Cytokinins enhance the metabolic activity of in vitro-grown catmint (Nepeta nuda L.).

The phytohormones cytokinins are essential mediators of developmental and environmental signaling, primarily during cell division and endophytic interactions, among other processes. Considering the limited understanding of the regulatory mechanisms that affect the growth and bioactivity of the medicinal plant Nepeta nuda (Lamiaceae), our study aimed to explore how cytokinins influence the plant's metabolic status. Exogenous administration of active cytokinin forms on in vitro N. nuda internodes stimulated intensive callus formation and de novo shoot regeneration, leading to a marked increase in biomass. This process involved an accumulation of oxidants, which were scavenged by peroxidases using phenolics as substrates. The callus tissue formed upon the addition of the cytokinin 6-benzylaminopurine (BAP) acted as a sink for sugars and phenolics during the allocation of nutrients between the culture medium and regenerated plants. In accordance, the cytokinin significantly enhanced the content of polar metabolites and their respective in vitro biological activities compared to untreated in vitro and wild-grown plants. The BAP-mediated accumulation of major phenolic metabolites, rosmarinic acid (RA) and caffeic acid (CA), corresponded with variations in the expression levels of genes involved in their biosynthesis. In contrast, the accumulation of iridoids and the expression of corresponding biosynthetic genes were not significantly affected. In conclusion, our study elucidated the mechanism of cytokinin action in N. nuda in vitro culture and demonstrated its potential in stimulating the production of bioactive compounds. This knowledge could serve as a basis for further investigations of the environmental impact on plant productivity.

Patterns of Genetic Variation of Nepeta nuda L. from the Central Balkans: Understanding Drivers of Chemical Diversity.

Nepeta nuda L., a notable medicinal species in the tradition of the Balkan region, is a rich source of bioactive iridoids and phenolics previously described as high-resolution taxonomical classifiers for the genus Nepeta. However, their potential in investigating intra-species differentiation is here described for the first time. The aim was to recognize the sources of natural chemical diversity and their association with the genetic variability both within and among N. nuda populations in the Central Balkans. Chemical diversity was assessed from methanol extracts and essential oils through untargeted and targeted metabolomics using state-of-the-art analytical tools, covering a broad spectrum of compounds that represent the N. nuda metabolome. We found that chemodiversity primarily resides within populations of N. nuda, and similar results were obtained at the DNA level using microsatellite markers. The low genetic and chemical differentiation of the studied N. nuda populations implies that their metabolomic profiles may be less influenced by geographic distance and variable environmental conditions within the Central Balkans, as they are under the pivotal control of their genetic backgrounds. Screening the distribution of the major bioactive compounds belonging to phenolics (phenolic acids and flavonoids) and iridoids (both aglycones and glycosylated forms), within and among N. nuda populations, is able to guarantee mass spectrometry-based tools for the selection of elite representative genotypes with practical importance. The knowledge acquired will allow us to delve deeper into the molecular background of N. nuda chemical diversity, which is the course of our further work.

Native and endemic Iranian Nepeta spp.: powerful antimicrobial agents.

Pathogenic microorganisms are more or less successfully treated by synthetic chemical compounds, whose residues often cause serious health problems. Plant specialized metabolites with antimicrobial properties have for a long time been the focus of both medicine and pharmacology. This study was conducted to evaluate the in vitro antimicrobial activity of methanol extracts of selected endemic and native Iranian Nepeta species against some of the most important pathogenic bacteria and fungi. The results indicated that N. kotschyi leaf extract was the most efficient against the tested bacteria, with Pseudomonas aeruginosa being the most sensitive and fungal species were more susceptible to the extracts than bacterial strains. Nepeta spp. extracts showed a strong antifungal activity against micromycetes, except for quite resistant Aspergillus niger. Antibacterial MIC values (mg.mL-1) ranged from 0.01 (N. kotschyi) to 0.20 (N. crassifolia), while antifungal MIC values ranged from 0.02 (N. crassifolia, N. kotschyi, N. menthoides, and N. cataria) to 0.13 (N. crassifolia and N. menthoides). When compared to positive controls, in most cases the extracts performed much better. The recorded antimicrobial activity candidates the selected 4 endemic and native Iranian Nepeta spp. as prospective and promising antimicrobial agents to be used in both pharmacology and biotechnology.

Uncovering the Interrelation between Metabolite Profiles and Bioactivity of In Vitro- and Wild-Grown Catmint (Nepeta nuda L.).

Nepeta nuda L. is a medicinal plant enriched with secondary metabolites serving to attract pollinators and deter herbivores. Phenolics and iridoids of N. nuda have been extensively investigated because of their beneficial impacts on human health. This study explores the chemical profiles of in vitro shoots and wild-grown N. nuda plants (flowers and leaves) through metabolomic analysis utilizing gas chromatography and mass spectrometry (GC-MS). Initially, we examined the differences in the volatiles' composition in in vitro-cultivated shoots comparing them with flowers and leaves from plants growing in natural environment. The characteristic iridoid 4a-α,7-ß,7a-α-nepetalactone was highly represented in shoots of in vitro plants and in flowers of plants from nature populations, whereas most of the monoterpenes were abundant in leaves of wild-grown plants. The known in vitro biological activities encompassing antioxidant, antiviral, antibacterial potentials alongside the newly assessed anti-inflammatory effects exhibited consistent associations with the total content of phenolics, reducing sugars, and the identified metabolic profiles in polar (organic acids, amino acids, alcohols, sugars, phenolics) and non-polar (fatty acids, alkanes, sterols) fractions. Phytohormonal levels were also quantified to infer the regulatory pathways governing phytochemical production. The overall dataset highlighted compounds with the potential to contribute to N. nuda bioactivity.

Solvent System-Guided Extraction of Centaurium spicatum (L.) Fritch Provides Optimized Conditions for the Biological and Chemical Characteristics of the Herbal Extracts.

Spiked centaury (Centaurium spicatum) is a well-known medicinal plant from the Mediterranean region with various bioactivities, but there are no studies addressing the use of different solvent systems to improve its pharmacological potential. Nine extraction procedures were adapted to study the effects of solvent composition on the content of bioactive compounds in C. spicatum extracts and on corresponding bioactivities. Targeted metabolomics was performed to obtain information on the chemical composition of extracts. Ethanol-water-based extraction procedures were the most efficient in isolating polyphenols, while less polar butanol extract contained the highest amount of iridoids. Antioxidant potential analysis revealed stronger activity in extracts with higher polyphenol content. Bacillus cereus and Staphylococus aureus were designated as the most sensitive bacterial strains to the activity of extracts, while among the micromycetes tested, Penicillium funiculosum was the most susceptible strain. Butanol extract showed antivirulence potential on Candida albicans morphological transition from yeast to hyphal form, and selected extracts were effective against biofilm formation in two Candida species. All the extracts tested in this study showed no cytotoxic activity to immortalize human skin keratinocyte cell line (HaCaT), whereas extracts obtained by ethanol-water extraction stand out for their potent wound healing effects. Moreover, the influence of the extraction solvent system on various bioactivities of C. spicatum is reported herein for the first time. Overall, the results presented in this study promote the use of C. spicatum as a source of natural products with potential antioxidant, wound healing, and antimicrobial applications that are potentially safe for human use.

Leaves micromorphology, chemical profile, and bioactivity of in vitro-propagated Nepeta cyrenaica (Lamiaceae).

INTRODUCTION: The endemic species Nepeta cyrenaica Quézel & Zaffran, native to northeastern Libya, is valued as an important honey-bearing plant. OBJECTIVES: This study was aimed to examine the micromorphology, phytochemistry, and bioactivity of in vitro-propagated N. cyrenaica for the first time. MATERIALS AND METHODS: The leaf indumentum was examined using light and scanning electron microscopy and further characterised for histochemistry. The chemical composition of essential oil (EO) was performed using GC-MS analysis, while dichloromethane (DCM), methanol (ME), ethanol (ET), and aqueous (AQ) extracts were analysed using qualitative and quantitative LC/MS analyses. The antioxidant activities of EO and extracts were assessed using three parallel assays, while enzyme-inhibiting effects were evaluated against four enzymes. RESULTS: The leaves bear various types of glandular trichomes, with lipophilic secretion predominating. The main EO component of EO was 1,8-cineole. A considerable number of phenolics and iridoids were tentatively identified in the ME extract. Quantitative LC/MS analysis confirmed that ferulic acid, rosmarinic acid, and epigallocatechin gallate were present in the highest amount in the extracts, in which three iridoids were also quantified. Although the ME extract contained the highest amount of polyphenolics and iridoids, the DCM extract showed the best overall biological potential. Additionally, EO exerted the strongest acetylcholinesterase and tyrosinase inhibition. CONCLUSION: This study demonstrated that the endemic N. cyrenaica can be efficiently grown under in vitro conditions, where it develops various glandular trichomes that are thought to secrete and/or accumulate bioactive compounds with valuable medicinal potential.

Variation in the chemical profiles of three foxglove species in the central Balkans.

The aim of this study was to determine intra- and interspecies variation in the qualitative and quantitative composition of methanol-soluble metabolites in the leaves of three Digitalis species (D. lanata, D. ferruginea, and D. grandiflora) from the central Balkans. Despite the steady use of foxglove constituents for human health as valuable medicinal products, populations of the genus Digitalis (Plantaginaceae) have been poorly investigated to describe their genetic and phenetic variation. Following untargeted profiling using UHPLC-LTQ Orbitrap MS, by which we identified a total of 115 compounds, 16 compounds were quantified using the UHPLC(-)HESI-QqQ-MS/MS approach. In total, 55 steroid compounds, 15 phenylethanoid glycosides, 27 flavonoids, and 14 phenolic acid derivatives were identified across the samples with D. lanata and D. ferruginea showing a great similarity, while 15 compounds were characteristic only for D. grandiflora. The phytochemical composition of methanol extracts, considered here as complex phenotypes, are further examined along multiple levels of biological organization (intra- and interpopulation) and subsequently subjected to chemometric data analysis. The quantitative composition of the selected set of 16 chemomarkers belonging to the classes of cardenolides (3 compounds) and phenolics (13 compounds) pointed to considerable differences between the taxa studied. D. grandiflora and D. ferruginea were found to be richer in phenolics as compared to cardenolides, which otherwise predominate in D. lanata over other compounds. PCA revealed lanatoside C, deslanoside, hispidulin, and p-coumaric acid to be the main compounds contributing to the differences between D. lanata on one side and D. grandiflora and D. ferruginea on the other, while p-coumaric acid, hispidulin, and digoxin contribute to the diversification between D. grandiflora and D. ferruginea. However, quantitative variation in the metabolite content within species was faint with mild population diversification visible in D. grandiflora and particularly in D. ferruginea. This pointed to the highly conserved content and ratio of targeted compounds within the analyzed species, which was not severely influenced by the geographic origin or environmental conditions. The presented metabolomics approach might have, along with morphometrics and molecular genetics studies, a high information value for further elucidation of the relationships among taxa within the genus Digitalis.

Solanum dulcamara L. Berries: A Convenient Model System to Study Redox Processes in Relation to Fruit Ripening.

The present study provides, for the first time, a physicochemical and biochemical characterization of the redox processes associated with the ripening of Solanum dulcamara L. (bittersweet) berries. Electron Paramagnetic Resonance Spectroscopy (EPRS) and Imaging (EPRI) measurements of reactive oxygen species (ROS) were performed in parallel with the tissue-specific metabolic profiling of major antioxidants and assessment of antioxidant enzymes activity. Fruit transition from the mature green (MG) to ripe red (RR) stage involved changes in the qualitative and quantitative content of antioxidants and the associated cellular oxidation and peroxidation processes. The skin of bittersweet berries, which was the major source of antioxidants, exhibited the highest antioxidant potential against DPPH radicals and nitroxyl spin probe 3CP. The efficient enzymatic antioxidant system played a critical protective role against the deleterious effects of progressive oxidative stress during ripening. Here, we present the EPRI methodology to assess the redox status of fruits and to discriminate between the redox states of different tissues. Interestingly, the intracellular reoxidation of cell-permeable nitroxide probe 3CP was observed for the first time in fruits or any other plant tissue, and its intensity is herein proposed as a reliable indicator of oxidative stress during ripening. The described noninvasive EPRI technique has the potential to have broader application in the study of redox processes associated with the development, senescence, and postharvest storage of fruits, as well as other circumstances in which oxidative stress is implicated.

Analysis of the Qualitative and Quantitative Content of the Phenolic Compounds of Selected Moss Species under NaCl Stress.

The response to salt stress analysed by quantitative and qualitative analyses in three selected moss species was studied. Non-halophytic funaroid Physcomitrium patens and two halophytic mosses, funaroid Entosthodon hungaricus and pottioid Hennediella heimii were exposed to salt stress under controlled in vitro conditions. The results clearly showed various phenolics to be present and included to some extent as a non-enzymatic component of oxidative, i.e., salt stress. The common pattern of responses characteristic of phenolic compounds was not present in these moss species, but in all three species the role of phenolics to stress tolerance was documented. The phenolic p-coumaric acid detected in all three species is assumed to be a common phenolic included in the antioxidative response and salt-stress tolerance. Although the stress response in each species also included other phenolics, the mechanisms were different, and also dependent on the stress intensity and duration.

UHPLC-MS Phytochemical Profiling and Insight into Bioactivity of Rabelera holostea (Greater Stitchwort) Extract.

Rabelera holostea (L.) M. T. Sharples & E. A. Tripp (Greater Stitchwort), formerly known as Stellaria holostea L., is widespread in the warm temperate areas of Europe and Western Asia, the Caucasus region, as well as in some countries of North Africa. Nowadays it is considered as a weed, but earlier it was often used raw in salads or for the treatment of various inflammatory disorders. The goal of this study was to determine the constituents of the methanol extract of R. holostea aerial parts and its biological potential in terms of antioxidant, antimicrobial, and anti-inflammatory properties. Until now, the constituents and biological activities of this plant were not reported in detail. A comprehensive phytochemical profiling of the extract has shown that phenolic acids, such as ferulic, chlorogenic, and p-coumaric acid, flavonoids and flavonoid glucosides, such as chrysoeriol, rutin, and naringin, are the most abundant compounds. The antioxidant activity of R. holostea extract towards DPPH and ABTS radicals, but also the total antioxidant capacity and the inhibition of lipid peroxidation were moderate. The antimicrobial potential was pronounced mostly towards some fungi such as F. oxysporum (MIC 1.25 mg/mL), whereas the capacity of R. holostea to affect the growth of bacteria was much less pronounced. R. holostea extract was most inclined to anti-inflammatory activity. At a concentration of 50 µg/mL, it significantly inhibited both cyclooxygenase enzymes (COX-1 and COX-2) by 71.24% and 72.83%, respectively. Molecular docking studies indicated that chlorogenic acid and chrysoeriol are the main contributors to COX-1 and COX-2 inhibitory activity.

Functional iridoid synthases from iridoid producing and non-producing Nepeta species (subfam. Nepetoidae, fam. Lamiaceae).

Iridoids, a class of atypical monoterpenes, exhibit exceptional diversity within the Nepeta genus (subfam. Nepetoidae, fam. Lamiaceae).The majority of these plants produce iridoids of the unique stereochemistry, with nepetalactones (NLs) predominating; however, a few Nepeta species lack these compounds. By comparatively analyzing metabolomics, transcriptomics, gene co-expression, and phylogenetic data of the iridoid-producing N. rtanjensis Diklic & Milojevic and iridoid-lacking N. nervosa Royle & Bentham, we presumed that one of the factors responsible for the absence of these compounds in N. nervosa is iridoid synthase (ISY). Two orthologues of ISY were mined from leaves transcriptome of N. rtanjensis (NrPRISE1 and NrPRISE2), while in N. nervosa only one (NnPRISE) was identified, and it was phylogenetically closer to the representatives of the Family 1 isoforms, designated as P5ßRs. Organ-specific and MeJA-elicited profiling of iridoid content and co-expression analysis of IBG candidates, highlighted NrPRISE2 and NnPRISE as promising candidates for ISY orthologues, and their function was confirmed using in vitro assays with recombinant proteins, after heterologous expression of recombinant proteins in E. coli and their His-tag affinity purification. NrPRISE2 demonstrated ISY activity both in vitro and likely in planta, which was supported by the 3D modeling and molecular docking analysis, thus reclassification of NrPRISE2 to NrISY is accordingly recommended. NnPRISE also displays in vitro ISY-like activity, while its role under in vivo conditions was not here unambiguously confirmed. Most probably under in vivo conditions the NnPRISE lacks substrates to act upon, as a result of the loss of function of some of the upstream enzymes of the iridoid pathway. Our ongoing work is conducted towards re-establishing the biosynthesis of iridoids in N. nervosa.

Functional Characterization of Genes Coding for Novel ß-D-Glucosidases Involved in the Initial Step of Secoiridoid Glucosides Catabolism in Centaurium erythraea Rafn.

Secoiridoid glucosides (SGs) are monoterpenoids derived from the iridoid cyclopentane-C-pyran skeleton with ß-D glucose linked at C1 position. Coordinated metabolic processes, such as biosynthesis and catabolism of SGs, ensure constitutive presence of these bitter tasting compounds in plant tissues, which plays a decisive role in the defense against pathogens and herbivores. These compounds are susceptible to hydrolysis mediated by enzymes ß-glucosidases, and the resulting aglycones are subsequently directed toward different metabolic pathways in plants. Function of two ß-D-glucosidases (named CeBGlu1 and CeBGlu2) from centaury (Centaurium erythraea Rafn; fam. Gentianaceae), belonging to the glycoside hydrolase 1 (GH1) family, was confirmed using in vitro assays with recombinant proteins, following their heterologous expression in E. coli and His-tag affinity purification. Although they show slightly differential substrate preference, both isoforms display high specificity toward SGs and the organ-specific distribution of transcripts was positively correlated with the content of SGs in diploid and tetraploid C. erythraea plants. Transient overexpression of CeBGlu1 and CeBGlu2 in C. erythraea leaves induced changes in metabolite profiles. The effectiveness of transgene overexpression has been altered by plant ploidy. UHPLC/DAD/(±)HESI - MS2 profiling of leaves of diploid and tetraploid C. erythraea genotypes revealed that the amounts of major SGs; sweroside, swertiamarin, and gentiopicrin was decreased in agroinfiltrated leaves, especially when CeBGlu1 and CeBGlu2 were co-expressed with transgene silencing suppressor p19. The work demonstrates that in planta metabolic engineering adopting transient overexpression of CeBGlu1 and CeBGlu2 is a suitable tool for the modulation of SGs content and glucosides/aglycones ratio, which might have substantial effects on overall phytochemistry of C. erythraea.

Catmint (Nepeta nuda L.) Phylogenetics and Metabolic Responses in Variable Growth Conditions.

Nepeta nuda (catmint; Lamiaceae) is a perennial medicinal plant with a wide geographic distribution in Europe and Asia. This study first characterized the taxonomic position of N. nuda using DNA barcoding technology. Since medicinal plants are rich in secondary metabolites contributing to their adaptive immune response, we explored the N. nuda metabolic adjustment operating under variable environments. Through comparative analysis of wild-grown and in vitro cultivated plants, we assessed the change in phenolic and iridoid compounds, and the associated immune activities. The wild-grown plants from different Bulgarian locations contained variable amounts of phenolic compounds manifested by a general increase in flowers, as compared to leaves, while a strong reduction was observed in the in vitro plants. A similar trend was noted for the antioxidant and anti-herpesvirus activity of the extracts. The antimicrobial potential, however, was very similar, regardless the growth conditions. Analysis of the N. nuda extracts led to identification of 63 compounds including phenolic acids and derivatives, flavonoids, and iridoids. Quantification of the content of 21 target compounds indicated their general reduction in the extracts from in vitro plants, and only the ferulic acid (FA) was specifically increased. Cultivation of in vitro plants under different light quality and intensity indicated that these variable light conditions altered the content of bioactive compounds, such as aesculin, FA, rosmarinic acid, cirsimaritin, naringenin, rutin, isoquercetin, epideoxyloganic acid, chlorogenic acid. Thus, this study generated novel information on the regulation of N. nuda productivity using light and other cultivation conditions, which could be exploited for biotechnological purposes.

Genetic and Environmental Factors Jointly Impact Leaf Phenolic Profiles of Iris variegata L.

A plant's main mechanism to diminish the effects caused by high free radical levels generated during high irradiance is the synthesis of various secondary metabolites. In addition to interspecies differences, their concentrations may be influenced by genetic, ontogenic, morphogenetic or environmental factors. We investigated the influence of genetic (genotypes from different natural habitats) and environmental (contrasting light regimes as well as successive parts of the vegetation period) variability on the accumulation of 10 selected phenolic compounds (phenolic acids, flavonoids, and xanthones) in Iris variegata genotypes. Genotypes originated from either sun-exposed or shaded natural habitats were transplanted to two experimental light treatments (high light intensity with a higher R/FR ratio and low light intensity with a lower R/FR ratio). Significant impacts of both genetic and environmental seasonal variability (spring, summer and fall during the vegetation period) on phenolic compound profiles were detected. Their highest amounts were detected in spring. The magnitude of difference between light treatments (high vs. low light intensity) and the direction of this change varied depending on the secondary compound class. Phenotypic correlations among the 10 analyzed secondary metabolites differed across the experimental light treatments and their number decreased from spring to fall.

New Evidence for Artemisia absinthium L. Application in Gastrointestinal Ailments: Ethnopharmacology, Antimicrobial Capacity, Cytotoxicity, and Phenolic Profile.

Artemisia absinthium L. (Asteraceae) is traditionally used for gastrointestinal ailments and disorders linked to numerous risk factors including microbial infections. We aimed to provide contemporary evidence for its ethnopharmacological use and determine its antimicrobial capacity and mode of action, cytotoxicity, and phenolic constituents. Ethnopharmacological survey was conducted using semistructured interviews. Antimicrobial and antibiofilm capacities were determined by microdilution/crystal violet assay, respectively. Modes of action tested include estimation of exopolysaccharide production (congo red binding assay) and interference with membrane integrity (crystal violet uptake and nucleotide leakage assay). Cytotoxicity was determined using crystal violet assay. Polyphenolic profiling was done by advanced liquid chromatography/mass spectrometry (UHPLC-LTQ OrbiTrap MS). Artemisia absinthium in Serbia is traditionally used for gastrointestinal disorders, among others. Further study revealed high antifungal capacity of herb ethanolic extract towards range of Candida species (MIC 0.5-1 mg/mL) along with promising antibacterial activities (MIC 0.25-4 mg/mL). Interference with membrane integrity could be observed as a possible antimicrobial mechanism. Antibiofilm potential can be considered as high (towards C. krusei) to limited (towards P. aeruginosa) and moderate based on reduction in exopolysaccharide content. In concentrations up to 400 µg/mL, no cytotoxicity was observed towards HaCaT and HGF-1 cell lines. Polyphenolic analysis revealed twenty-one different constituents. A. absinthium usage as a gastrointestinal ailment remedy has been confirmed in vitro by its antimicrobial capacity towards microorganisms whose presence is linked to the diseases and associated complications and noncytotoxic nature of the natural product. The observed activities could be attributed to the present phenolic compounds.

Comparative Metabolite and Gene Expression Analyses in Combination With Gene Characterization Revealed the Patterns of Flavonoid Accumulation During Cistus creticus subsp. creticus Fruit Development.

Cistus creticus L. subsp. creticus (rockrose) is a shrub widespread in Greece and the Mediterranean basin and has been used in traditional medicine as herb tea for colds, for healing and digestive hitches, for the treatment of maladies, as perfumes, and for other purposes. Compounds from its flavonoid fraction have recently drawn attention due to antiviral action against influenza virus and HIV. Although several bioactive metabolites belonging to this group have been chemically characterized in the leaves, the genes involved in their biosynthesis in Cistus remain largely unknown. Flavonoid metabolism during C. creticus fruit development was studied by adopting comparative metabolomic and transcriptomic approaches. The present study highlights the fruit of C. creticus subsp. creticus as a rich source of flavonols, flavan-3-ols, and proanthocyanidins, all of which displayed a decreasing trend during fruit development. The majority of proanthocyanidins recorded in Cistus fruit are B-type procyanidins and prodelphinidins, while gallocatechin and catechin are the dominant flavan-3-ols. The expression patterns of biosynthetic genes and transcription factors were analyzed in flowers and throughout three fruit development stages. Flavonoid biosynthetic genes were developmentally regulated, showing a decrease in transcript levels during fruit maturation. A high degree of positive correlations between the content of targeted metabolites and the expression of biosynthetic genes indicated the transcriptional regulation of flavonoid biosynthesis during C. creticus fruit development. This is further supported by the high degree of significant positive correlations between the expression of biosynthetic genes and transcription factors. The results suggest that leucoanthocyanidin reductase predominates the biosynthetic pathway in the control of flavan-3-ol formation, which results in catechin and gallocatechin as two of the major building blocks for Cistus proanthocyanidins. Additionally, there is a decline in ethylene production rates during non-climacteric Cistus fruit maturation, which coincides with the downregulation of the majority of flavonoid- and ethylene-related biosynthetic genes and corresponding transcription factors as well as with the decline in flavonoid content. Finally, functional characterization of a Cistus flavonoid hydroxylase (F3'5'H) was performed for the first time.

Antimicrobial and Immunomodulating Activities of Two Endemic Nepeta Species and Their Major Iridoids Isolated from Natural Sources.

Two Balkan Peninsula endemics, Nepeta rtanjensis and N. argolica subsp. argolica, both characterized by specialized metabolite profiles predominated by iridoids and phenolics, are differentiated according to the stereochemistry of major iridoid aglycone nepetalactone (NL). For the first time, the present study provides a comparative analysis of antimicrobial and immunomodulating activities of the two Nepeta species and their major iridoids isolated from natural sources-cis,trans-NL, trans,cis-NL, and 1,5,9-epideoxyloganic acid (1,5,9-eDLA), as well as of phenolic acid rosmarinic acid (RA). Methanol extracts and pure iridoids displayed excellent antimicrobial activity against eight strains of bacteria and seven strains of fungi. They were especially potent against food-borne pathogens such as L. monocytogenes, E. coli, S. aureus, Penicillium sp., and Aspergillus sp. Targeted iridoids were efficient agents in preventing biofilm formation of resistant P. aeruginosa strain, and they displayed additive antimicrobial interaction. Iridoids are, to a great extent, responsible for the prominent antimicrobial activities of the two Nepeta species, although are probably minor contributors to the moderate immunomodulatory effects. The analyzed iridoids and RA, individually or in mixtures, have the potential to be used in the pharmaceutical industry as potent antimicrobials, and in the food industry to increase the shelf life and safety of food products.

Antagonistic Interaction between Phosphinothricin and Nepeta rtanjensis Essential Oil Affected Ammonium Metabolism and Antioxidant Defense of Arabidopsis Grown In Vitro.

Phosphinothricin (PPT) is one of the most widely used herbicides. PTT targets glutamine synthetase (GS) activity in plants, and its phytotoxicity is ascribed to ammonium accumulation and reactive oxygen species bursts, which drives rapid lipid peroxidation of cell membranes. In agricultural fields, PPT is extensively sprayed on plant foliage; however, a portion of the herbicide reaches the soil. According to the present study, PPT absorbed via roots can be phytotoxic to Arabidopsis, inducing more adverse effects in roots than in shoots. Alterations in plant physiology caused by 10 days exposure to herbicide via roots are reflected through growth suppression, reduced chlorophyll content, perturbations in the sugar and organic acid metabolism, modifications in the activities and abundances of GS, catalase, peroxidase, and superoxide dismutase. Antagonistic interaction of Nepeta rtanjensis essential oil (NrEO) and PPT, emphasizes the existence of complex control mechanisms at the transcriptional and posttranslational level, which result in the mitigation of PPT-induced ammonium toxicity and in providing more efficient antioxidant defense of plants. Simultaneous application of the two agents in the field cannot be recommended; however, NrEO might be considered as the PPT post-treatment for reducing harmful effects of herbicide residues in the soil on non-target plants.

Alterations in nepetalactone metabolism during polyethylene glycol (PEG)-induced dehydration stress in two Nepeta species.

A number of Nepeta species (fam. Lamiaceae) are interesting medicinal crops for arid and semi-arid areas, due to their ability to maintain essential developmental and physiological processes and to rationalize their specialized metabolism under water deficit growth conditions. The present research is, to our knowledge, the first attempt to investigate the molecular background of the dehydration-induced changes in specialized metabolism of Nepeta species, which will help to understand relations between dehydration stress on one hand and biomass production and yield of nepetalactone (NL) on the other. During the 6 days exposure of Nepeta rtanjensis Diklic & Milojevic and Nepeta argolica Bory & Chaub. ssp. argolica plants to PEG-induced dehydration stress under experimental in vitro conditions, decrease in transcript levels of the majority of 10 NL biosynthetic genes, and some of the 5 transcription factors (TFs) were recorded, simultaneously with the initial reduction in NL content. The two model species evidently employ similar strategies in response to severe dehydration stress; however N. rtanjensis is highlighted as the species more efficient in maintaining NL amounts in tissues. The results suggest trichome-specific and co-ordinately regulated NL biosynthesis at the level of gene expression, with trichome enriched MYC2 and YABBY5 TFs being the potential positive regulators. Manipulation of such TFs can be effective for engineering the NL biosynthetic pathway, and for the increased production of cis,trans-NL in N. argolica ssp. argolica and trans,cis-NL in N. rtanjensis.

Secoiridoids Metabolism Response to Wounding in Common Centaury (Centaurium erythraea Rafn) Leaves.

Centaurium erythraea Rafn produces and accumulates various biologically active specialized metabolites, including secoiridoid glucosides (SGs), which help plants to cope with unfavorable environmental conditions. Specialized metabolism is commonly modulated in a way to increase the level of protective metabolites, such as SGs. Here, we report the molecular background of the wounding-induced changes in SGs metabolism for the first time. The mechanical wounding of leaves leads to a coordinated up-regulation of SGs biosynthetic genes and corresponding JA-related transcription factors (TFs) after 24 h, which results in the increase of metabolic flux through the biosynthetic pathway and, finally, leads to the elevated accumulation of SGs 96 h upon injury. The most pronounced increase in relative expression was detected for secologanin synthase (CeSLS), highlighting this enzyme as an important point for the regulation of biosynthetic flux through the SG pathway. A similar expression pattern was observed for CeBIS1, imposing itself as the TF that is prominently involved in wound-induced regulation of SGs biosynthesis genes. The high degree of positive correlations between and among the biosynthetic genes and targeted TFs expressions indicate the transcriptional regulation of SGs biosynthesis in response to wounding with a significant role of CeBIS1, which is a known component of the jasmonic acid (JA) signaling pathway.