by the crop plants. This path of contamination pertains even when there is only one cigarette butt per square meter. Even such minor pollution results - at least in the case of basil and peppermint - in considerable high nicotine contaminations, which exceed the maximum residue level by more than 20-fold.The data reported here clearly outline the large practical relevance of this soil-borne contamination path and imply that unthoughtful disposal of cigarette butts in the field by farm workers may be the reason for the widespread occurrence of nicotine contamination in plant-derived commodities. Therefore, such misbehavior needs to be prevented using education and sensitization, and by including this issue into the guidelines of good agricultural practice.

Horizontal natural product transfer: A potential source of alkaloidal contaminants in phytopharmaceuticals

BackgroundIt was recently shown that nicotine and pyrrolizidine alkaloids that leach out from decomposing plant material (donor plants) are subsequently taken up by the roots of acceptor plants and translocated into their leaves. Furthermore, it is well established that plant roots take up xenobiotics, generally by simple diffusion, and that this passive import depends on the physico-chemical properties of the substances. HypothesisBased on the well-known uptake of xenobiotics, we assumed that in analogy, the uptake of alkaloids, which are leached out from plant material (donor plants) represents a quite general feature of plant biology. MethodsUsing barley as a model plant, we analyzed the uptake of alkaloids by applying them to Hordeum vulgare seedlings. Based on HPLC analyses, the presence of the particular alkaloids in the acceptor plants was determined. ResultsWe demonstrated that numerous alkaloids of different structural types are able to diffuse through biomembranes and are taken up by acceptor plants. In contrast, an uptake of quaternary alkaloids, with a permanent positive charge, could not be detected. ConclusionAs most alkaloidal plants generally die back afield, and the corresponding natural products are leached out into the soil. Our findings have substantial relevance for all plant-derived commodities, especially for the production of phytopharmaceuticals and the related safety issues. Moreover, the evidence that plants are inherently able to take up alkaloids from the soil, which are derived from other plants, will alter our appraisal of plant–plant interactions. In this context, the classical definition of xenobiotics, which are considered as “non-natural” substances, might be also extended by including natural products leached out into the soil.

Interspecific transfer of pyrrolizidine alkaloids: An unconsidered source of contaminations of phytopharmaceuticals and plant derived commodities

Many plant derived commodities contain traces of toxic pyrrolizidine alkaloids (PAs). The main source of these contaminations seems to be the accidental co-harvest of PA-containing weeds. Yet, based on the insights of the newly described phenomenon of the horizontal transfer of natural products, it is very likely that the PA-contaminations may also be due to an uptake of the alkaloids from the soil, previously being leached out from rotting PA-plants. The transfer of PAs was investigated using various herbs, which had been mulched with dried plant material from Senecio jacobaea. All of the acceptor plants exhibited marked concentrations of PAs. The extent and the composition of the imported PAs was dependent on the acceptor plant species.These results demonstrate that PAs indeed are leached out from dried Senecio material into the soil and confirm their uptake by the roots of the acceptor plants and the translocation into the leaves.

Phytotoxic Effects of Cyanamide on Seed Germination and Seedling Growth of Weed and Crop Species

We examined the response of plants of various crop and weed species to cyanamide in order to evaluate allelochemical-mediated interactions between the species. We studied germination and seedling growth in the common weeds Galium aparine L. and Amaranthus retroflexus L., and the crops Zea mays L., Triticum aestivum L., Lactuca sativa L., Solanum lycopersicum L. and Sinapis alba L. as acceptor plants. Concentration-dependent phytotoxic effects of cyanamide were noted during seed germination and in the root and shoot growth of the tested plants. The monocotyledonous plants generally were less sensitive to cyanamide treatment. Seed germination and seedling growth of the dicotyledonous plants were strongly inhibited by the allelochemical at both tested concentrations (1.2 mM, 3 mM). We conclude that cyanamide has potential for use as a natural herbicide only in specific field systems of cyanamide-tolerant monocotyledonous crops accompanied by cyanamide-sensitive dicotyledonous weeds.

Allelopathic activity of some grass species on Phleum pratense seed germination subject to their density

Efficient utilization of allelopathy in the agricultural practice requires searching for some species and developmental stages when the allelopathic substances are generated in bioactive concentrations. That also requires the knowledge of allelopathy mechanisms and primarily its separation from the other aspects of plant activity, mainly from competition for environmental resources. This task, however, has remained vital in the studies on plant interference, being extremely difficult to perform under field conditions. Therefore, the studies were conducted in the laboratory. To determine the activity of an allelopathic agent of the selected grass species, the density dependent phytotoxicity model was employed. The model is based on the fact that an increase of acceptor plants density evokes a decrease of their response to the allelopathic compounds, whereas the negative effects of the competition become more intense. A higher rate of acceptor plants growth accompanying their density increase in the given object does not agree with the competition rules and thus, it may imply an allelopathic background of the observed changes. In the presented studies, the allelopathic properties of grasses - donors were evaluated by studying the effect of two densities of the emerging seeds and two- and four weeks aged seedlings of <i>F. arundinacea</i>, <i>L. multiflorum</i>, <i>L. perenne</i> and <i>P. pratensis</i>. The tested species - acceptor <i>Ph. pratensis</i> was sown in the density of 10 and 20 seeds in a pan. The results revealed that the germination of acceptor seeds was differentiated depending on their density in the pan, and on the species, density and the age of the donor. Inhibition of <i>Ph. pratense</i> seed germination in objects with a lover density may prove allelopathic effects of the studied donor grasses.

Expression Patterns of Glutathione Transferase Gene (GstI) in Maize Seedlings Under Juglone-Induced Oxidative Stress

Juglone (5-hydroxy-1,4-naphthoquinone) has been identified in organs of many plant species within Juglandaceae family. This secondary metabolite is considered as a highly bioactive substance that functions as direct oxidant stimulating the production of reactive oxygen species (ROS) in acceptor plants. Glutathione transferases (GSTs, E.C.2.5.1.18) represent an important group of cytoprotective enzymes participating in detoxification of xenobiotics and limiting oxidative damages of cellular macromolecules. The purpose of this study was to investigate the impact of tested allelochemical on growth and development of maize (Zea mays L.) seedlings. Furthermore, the effect of juglone-induced oxidative stress on glutathione transferase (GstI) gene expression patterns in maize seedlings was recorded. It was revealed that 4-day juglone treatment significantly stimulated the transcriptional activity of GstI in maize seedlings compared to control plants. By contrast, at the 6th and 8th day of experiments the expression gene responses were slightly lower as compared with non-stressed seedlings. Additionally, the specific gene expression profiles, as well as the inhibition of primary roots and coleoptile elongation were proportional to juglone concentrations. In conclusion, the results provide strong molecular evidence that allelopathic influence of juglone on growth and development of maize seedlings may be relevant with an induction of oxidative stress in acceptor plants.

Development of an in vitro System for the Evaluation of Allelopathic Activities of Asparagus Calluses

An aseptic bioassay system to estimate allelopathy in asparagus calluses was established. Allelopathic activities of calluses were measured according to the callus packed cell volume (PCV) by means of the sandwich method, and remarkable inhibition of acceptor plants was observed by the addition of over 0.1 mL PCV. When female and male calluses were separately used for the sandwich method, there was no sexual difference in allelopathic activity. To detect the allelopathic activities of the callus cells in detail, we improved the plant box method that substituted the calluses for the seedlings, and serious inhibition of acceptor plants was observed with the addition of over 0.35 mL PCV as donor material. Allelopathic activities were accelerated by irradiating the calluses with light-emitting diodes (LEDs) prior to performing the aseptic bioassay. It also seemed that the light quality would affect their activities. The allelopathic activity appeared intensely with irradiation of the calluses with a blue LED. The aseptic bioassay system that employed LED-irradiated calluses established here could accelerate effectively the isolation and identification of allelochemical(s).

プラントボックス法における活性炭を利用したアレロパシーの証明法

プラントボックス法における活性炭を利用したアレロパシーの証明法

Translocation of exogen phenoloids and alkaloids in acceptor plants -histo- and phytochemical characteristics

Phenoloids with allelopathic effect (juglone, ctaechin, tannin, gallic acid t-cinnamic acid, caffeic acid, coumarin, thymol, salicin in 1mM concentration) cannot be detected after the absorption from the acceptor plant (bean) by the applied selective analytical method (TLC densitometry). Their localisation can be determined by histochemical reagens (ferrichloride, potassium bichromate, sodium hydroxide). In the foliage leaves and excised bean plants they are present mainly in the parenchymatuos elements of the vascular tissue already on the 3rd day, at the beginning of wilting. Some substances (tropanes) out of the studied allelopathic alkaloids (atropine, scopolamine, belladonnin, tropine and caffeine) can be detected only in small amounts (7-8%) or only in traces in the leaf. Others (e.g. caffeine) accumulate in substantial amount (almost 200%) in their original form. Alkaloids,as well as phenoloids, can be detected in lesf tissue by histochemical methods (Dragendorff and Meyer reagents).