Extracts from medicinal plants are widely used in the treatment and prevention of different diseases. Micromeria frivaldszkyana is a Balkan endemic species with reported antioxidant and antimicrobial characteristics; however, its phytochemical composition is not well defined. Here, we examined the metabolome of M. frivaldszkyana by chromatography–mass spectrometry (GC-MS), ultra-performance liquid chromatography–mass spectrometry (UPLC-MS-MS), and inductively coupled plasma mass spectrometry (ICP-MS). Amino acids, organic acids, sugars, and sugar alcohols were the primary metabolites with the highest levels in the plant extract. Detailed analysis of the sugar content identified high levels of sucrose, glucose, mannose, and fructose. Lipids are primary plant metabolites, and the analysis revealed triacylglycerols as the most abundant lipid group. Potassium (K), magnesium (Mg), zinc (Zn), and calcium (Ca) were the elements with the highest content. The results showed linarin, 3-caffeoil-quinic acid, and rosmarinic acid, as well as a number of polyphenols, as the most abundant secondary metabolites. Among the flavonoids and polyphenols with a high presence were eupatorin, kaempferol, and apigenin—compounds widely known for their bioactive properties. Further, the acute toxicity and potential anti-inflammatory activity of the methanolic extract were evaluated in Wistar rats. No toxic effects were registered after a single oral application of the extract in doses of between 200 and 5000 mg/kg bw. A fourteen-day pre-treatment with methanolic extract of M. frivaldszkyana in doses of 250, 400, and 500 mg/kg bw induced anti-inflammatory activity in the 1st, 2nd, and 3rd hours after carrageenan injection in a model of rat paw edema. This effect was also present in the 4th hour only in the group treated with a dose of 500 mg/kg. In conclusion, M. frivaldszkyana extract is particularly rich in linarin, rosmarinic acid, and flavonoids (eupatorin, kaempferol, and apigenin). Its methanolic extract induced no toxicity in male Wistar rats after oral application in doses of up to 5000 mg/kg bw. Additionally, treatment with the methanolic extract for 14 days revealed anti-inflammatory potential in a model of rat paw edema on the 1st, 2nd, and 3rd hours after the carrageenan injection. These results show the anti-inflammatory potential of the plant, which might be considered for further exploration and eventual application as a phytotherapeutic agent.

Marrubium species have been used since ancient times as food additives and curative treatments. Their phytochemical composition and various pharmacological activities were the focus of a number of scientific investigations but no comprehensive metabolome profiling to identify the numerous primary and secondary metabolites has been performed so far. This study aimed to generate a comprehensive picture of the total metabolite content of two Marrubium species—M. peregrinum and M. friwaldskyanum—to provide detailed information about the main primary and secondary metabolites. In addition, the elemental composition was also evaluated. For this purpose, non-targeted metabolomic analyses were conducted using GC-MS, UPLC-MS/MS and ICP-MS approaches. Nearly 500 compounds and 12 elements were detected and described. The results showed a strong presence of phenolic acids, flavonoids and their glucosides, which are generally of great interest due to their various pharmacological activities. Furthermore, tissue-specific analyses for M. friwaldskyanum stem, leaves and flowers were carried out in order to outline the sources of potentially important bioactive molecules. The results generated from this study depict the Marrubium metabolome and reveal its dual scientific importance—from one side, providing information about the metabolites that is fundamental and vital for the survival of these species, and from the other side, defining the large diversity of secondary substances that are a potential source of phytotherapeutic agents.

This study addresses the role of cytosine methylation in the fine-tuning of flowering time under water deficit in Arabidopsis thaliana. A drm1 drm2 cmt3 (ddc) triple methylation mutant was used together with the Col-0 wild type. The plants were grown under long-day conditions with water deficit induced by cessation of watering starting 12 days after seeding. Col-0 showed a 1-day delay in flowering as a result of the treatment. In contrast, ddc showed a 2-day delay regardless of the experimental conditions. We found that the two b-box domain proteins, BBX16/COL7 and BBX17/COL8, became overexpressed in the ddc background and in Col-0 under water deficit 24 days after seeding. Additionally, the NF-YA2 transcription factor became correspondingly down-regulated. Our results support a model where BBX16/COL7 and BBX17/COL8 interact with CONSTANS to delay the induction of FT under long-day conditions. NF-YA2, which is also recognized as a promoter of FT expression, with its down-regulation causes additional delay of FT-induced flowering. The plants overcome the BBX/NF-YA inhibition easily, resulting in a relatively small delay in flowering. The expression patterns of the three genes suggest the involvement of cytosine methylation in their regulation; however, no differential methylation could be found in cis that can explain these effects. The results therefore suggest a trans acting mechanism. Considering that the activities of BBX16/COL7 and BBX17/COL8 in different physiological conditions are not elucidated, this paper provides a background for future experiments targeting the role of these genes in the fine-tuning of flowering time in A. thaliana.

This study addresses the role of cytosine methylation in the fine tuning of flowering time under conditions of water deficit in Arabidopsis thaliana . For this purpose, the drm1 drm2 cmt3 triple methylation mutant in the Col-0 background was used. The plants were grown under long day conditions with water deficit induced by cessation of watering starting at 12 days after seeding. Col-0 showed 1 day delay in flowering as a result of the treatment. ddc on the other hand, showed 2 days of delay regardless of the experimental conditions. We found that the two b-box domain proteins BBX16/COL7 and BBX17/ COL8 became overexpressed in the ddc background and in Col-0 under water deficit at 24 days after seeding. Additionally, the NF-YA2 transcription factor became correspondingly down-regulated. Our results suggest a model, where BBX16/COL7 and BBX17/ COL8 interact with CONSTANS to delay the induction of FT under long day conditions. NF-YA2 , which is also recognized as a promoter of FT expression, with its down-regulation causes additional delay of FT induced flowering. In the Col-0 background, the weak FRIGIDA allele fails to induce sufficient expression of FLC for additional suppression of FT . The plants overcome easily the BBX/NF-YA inhibition resulting in a relatively small delay in flowering. The expression patterns of the three genes suggest involvement of cytosine methylation in their regulation, however no differential methylation could be found in cis that can explain these effects. The results therefore, suggest a trans acting mechanism. Considering that the activities of BBX16 / COL7 and BBX17 / COL8 in different physiological conditions are not elucidated, this paper provides a background for future experiments targeting the role of these genes in the fine tuning of flowering time in Arabidopsis thaliana .

Water deficit is a major constraint on pepper (Capsicum annuum) yield, yet the genetic architecture of reproductive-stage drought tolerance remains poorly resolved. We phenotyped a Balkan C. annuum diversity panel (n=133) and an interspecific backcross inbred line population (n=76) under well-watered (WW) and water-stress (WS) conditions. WS was applied from anthesis of the second truss as a stepwise reduction in irrigation volume relative to WW (30% for 7 d, then 60% thereafter), maintained for 90 d across the reproductive period. We assessed yield components, soluble solids, and stress-tolerance and stress-susceptibility indices. Genome-wide association study (GWAS) identified 104 single nucleotide polymorphism (SNP)–trait associations (P<1×10−5), and quantitative trait locus (QTL) mapping detected 38 significant QTLs (1000 permutations, α=0.01), with the QTL intervals defined at LOD ≥8. Integrating GWAS and QTL mapping under WS revealed overlapping loci on chromosomes 5 and 6, harboring two consensus intergenic SNPs associated with yield components and soluble solids. Haplotype analysis linked chromosome 5 alleles to higher fruit number and soluble solids. At chromosome 6, the G allele at SNP 6_28348737 was enriched in tolerant lines for fruit number. These regions harbor candidate genes for reproductive development and stress response, possibly through cis-regulatory variation. Together, these results advance understanding of the genetic basis of pepper yield under drought and identify candidate breeding markers.