Salicylic and methyl salicylic acid affect quality and phenolic profile of apple fruits three weeks before the harvest

Effects of spraying over apple trees (Malus domestica‘Topaz’) with methyl salicylic acid (MeSA) and SA during fruit maturation were investigated for quality parameters (weight, firmness, hue angle, red blush, yield) and phenolic profile of the peel and pulp (HPLC–mass spectrometry). These treatment effects were also investigated for activities of the phenylpropanoid pathway enzymes phenylalanine ammonia-lyase, chalcone synthase and isomerase (combined), and flavanone-3βhydroxylase. The MeSA and SA treatments resulted in poor fruit peel coloration, with higher hue angles and 20% and 10% lower red blush, respectively. Anthocyanin levels were also significantly lower (56%) for MeSA treatment. MeSA stimulated activities of phenylalanine ammonia-lyase and chalcone synthase/isomerase, which resulted in higher levels of flavanols (to 34%), flavonols (to 33%), and hydroxycinnamic acids (to 29%), versus control. Therefore, while these salicylate treatments improve levels of some beneficial polyphenols, they also have negative effects on the external quality characteristics of the fruit

Biochemical characterization of black and green mutant elderberry during fruit ripening

The content of sugars, organic acids, phenolic compounds and selected enzyme activities in the anthocyanin pathway were analyzed in NIGRA (Sambucus nigra var. nigra—black fruits) and VIRIDIS (S. nigra var. viridis—green fruits) fruits over four stages of ripening. The share of glucose and fructose in green fruits was higher than in colored fruits, and the sugar content increased significantly until the third developmental stage. Ripe NIGRA berries had 47% flavonol glycosides, 34% anthocyanins, 3% hydroxycinnamic acids and 14% flavanols, whereas the major phenolic group in the VIRIDIS fruits, making up 88% of the total analyzed polyphenols, was flavonols. NIGRA fruits were rich in anthocyanins (6020 [mi]g g$^{-1}$ FW), showing strong activation of the late anthocyanin pathway (dihydroflavonol 4-reductase, anthocyanidin synthase). In both color types, phenylalanine ammonia lyase and chalcone synthase/chalcone isomerase activities were highest in the first stage and decreased during ripening. In VIRIDIS fruit, no anthocyanins and only one flavanol (procyanidin dimer) were found. This was most likely caused by a lack of induction of the late anthocyanin pathway in the last period of fruit ripening. The VIRIDIS genotype may be useful in studying the regulatory structures of anthocyanin biosynthesis and the contribution of distinct flavonoid classes to the health benefits of elderberries

The (Bio)chemical Base of Flower Colour in Bidens ferulifolia

Bidens ferulifolia is a yellow flowering plant, originating from Mexico, which is increasingly popular as an ornamental plant. In the past few years, new colour combinations ranging from pure yellow over yellow-red, white-red, pure white and purple have emerged on the market. We analysed 16 Bidens ferulifolia genotypes to provide insight into the (bio)chemical base underlying the colour formation, which involves flavonoids, anthochlors and carotenoids. In all but purple and white genotypes, anthochlors were the prevalent pigments, primarily derivatives of okanin, a 6′-deoxychalcone carrying an unusual 2′3′4′-hydroxylation pattern in ring A. The presence of a cytochrome-P450-dependent monooxygenase introducing the additional hydroxyl group in position 3′ of both isoliquiritigenin and butein was demonstrated for the first time. All genotypes accumulate considerable amounts of the flavone luteolin. Red and purple genotypes additionally accumulate cyanidin-type anthocyanins. Acyanic genotypes lack flavanone 3-hydroxylase and/or dihydroflavonol 4-reductase activity, which creates a bottleneck in the anthocyanin pathway. The carotenoid spectrum was analysed in two Bidens genotypes and showed strong variation between the two cultivars. In comparison to anthochlors, carotenoids were present in much lower concentrations. Carotenoid monoesters, as well as diesters, were determined for the first time in B. ferulifolia flower extracts

Experiences with Pluvial Flood Risk Mapping in Croatia at Multiple Spatial Scales

This study presents our experience with pluvial flood modelling and risk assessment in Croatia. We focus on the methodology that is suitable for investigating different levels of flood risk - from the catchment level to the city level to the local sub-basin level. We discuss the availability and selection of spatial input data, such as digital terrain models (DTMs) and land use models, and how they can be processed for flood hazard analysis and flood risk assessments. We highlight the differences between flood simulation results using DTMs at different resolutions and discuss the optimal resolution. We also examine different types of open land cover/land use data available for the EU, such as the Copernicus Land Monitoring Service data. We describe various aspects related to integrated 2D hydrological-hydraulic modelling and the rain-on-grid approach, and we compare the results at different scales. In terms of flood risk assessment, we explain two different types of analyses, particularly the differences between the object-based and the pixel-based approach. Lastly, we provide flood hazard and flood risk maps for a pilot area in Croatia and offer some recommendations for further research. Our study emphasizes the importance of using an integrated and comprehensive approach that considers different sources of flooding, as well as the potential compound effects of these sources, to develop more realistic and accurate flood risk assessments

Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica

Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2′-O-glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic M. × domestica plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3′-hydroxylase (F3′H), which catalyzes B-ring hydroxylation of flavonoids. We isolated two F3′H cDNA clones from apple leaves and tested recombinant Malus F3′Hs for their substrate specificity. From the two isolated cDNA clones, only F3′HII encoded a functionally active enzyme. In the F3′HI sequence, we identified two putatively relevant amino acids that were exchanged in comparison to that of a previously published F3′HI. Site directed mutagenesis, which exchanged an isoleucine into methionine in position 211 restored the functional activity, which is probably because it is located in an area involved in interaction with the substrate. In contrast to high activity with various flavonoid substrates, the recombinant enzymes did not accept phloretin under assay conditions, making an involvement in the dihydrochalcone biosynthesis unlikely

Alteration of the phenylpropanoid pathway by watercore disorder in apple (Malus x domestica)

Watercore is a physiological disorder of apple expressed as fluid deposition in intercellular spaces. We analysed the watercore prone cultivar \u27Fuji\u27 to establish a link between enzymatic response and fluid accumulation in the fruit. Individual phenolic content, selected enzyme activities of the phenylpropanoid pathway, sugars and organic acids were quantified in watercore affected flesh and peel. In addition, transcriptome sequencing was performed to obtain a first insight into molecular mechanisms underlying the physiological disorder. Sampled material included: peel (HP) and flesh from healthy fruit (H), peel from watercore affected fruit (WP), healthy flesh from fruit with watercore (HW) and watercore flesh (WW) from fruit with watercore. A sorbitol increase in watercore affected apple was observed not only in the flesh but also in the peel. Moreover, two phenolic groups (hydroxycinnamic acids and flavonols) were significantly affected by watercore and their content was higher in WP in comparison to HP. Dihydrochalcone content was the highest in WW flesh. This was supported by RNA-seq data, which indicated a generally increased expression of genes from the early flavonoid pathway, sorbitol metabolism and polyphenol oxidases. Among the phenylpropanoid pathway enzymes analysed, phloretin 2′-O-glycosyltransferase was the only one exhibiting altered activity