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.

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.

Numerous members of the Nepeta genus (family Lamiaceae, subfamily Nepetoideae) are medicinal herbs and sources of important bioactive compounds. Most Nepeta species produce iridoids, which are monoterpenoids that deter herbivores and pathogens and are potential biopesticides. In Nepeta , some species produce iridoid aglycones and glycosylated iridoids (referred to as chemotype A), some produce only glycosylated iridoids (chemotype B), and some produce neither iridoid aglycones nor glycosylated iridoids (chemotype C). Here, we show that the observed diversity in iridoids is, at least partially, attributed to evolutionary gains and losses of key biosynthetic genes. Based on reconstructed phylogenetic relationships, we propose a scenario in which partial or complete loss of the ability to synthesize iridoids with specific stereochemistries in the taxa with chemotypes B and C resulted from independent evolutionary events. These observations improve our understanding of metabolic diversity in the Nepeta genus and may inform efforts to produce specific iridoids in plants.

Centaurium tenuiflorum (fam. Gentianaceae) is an annual herb rich in highly-valuable bioactive metabolites, predominately xanthones and secoiridoids. The metabolomes of the aerial parts of slender centaury resemble those of C. erythraea, a species widely used in traditional medicine for treating mild dyspeptic and gastrointestinal complaints and temporary loss of appetite. This research aimed to develop an efficient clonal propagation protocol for two C. tenuiflorum chemotypes corresponding to the white-flowered (CtW) and pink-flowered (CtP) phenotypes, which is a prerequisite for the development and optimisation of biotechnological processes to produce bioactive metabolites. No spontaneous regeneration of CtP and CtW shoots occurred in either the solid or liquid half-strength MS culture media, and both were accompanied by tissue necrosis and browning. Supplementation of the culture medium or the pretreatment of the explants with anti-browning agents, i.e. activated charcoal, PVP, ascorbic acid, caffeic acid, citric acid or silver nitrate, all at various concentrations, did not enhance the regeneration efficacy or prevent the tissue browning. In contrast, the addition of plant growth regulators promoted high-frequency shoot initiation from both the root and leaf explants, and effectively induced rooting. The optimal conditions for the clonal propagation of both the CtP and CtW genotypes was a liquid basal medium containing 1 mg L-1 TDZ and 1 mg L-1 IAA at the proliferation stage, and a solid basal medium containing 1 mg L-1 IAA at the rooting stage. Although no significant differences between the regenerative capacity of the CtP and CtW genotypes were observed, targeted metabolomics data clearly distinguish the two chemotypes. The present study highlights the active production of xanthones and secoiridoids in the C. tenuiflorum regenerated shoots and their intensive exudation into the surrounding culture medium. Among the ten targeted metabolites, polyphenolics from the groups of xanthones and phenolic acids, highly abundant in both the C. tenuiflorum tissue and the culture media, were recognised as the main contributors to the tissue browning. The study demonstrates an efficient approach for recovering xanthones, phenolic acids, and secoiridoids from liquid culture media and highlights its potential as a promising source of bioactive compounds.