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 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.

Impact of drought stress on specialised metabolism: Biosynthesis and the expression of monoterpene synthases in sage (Salvia officinalis).

In previous experiments, we demonstrated that the amount of monoterpenes in sage is increased massively by drought stress. Our current study is aimed to elucidate whether this increase is due, at least in part, to elevated activity of the monoterpene synthases responsible for the biosynthesis of essential oils in sage. Accordingly, the transcription rates of the monoterpene synthases were analyzed. Salvia officinalis plants were cultivated under moderate drought stress. The concentrations of monoterpenes as well as the expression of the monoterpene synthases were analyzed. The amount of monoterpenes massively increased in response to drought stress; it doubled after just two days of drought stress. The observed changes in monoterpene content mostly match with the patterns of monoterpene synthase expressions. The expression of bornyl diphosphate synthase was strongly up-regulated; its maximum level was reached after two days. Sabinene synthase increased gradually and reached a maximum after two weeks. In contrast, the transcript level of cineole synthase continuously declined. This study revealed that the stress related increase of biosynthesis is not only due to a "passive" shift caused by the stress related over-reduced status, but also is due - at least in part-to an "active" up-regulation of the enzymes involved.

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.

13,14-dihydrocoptisine--the genuine alkaloid from Chelidonium majus.

The genuine major benzylisoquinoline alkaloid occurring in the traditional medicinal plant greater celandine (Chelidonium majus L.) is 13,14-dihydrocoptisine and not - as described previously - coptisine. Structure of 13,14-dihydrocoptisine was elucidated. The discrepancy between the alkaloid pattern of the living plants and that of detached and dried leaves is due to the rapid and prompt conversion of 13,14-dihydrocoptisine to coptisine in the course of tissue injuries. Indeed, apart from the major alkaloid, some minor alkaloids might also be converted; this however is not in the centre of focus of this paper. This conversion is initiated by the change of pH. In vivo 13,14-dihydrocoptisine is localized in the acidic vacuoles, where it is stable. In contrast, in the neutral milieu, which results when vacuoles are destroyed in the course of tissue injuries, the genuine alkaloid is oxidized to yield coptisine. Accordingly, when alkaloids from C.majus should be analyzed, any postmortal conversion of 13,14-dihydrocoptisine has to be prevented.

Biochemical heterogeneity of malt is caused by both biological variation and differences in processing: I. Individual grain analyses of biochemical parameters in differently steeped barley (Hordeum vulgare L.) malts.

Using individual grain analyses, the degree of inherent biological variation in germinating barley seeds has been established. Even under homogenous laboratory conditions, the activities of the germination-related enzymes α-amylase, ß-amylase and ß-glucanase varied by a factor of two to three. The comparison with single grain analyses of different industrially produced malts (steeping systems without aeration, with air suction and pressurised aeration) revealed that the heterogeneity of these malts nearly tripled. This increase may be due to the gradients in O2 and CO2 that arise in large industrial steeping vessels. The most homogenous malting in the industrial systems was achieved without any aeration during steeping. Therefore, to improve homogeneity, the common practise of steep aeration should be omitted. Germination progression was quite different within the three exhaustively aerated attempts, which indicated that gaseous composition was not the only factor affecting germination progression.

Effects of oregano on performance and immunmodulating factors in weaned piglets.

Many health effects can be attributed to the Mediterranean herb oregano (Origanum vulgare L.) and several studies demonstrated the improving effect on performance, changes in blood count, antibacterial, antifungal and immunmodulating abilities. The majority of these investigations were carried out with processed essential oil, while whole plant material was only used in a few studies. Thus, the aim of the present experiment was to test the effect of increasing proportions of dried oregano in piglet feed on health and performance, with a special focus on immune modulation. A total of 80 male castrated weaned piglets (body weight [BW] 7.9 kg ± 1.0 kg) were used in a feeding experiment lasting 5 weeks. They were assigned to 4 experimental groups: a control diet, and three diets with an oregano supplementation at 2 g, 4 g and 8 g per kg feed, respectively, corresponding to 23.5 mg, 46.9 mg and 93.9 mg carvacrol/kg DM. After 3 weeks, half of each group was challenged with 5 µg lipopolysaccharides (LPS) per kg BW. Blood samples were collected 2 h after LPS stimulation and analysed for T-cell phenotypes, granulocyte activity, clinical-chemistry as well as white and red blood count. The results indicate no effects of oregano on performance. In contrast, oregano altered the lymphocyte proportion and the ratio of CD4(+) and CD8(+) T-cells as well as the triglyceride concentration in the serum of non-stimulated and in LPS-stimulated piglets. In conclusion, whole plant supplementation of oregano to piglet feed altered immune-related parameters, but did not modulate the acute inflammatory response induced by LPS stimulation.

Stress enhances the synthesis of secondary plant products: the impact of stress-related over-reduction on the accumulation of natural products.

Spice and medicinal plants grown under water deficiency conditions reveal much higher concentrations of relevant natural products compared with identical plants of the same species cultivated with an ample water supply. For the first time, experimental data related to this well-known phenomenon have been collected and a putative mechanistic concept considering general plant physiological and biochemical aspects is presented. Water shortage induces drought stress-related metabolic responses and, due to stomatal closure, the uptake of CO2 decreases significantly. As a result, the consumption of reduction equivalents (NADPH + H(+)) for CO2 fixation via the Calvin cycle declines considerably, generating a large oxidative stress and an oversupply of reduction equivalents. As a consequence, metabolic processes are shifted towards biosynthetic activities that consume reduction equivalents. Accordingly, the synthesis of reduced compounds, such as isoprenoids, phenols or alkaloids, is enhanced.

Sulfate determines the glucosinolate concentration of horseradish in vitro plants (Armoracia rusticana Gaertn., Mey. & Scherb.).

BACKGROUND: Horseradish plants (Armoracia rusticana) contain high concentrations of glucosinolates. Former studies have revealed that Armoracia plants cultivated in vitro have markedly lower glucosinolate concentrations than those grown in soils. Yet, these studies neglected that the sulfate concentration in the growth medium may have had a strong impact on glucosinolate metabolism. Accordingly, in this study horseradish in vitro plants were cultivated with differing sulfate concentrations and the glucosinolate concentrations were quantified by ion pair HPLC. RESULTS: Cultivation in 1.7 mmol L(-1) sulfate (as used in the prior studies) resulted in the accumulation of 16.2 µmol g(-1) DW glucosinolates, while the glucosinolate concentration increased to more than 23 µmol g(-1) DW when 23.5 mmol L(-1) sulfate was used in the medium. Correspondingly, the glucosinolate concentration decreased to 1.6 µmol g(-1) DW when sulfate concentration was lowered to 0.2 mmol L(-1). CONCLUSION: Since the glucosinolate accumulation in relation to the sulfate concentration follows a typical saturation curve, we deduce that the availability of sulfate determines the glucosinolate concentration in horseradish in vitro plants.

Malting revisited: Germination of barley (Hordeum vulgare L.) is inhibited by both oxygen deficiency and high carbon dioxide concentrations.

During malting, barley (Hordeum vulgare L.) seeds are germinated to promote the mobilisation of storage compounds. Germination is strongly influenced by O2 and CO2; however, any distinction between the particular effects is missing. Since, in this study, the ambient O2 concentration was maintained when high CO2 concentrations were applied, for the first time the impacts of CO2 and of O2 deficiency could be distinguished unambiguously. Germination was inhibited by both O2 deficiency and high CO2 (80%) concentrations, documented by the lack of any growth of coleoptiles and any increase of α-amylase and ß-glucanase activity. In contrast, the related impacts of O2 starvation and high CO2 on fermentation differ strongly, demonstrated by quite different patterns of ethanol emission. Additionally, the stress metabolism - monitored by the means of GABA accumulation - was also differently impacted. The elucidation of the underlying, so far unknown, mechanisms will provide novel opportunities to improve malting.

Stress metabolism in green coffee beans (Coffea arabica L.): expression of dehydrins and accumulation of GABA during drying.

In order to produce tradeable standard green coffee, processed beans must be dried. The drying procedure affects the abundance of relevant aroma substances, e.g. carbohydrates. Using molecular tools, the corresponding metabolic basis is analyzed. A decrease in water potential of the still living coffee seeds induces massive drought stress responses. As a marker for these stress reactions, accumulation of a general stress metabolite, GABA (gamma-aminobutyric acid), and associated gene expression of drought stress-associated dehydrins were monitored. The results of this study indicate that metabolism in drying coffee beans is quite complex since several events trigger accumulation of GABA. The first peak of GABA accumulation during drying is correlated with expression of isocitrate lyase and thus with ongoing germination processes in coffee seeds. Two subsequent peaks of GABA accumulation correspond to maxima of dehydrin gene expression and are thought to be induced directly by drought stress in the embryo and endosperm tissue, respectively. Apart from the significance for understanding basic seed physiology, metabolic changes in coffee seeds during processing provide valuable information for understanding the role and effect of the steps of green coffee processing on the quality of the resulting coffee.

The glucosinolate-myrosinase system in nasturtium (Tropaeolum majus L.): variability of biochemical parameters and screening for clones feasible for pharmaceutical utilization.

Leaves of Tropaeolum majus L. contain high amounts of the glucosinolate glucotropaeolin. They are used in traditional medicine to treat infections of the urinary tract. When Tropaeolum leaves are consumed, glucotropaeolin is hydrolyzed to yield mustard oils, which are absorbed in the intestine and excreted in the urine, exhibiting their antimicrobial activity. For a corresponding phytopharmacon, a sufficiently high glucotropaeolin concentration is required and any degradation of glucosinolates while drying must be minimized, i.e. the post mortal cleavage by myrosinases, which are activated by ascorbic acid. In extensive screenings, the dominant parameters determining the glucotropaeolin content in the dried leaves were quantified. It turned out that the glucotropaeolin concentration in the dried leaves represented the most suitable screening parameter. The screening of several hundred Tropaeolum plants resulted in the selection of eight high-yield varieties, from which in vitro plants had been generated and propagated as a source for large field trials.

A novel approach for reliable activity determination of ascorbic acid depending myrosinases.

Up to now, a wide array of methods for the determination of myrosinase activity has been described. These vary from the simple photometric estimation to highly sophisticated assays using radioactively labelled substrates. However, ascorbic acid--an effective activator of myrosinases--interferes with most of these enzyme tests. Unfortunately, in the past, such interferences were disregarded in many scientific examinations of myrosinases. Whereas such failings have less effects when the activation of myrosinases is not very distinctive, they are quite relevant in all cases where myrosinases are completely inactive in the absence of ascorbic acid. In this paper, the current methods for myrosinase determination are reviewed critically with special emphasis on putative interferences with ascorbic acid. Thereafter, an alternative and interference-free HPLC-based quantification method of the enzymatically produced glucose is presented. Due to the benzoylation of glucose, it becomes possible to quantify even those exiguous glucose concentrations, which are indispensable for correct determination of kinetic enzyme data in the presence of ascorbic acid. Using this new method, the activity of Tropaeolum majus myrosinase towards glucotropaeolin was analyzed. This enzyme shows a distinctive activation by ascorbic acid with maximal activation at a concentration of about 2 mM.