Background: Flavonoids are plant specialised metabolites, which derive from phenylalanine and acetate metabolism. They possess a variety of beneficial characteristics for plants and humans. Several modification steps in the synthesis of tricyclic flavonoids cause for the amazing diversity of flavonoids in plants. The 2-oxoglutarate-dependent dioxygenases (2-ODDs) flavanone 3-hydroxylase (F3H, synonym FHT), flavonol synthase (FLS) and anthocyanidin synthase (ANS, synonym leucoanthocyanidin dioxygenase (LDOX)), catalyse oxidative modifications to the central C ring. They are highly similar and have been shown to catalyse, at least in part, each other’s reactions. FLS and ANS have been identified as bifunctional enzymes in many species, including Arabidopsis thaliana, stressing the capability of plants to bypass missing or mutated reaction steps on the way to flavonoid production. However, little is known about such bypass reactions and the flavonoid composition of plants lacking all three central flavonoid 2-ODDs. Results: To address this issue, we generated a f3h/fls1/ans mutant, as well as the corresponding double mutants and investigated the flavonoid composition of this mutant collection. The f3h/fls1/ans mutant was further characterised at the genomic level by analysis of a nanopore DNA sequencing generated genome sequence assembly and at the transcriptomic level by RNA-Seq analysis. The mutant collection established, including the novel double mutants f3h/fls1 and f3h/ans, was used to validate and analyse the multifunctionalities of F3H, FLS1, and ANS in planta. Metabolite analyses revealed the accumulation of eriodictyol and additional glycosylated derivatives in mutants carrying the f3h mutant allele, resulting from the conversion of naringenin to eriodictyol by flavonoid 3’-hydroxylase (F3’H) activity. Conclusions: We describe the in planta multifunctionality of the three central flavonoid 2-ODDs from A. thaliana and identify a bypass in the f3h/fls1/ans triple mutant that leads to the formation of eriodictyol derivatives. As (homo-)eriodictyols are known as bitter taste maskers, the annotated eriodictyol (derivatives) and in particular the observations made on their in planta production, could provide valuable insights for the creation of novel food supplements.

Primula veris subsp. veris L. is a perennial herbaceous and medicinal plant species the roots and flowers of which are a source of valuable pharmaceutical raw materials. The plant tissues are used to produce expectorant and diuretic drugs due to their high content of triterpene saponins and phenolic glycosides. Underground roots of P. veris can be obtained only through a destructive process during the plant’s harvesting. In the present study, an in vitro adventitious root production protocol was developed as an alternative way of production, focused on four species-specific secondary metabolites. Root explants were cultured in Murashing & Skoog liquid medium supplemented with 5.4 μM α-naphthaleneacetic acid, 0.5 μM kinetin, L-proline 100 mg/L, and 30 g/L sucrose, in the dark and under agitation. The effect of temperature (10, 15 and 22 °C) on biomass production was investigated. The content of two flavonoid compounds (primeverin and primulaverin), and two main triterpene saponins (primulic acid I and II) were determined after 60 days of culture and compared with 1.5-year-old soil-grown plants. The accumulated content (mg/g DW) of bioactive compounds of in vitro adventitious roots cultured under 22 °C was significantly higher than the other two temperatures of the study, being 9.71 mg/g DW in primulaverin, 0.09 mg/g DW in primeverin, 6.09 mg/g DW in primulic acid I, and 0.51 mg/g DW in primulic acid II. Compared to the soil-grown roots (10.23 mg/g DW primulaverin, 0.28 mg/g DW primeverin, 17.01 mg/g DW primulic acid I, 0.09 mg/g DW primulic acid II), the in vitro grown roots at 22 °C exhibited a 5.67-fold higher content in primulic acid II. However, primulic acid I and primeverin content were approximately three-fold higher in soil-grown roots, while primulaverin content were at similar levels for both in vitro at 22 °C and soil-grown roots. From our results, tissue culture of P. veris subsp. veris could serve not only for propagation but also for production of species-specific secondary metabolites such as primulic acid II through adventitious root cultures. This would therefore limit the uncontrolled collection of this plant from its natural environment and provide natural products free from pesticides in a sustainable way.

The present study investigated the antifungal potential of hydroalcoholic extracts of propolis against the causal agent of grey mould, Botrytis cinerea, by in vitro and in vivo assays. Five different propolis from different Italian regions were subjected to hydroalcoholic extraction using different ethanol concentrations and extraction methods. The preliminary bio-assay showed significant inhibitory effects on B. cinerea mycelial growth of propolis extracts obtained using 90% ethanol and subjected to sonication. The calculation of EC50 values, based on the demonstrated efficacy of non-volatile and volatile metabolites of propolis extracts, was useful to understand the main fraction involved in the antifungal activity of the samples and to perform the in vivo assay on grape and blueberry fruits. Three of the propolis extracts showed a high amount of genistein. Conversely, the other two propolis showed a fair amount of apigenin, caffeic acid, chrysin, ferulic acid, kaempferol, luteolin, p-coumaric acid and quercetin. From the volatile analysis of propolis, the main compounds detected were α-cadinol, α-eudesmol, calamenene, cadinol, benzyl acetate, benzyl benzoate and benzyl alcohol. The results suggest that propolis extracts have potential as an effective postharvest antifungal treatment, with varying degrees of efficacy depending on the extraction method and the type of propolis metabolites.

In the Trentino Alto Adige region, disposal of grape stalks (GS) represents a major cost for wineries, despite their content of phenolic and tannin-rich compounds with potential functional value in ruminant nutrition. This study evaluated whether dietary GS supplementation could influence milk microbiota and cheese quality, supporting local circular-economy strategies. GS from three red cultivars (L-GS, CS-GS, M-GS) were dried, milled, and assessed for safety; their average total polyphenol content was approximately 15 g/kg DM. 3 Holstein cows underwent a 7-week trial consisting of alternating control (CTRL) and GS-supplemented periods (2% DM). Weekly milk samples (n = 21) and corresponding mini-cheeses (n = 21) were analyzed. GS supplementation did not affect milk coliforms (2.3–2.5 log CFU/mL), while total mesophilic counts were significantly lower in M-GS (2.8 ± 0.46 log CFU/mL) than in CTRL (4.5 ± 0.71; p < 0.05). Acinetobacter dominated the milk microbiota but decreased from 34.0% in CTRL to 18.0% in L-GS. Cheese total polyphenols were highest in CS-GS (224 ± 34 mg/kg). ¹H-NMR and VOCs profiling indicated cultivar-dependent shifts in carbohydrate-related metabolites and short-chain fatty acids. Overall, GS supplementation subtly modulated cheese biochemical and aromatic traits without impairing technological performance, supporting the valorization of winery by-products within integrated dairy-viticulture systems.

Mankai is a cultivated strain of Wolffia globose, an aquatic plant, one of the smallest plants on earth, belonging to the family Lemnaceae, known commonly as Duckweeds. Mankai is being considered as a high-quality substitute for animal protein, and a potential source of vitamin B12(1). In fact, it contains all 9 essential amino acids and its protein profile is extremely close to that of egg. Recently, it has been granted GRAS status, and chosen as a test food in a long-term intervention DIRECT-PLUS (ClinicalTrials.gov identifier (NCT number): NCT03020186) which aims to compare the effect of green-Mediterranean diet (composed with polyphenol enriched food including Mankai) + physical activity (PA) vs. Mediterranen diet + PA vs in 300 volunteers suffering from cardiometabolic disease. Mankai is also rich in iron and has been shown in humans to maintain Iron-Homeostasis and to reverse Anemia in Rats(2). However, molecular characterization of phenolic composition of Mankai plant has not previously been reported. Our initial measurements of total phenolic content determined by Folin-Ciocalteu assay classifies Mankai amongst the foods highest in polyphenols content (8606.7 mg/kg)(3). This poster presents the characterization of Mankai polyphenols according to Vrhovsek et al. 2012 [4] using UHPLC-ESI-MS/MS system, identifying 26 different polyphenols. One of the main advantages of Mankai is its hydroponic cultivation that optimizes yield throughout the year. Light source, water and mineral management can influence the composition of phenolic content. We quantified Mankai polyphenols in 30 different plant batches and 52 different treatments to assess how quality of light may play a major role in the accumulation of secondary plant compounds. Notably, led light seems to enhance the glycated form of luteolin and quercetin compared to sunlight and the aglycone form of luteolin and quercetin are higher in sunlight treatment. The present analysis confirms the high polyphenol status of Mankai, profiles its major polyphenol components and provides new information on how production process in terms of light quality determines polyphenol content.