Influence of Harvest Date and Postharvest Treatment on Carotenoid and Flavonoid Composition in French Marigold Flowers.

The aim of this study was to characterize carotenoids and flavonoids present in French marigold flowers (Tagetes patula "Durango Red") as well as to assess the effects of harvest dates and postharvest treatments on these compounds. Carotenoids and flavonoids were quantified using their respective standards or semiquantified using relevant related standards. Lutein and its derivatives largely dominated the flower carotenoids, while the flavonoids were diverse with patuletin leading the list. Of the different postharvest treatments, ensilage leads to the highest content of carotenoids (from 5.0 to 7.8 g kg-1 dw) and flavonoids (from 19 to 50 g kg-1 dw). The composition of individual flavonoids was greatly influenced by different postharvest treatments, while the influence of harvest dates was secondary. Ensilage and drying induced separate metabolic pathways leading to degradation of high-molecular glycosidic compounds, converting the compounds either to their aglycones or into the intermediate flavonoid glycosides. We conclude that according to the intended industrial application, different postharvest techniques can be employed to acquire desired flavonoids on a large scale.

Plant growth promotion of Miscanthus × giganteus by endophytic bacteria and fungi on non-polluted and polluted soils.

Putative endophytes of Miscanthus × giganteus were isolated, and screened in the laboratory, greenhouse and field for their plant growth promoting properties in this host. Pantoea ananatis and Pseudomonas savastanoi were the predominant bacteria in leaves whereas other pseudomonads prevailed in roots. Almost all fungal endophytes belonged to the Pezizomycotina and most were isolated from roots; Fusarium oxysporum was most abundant, followed by the genera Periconia, Exophiala, Microdochium and Leptodontidium. All endophytic groups produced phytohormones and some bacteria also produced siderophores, solubilised P and exhibited ACC-deaminase activity in vitro. In subsequent pot experiments with pre-selected endophytes, several isolates including pseudomonads, Variovorax paradoxus, Verticillium leptobactrum, Halenospora sp. and Exophiala sp. enhanced Miscanthus growth in gamma-sterilised soil. These promising Miscanthus-derived isolates were tested either as single or mixed inocula along with a mixed bacterial inoculum originating from poplar. No significant effects of inocula were detected in a pot experiment in non-sterilised soil. On two marginal field sites the mixture of bacterial endophytes from poplar had a consistently negative effect on survival and growth of Miscanthus. Contrarily, mixtures consisting of bacteria or fungi originating from Miscanthus promoted growth of their host, especially on the heavy metals-polluted site. The combination of bacteria and fungi was inferior to the mixtures consisting of bacteria or fungi alone. Our observations indicate extensive potential of mixed bacterial and fungal endophytic inocula to promote establishment and yield of Miscanthus grown on marginal and polluted land and emphasise the necessity to test particular microbial-plant host combinations. Morphotypes of fungi isolates from Miscanthus × giganteus.

Combined effects of fungal inoculants and the cytokinin-like growth regulator thidiazuron on growth, phytohormone contents and endophytic root fungi in Miscanthus × giganteus.

Aim of this study was to investigate main effects and interactions between symbiotic fungi and the cytokinin-like growth regulator thidiazuron (TDZ) in Miscanthus × giganteus. The arbuscular mycorrhiza fungus Rhizophagus intraradices (AMF) and the endophyte Piriformospora indica (PI) were chosen as model symbionts. The fungal inoculants and TDZ had no significant effect on plant growth but modulated phytohormone levels in the leaves. TDZ induced accumulation of salicylic acid in controls, but not in plants inoculated with fungi. Leaf concentrations of abscisic acid (ABA) derivatives, auxin (indole-3-acetic acid) precursors and catabolites and numerous cytokinins were increased by R. intraradices but lowered by P. indica. TDZ raised concentrations of ABA compounds, the non-indole auxin phenylacetic acid, jasmonate and some cytokinins, but decreased cis-zeatin and N6-(Δ2-isopentenyl)adenine levels. Inoculation with AMF reduced abundance of endogenous clampless endophytes. TDZ application strongly reduced formation of arbuscular mycorrhiza and increased occurrence of clamped mycelia (i.e. basidiomycetous endophytes). Our study provides a thorough outline of the phytohormone homeostasis under the combined influence of beneficial inoculants and a growth regulator, highlighting the necessity to study their interaction in the whole plant-microbial context.

Assessment of rosehips based on the content of their biologically active compounds.

In this study, an in-depth analysis of the unique set of rosehip samples from 71 Rosa genotypes was conducted with the aim to identify the most suitable ones for applications in the food and pharmaceutical industries based on the content of biologically active compounds. In the first part of our experiments, the antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl assay and the genotypes with the highest values were selected for the follow-up analysis. In the second part of experiments, the major classes of biologically active compounds in rosehips such as carotenoids, tocopherols, flavonoids, and triterpenoic acids were further quantified using liquid chromatography-based techniques. Large variation was observed among all the analyzed compounds with intraspecific variation often hiding interspecific or intersectional differences. The compounds studied herein thus do not provide a sharp tool for chemotaxonomic resolution of the genus Rosa. High intraspecific variation indicates the necessity to screen and utilize individual rose genotypes rather than representatives of the species when searching for sources of biologically active compounds. In the final stage of the study, 10 genotypes were selected for further cultivation and use, based on the highest concentrations of the analyzed biologically active compounds.

Distribution of P, K, Ca, Mg, Cd, Cu, Fe, Mn, Pb and Zn in wood and bark age classes of willows and poplars used for phytoextraction on soils contaminated by risk elements.

Fast-growing clones of Salix and Populus have been studied for remediation of soils contaminated by risk elements (RE) using short-rotation coppice plantations. Our aim was to assess biomass yield and distributions of elements in wood and bark of highly productive willow (S1--[Salix schwerinii × Salix viminalis] × S. viminalis, S2--Salix × smithiana clone S-218) and poplar (P1--Populus maximowiczii × Populus nigra, P2--P. nigra) clones with respect to aging. The field experiment was established in April 2008 on moderately Cd-, Pb- and Zn- contaminated soil. Shoots were harvested after four seasons (February 2012) and separated into annual classes of wood and bark. All tested clones grew on contaminated soils, with highest biomass production and lowest mortality exhibited by P1 and S2. Concentrations of elements, with exception of Ca and Pb, decreased with age and were higher in bark than in wood. The Salix clones were characterised by higher removal of Cd, Mn and Zn compared to the Populus clones. Despite generally higher RE content in young shoots, partly due to lower wood/bark ratios and higher RE concentrations in bark, the overall removal of RE was higher in older wood classes due to higher biomass yield. Thus, longer rotations seem to be more effective when phytoextraction strategy is considered. Of the four selected clones, S1 exhibited the best removal of Cd and Zn and is a good candidate for phytoextraction.

Potential and drawbacks of EDDS-enhanced phytoextraction of copper from contaminated soils.

Incubation and pot experiments using poplar (Populus nigra L. cv. Wolterson) were performed in order to evaluate the questionable efficiency of EDDS-enhanced phytoextraction of Cu from contaminated soils. Despite the promising conditions of the experiment (low contamination of soils with a single metal with a high affinity for EDDS, metal tolerant poplar species capable of producing high biomass yields, root colonization by mycorrhizal fungi), the phytoextraction efficiency was not sufficient. The EDDS concentrations used in this study (3 and 6 mmol kg(-1)) enhanced the mobility (up to a 100-fold increase) and plant uptake of Cu (up to a 65-fold increase). However, despite EDDS degradation and the competition of Fe and Al for the chelant, Cu leaching cannot be omitted during the process. Due to the low efficiency, further research should be focused on other environment-friendly methods of soil remediation.

Development and activity of Glomus intraradices as affected by co-existence with Glomus claroideum in one root system.

The co-existence of two arbuscular mycorrhizal fungal (AMF) species, Glomus intraradices and Glomus claroideum, in the root systems of plants was investigated in a greenhouse experiment aimed at reconstructing interactions during an early stage of primary succession on a coal-mine spoil bank in Central Europe. Two plant species, Tripleurospermum inodorum and Calamagrostis epigejos, were inoculated either with one or both AMF species. Fungal development, determined by trypan blue and alkaline phosphatase staining as well as by PCR amplification of rRNA genes with species-specific primers, and the expression of five genes with different metabolic functions in the intraradical structures of G. intraradices were followed after 6 and 9 weeks of cultivation. The two AMF closely co-existed in the root systems of both plants possibly through similar colonisation rates and competitivity. Inoculation with the two fungi, however, did not bring any additional benefit to the host plants in comparison with single inoculation; moreover, plant growth depression observed after inoculation with G. claroideum persisted also in mixed inoculation. The expression of all the assayed G. intraradices genes was affected either by host plant or by co-inoculation with G. claroideum. The effects of both factors depended on the time of sampling, which underlines the importance of addressing this topic in time-course studies.

Melatonin inhibits prostaglandin E2- and sodium nitroprusside-induced ion secretion in rat distal colon.

Although the gastrointestinal tract is a rich source of melatonin and possesses numerous melatonin-binding sites, the role of melatonin in this tissue has not yet been fully elucidated. In this work we focused on the role of melatonin in the modulation of ion transport in rat distal colon. Whereas melatonin had no effect on colonic secretion or caused only infrequent and small changes in the short circuit current (Isc) due to its solvent ethanol, this mediator significantly modulated the secretion elicited by some secretagogues. Out of the five substances tested (prostaglandin E(2); 5-hydroxytryptamine; bethanechol; histamine; sodium nitroprusside) melatonin inhibited the effect of prostaglandin E(2) (PGE(2)) and sodium nitroprusside (SNP). Melatonin concentration-dependently decreased PGE(2)-evoked Isc and this inhibitory effect was more obvious from the mucosal side. The basal level of cAMP in colonic mucosa was not influenced by melatonin, but this drug prevented a PGE(2)-induced increase in the level of cAMP. The neurotoxin tetrodotoxin blocked the inhibitory effect of melatonin on SNP-induced Isc. Our data suggests that melatonin takes part in the modulation of colonic ion transport. The modulatory effect of melatonin on PGE(2)-induced Isc occurs directly at the level of the epithelium, whereas the effect on SNP-induced Isc is indirect and located in tetrodotoxin-sensitive enteric neurons.

Insight into the circadian clock within rat colonic epithelial cells.

BACKGROUND & AIMS: The gastrointestinal tract exhibits diurnal rhythms in many physiologic functions. These rhythms are driven by food intake but are also preserved during food deprivation, suggesting the presence of endogenous circadian rhythmicity. The aim of the study was to provide insight into the circadian core clock mechanism within the rat colon. Moreover, the potency of a restricted feeding regime to shift the circadian clock in the colon was tested. The question of whether the colonic clock drives circadian expression in NHE3, an electroneutral Na(+)/H(+) exchanger, was also addressed. METHODS: Daily profiles in expression of clock genes Per1, Per2, Cry1, Bmal1, Clock, and Rev-erbalpha, and the NHE3 transporter were examined by reverse transcriptase-polymerase chain reaction and their mRNA levels, as well as PER1 and BMAL1 protein levels, were localized in the colonic epithelium by in situ hybridization and immunocytochemistry, respectively. RESULTS: Expression of Per1, Per2, Cry1, Bmal1, Clock, Rev-erbalpha, and NHE3, as well as PER1 and BMAL1 protein levels, exhibited circadian rhythmicity in the colon. The rhythms were in phase with those in the liver but phase-delayed relative to the master clock in the suprachiasmatic nucleus. Restricted feeding entrained the clock in the colon, because rhythms in clock genes as well as in NHE3 expression were phase-advanced similarly to the clock in the liver. CONCLUSIONS: The rat colon harbors a circadian clock. The colonic clock is likely to drive rhythmic NHE3 expression. Restricted feeding resets the colonic clock similarly to the clock in the liver.

Heterogeneous expression of melatonin receptor MT1 mRNA in the rat intestine under control and fasting conditions.

Melatonin is found in mammalian central nervous system and various peripheral tissues including gastrointestinal tract (GIT) where it participates in the regulation of intestinal motility, blood flow, immunomodulation, ion transport, cell proliferation and scavenging of free radicals. Some of these effects are achieved via melatonin binding to specific receptors, MT1 and MT2. As no thorough study on the expression of these receptors in the GIT has yet been done, the aim of this study was to determine the MT1 mRNA expression in the rat intestine under both control and fasting conditions. Our results suggest that MT1 mRNA is present in epithelial as well as subepithelial layer, with higher expression in the latter in all intestinal segments studied. The highest signal of the MT1 transcript along the rostro-caudal intestinal axis was found both in epithelial and subepithelial layers of the duodenum. Nevertheless, duodenal MT1 mRNA expression did not reach the level found in pituitary gland. In a 12:12-hr light:dark cycle a MT1 receptor expression in the subepithelial layer of rat distal colon did not manifest a significant diurnal rhythm. Short-term fasting increased the expression of MT1 transcript in the subepithelial layer of both the small and large intestine. During long-term fasting the increase persisted only in distal colon while a return to control levels was observed in small intestinal segments. In conclusion we demonstrated heterogeneous expression of MT1 receptor in the rat intestine and showed that its expression is up-regulated by nutritional deprivation.

Age-dependent effect of secretagogues during colonic maturation.

Intestinal secretory response is altered during colonic development. The aim of this report was to study the developmental changes of the Ca(2+)- and cAMP-induced regulatory pathways with special attention to the direct and indirect effect of secretagogues on the colonic epithelium. We investigated the effect of bethanechol, 5-hydroxytryptamine (5-HT), and histamine on Cl(-) secretion and stimulation of intracellular Ca(2+) ([Ca(2+)](i)) and cAMP in the distal colon of suckling, weanling and adult rats. In the presence of tetrodotoxin, immature colon of suckling and weanling rats displayed higher potency (EC(50)) of 5-HT to stimulate Cl(-) secretion, whereas the potency of histamine was not changed during development. The potency of bethanechol was reduced during weaning and partially restored in adulthood. 5-HT increased cAMP level similarly in both neonatal and adult colonic crypts, but the adults had higher basal level of cAMP than suckling rats. Also the effect of bethanechol on [Ca(2+)](i) was independent of colonic maturation. The results suggest that colonic Cl(-) secretion displays developmental changes of regulation depending on the non-neural secretagogue-signalling pathway and that these developmental changes seem to be localized somewhere outside colonocytes.