Biochemical Response and Gene Expression to Water Deficit of Croatian Grapevine Cultivars (Vitis vinifera L.) and a Specimen of Vitis sylvestris.

The biochemical response and gene expression in different grapevine cultivars to water deficit are still not well understood. In this study, we investigated the performance of four traditional Croatian Vitis vinifera L. cultivars ('Plavac mali crni', 'Istrian Malvasia', 'Grasevina', and 'Tribidrag'), and one wild (Vitis vinifera subsp. sylvestris) genotype exposed to water deficit (WD) for nine days under semi-controlled conditions in the greenhouse. Sampling for biochemical and gene expression analyses was performed at days six and nine from the beginning of WD treatment. The WD affected the accumulation of metabolites with a significant increase in abscisic acid (ABA), salicylic acid (SA), and proline in the leaves of the stressed genotypes when the WD continued for nine days. Lipid peroxidation (MDA) was not significantly different from that of the control plants after six days of WD, whereas it was significantly lower (297.40 nmol/g dw) in the stressed plants after nine days. The cultivar 'Istrian Malvasia' responded rapidly to the WD and showed the highest and earliest increase in ABA levels (1.16 ng mg-1 dw, i.e., 3.4-fold increase compared to control). 'Grasevina' differed significantly from the other genotypes in SA content at both time points analyzed (six and nine days, 47.26 and 49.63 ng mg-1 dw, respectively). Proline level increased significantly under WD (up to 5-fold at day nine), and proline variation was not genotype driven. The expression of aquaporin genes (TIP2;1 and PIP2;1) was down-regulated in all genotypes, coinciding with the accumulation of ABA. The gene NCED1 (9-cis-epoxycarotenoid dioxygenase) related to ABA was up-regulated in all genotypes under stress conditions and served as a reliable marker of drought stress. This work suggests that the stress response in metabolite synthesis and accumulation is complex, treatment- and genotype-dependent.

Croatian Native Grapevine Varieties' VOCs Responses upon Plasmopara viticola Inoculation.

The Plasmopara viticola pathogen causes one of the most severe grapevine diseases, namely downy mildew. The response to P. viticola involves both visible symptoms and intricate metabolomic alterations, particularly in relation to volatile organic compounds, and depends on the degree of resistance of a particular variety. There are numerous native grapevine varieties in Croatia, and they vary in susceptibility to this oomycete. As previously reported, in vitro leaf disc bioassay and polyphenolic compound analysis are complementary methods that can be used to separate native varieties into various resistance classes. This research used the Solid Phase Microextraction-Arrow Gas Chromatography-Mass Spectrometry method to identify the early alterations in the VOCs in the leaves after P. viticola inoculation. Based on the absolute peak area of sesquiterpenes, some discrepancies between the sampling terms were noticed. The presence of certain chemical compounds such as humulene, ylangene, and α-farnesene helped distinguish the non-inoculated and inoculated samples. Although specific VOC responses to P. viticola infection of native varieties from various resistance classes could not be identified, the response of less susceptible native varieties and resistant controls was associated with an increase in the absolute peak area of several compounds, including geranylacetone, ß-ocimene, and (E)-2-hexen-1-ol.

Leaf Polyphenolic Profile as a Determinant of Croatian Native Grapevine Varieties' Susceptibility to Plasmopara viticola.

Since grapevine is highly susceptible to various pathogens, enormous amounts of pesticides are applied each season to achieve profitable production. One of the most destructive grapevine diseases is downy mildew, and their interaction has been in the spotlight for more than a decade. When it comes to a metabolome level, phenolic compounds are relevant to investigate due to their involvement in the plant immune system and known antifungal properties. Croatian grapevine germplasm is highly heterogeneous due to its long history of cultivation in diversified geographical regions. Since it has been found that native varieties react differently to the infection of Plasmopara viticola, the intention of this study is to define if the chemical background of the leaves, i.e., polyphenolic composition, is responsible for these dissimilarities. Therefore, the leaves of 17 genotypes, among which 14 were native and 3 were controls, were analyzed using high-performance liquid chromatography (HPLC) in four terms: before inoculation and 24, 48, and 96 h post inoculation (hpi). During this early phase, significant differences were found neither between the terms nor between the non-inoculated and inoculated samples, except for resveratrol-3-O-glucoside. By applying principal component analysis (PCA) using initial leaf polyphenolic composition, varieties of V. vinifera were clearly separated into three different groups corresponding to their International Organization of Vine and Wine (OIV) classes of susceptibility to P. viticola. Results obtained in this research suggest that the initial constitutive polyphenolic composition of the cultivar leaves has a crucial influence on their susceptibility to P. viticola, and this finding can be used to improve the success of grapevine breeding programs toward downy mildew resistance.

Optimization of SPME-Arrow-GC/MS Method for Determination of Free and Bound Volatile Organic Compounds from Grape Skins.

(1) Background: Solid phase microextraction (SPME)-Arrow is a new extraction technology recently employed in the analysis of volatiles in food materials. Grape volatile organic compounds (VOC) have a crucial role in the winemaking industry due to their sensory characteristics of wine.; (2) Methods: Box-Behnken experimental design and response surface methodology were used to optimise SPME-Arrow conditions (extraction temperature, incubation time, exposure time, desorption time). Analyzed VOCs were free VOCs directly from grape skins and bound VOCs released from grape skins by acid hydrolysis.; (3) Results: The most significant factors were extraction temperature and exposure time for both free and bound VOCs. For both factors, an increase in their values positively affected the extraction efficiency for almost all classes of VOCs. For free VOCs, the optimum extraction conditions are: extraction temperature 60 °C, incubation time 20 min, exposure time 49 min, and desorption time 7 min, while for the bound VOCs are: extraction temperature 60 °C, incubation time 20 min, exposure time 60 min, desorption time 7 min.; (4) Conclusions: Application of the optimized method provides a powerful tool in the analysis of major classes of volatile organic compounds from grape skins, which can be applied to a large number of samples.

Solid-liquid Extraction of Phenolics from Red Grape Skins.

For the characterization of grape cultivars, the profile and content of flavonoids are important because these compounds have an impact on grape and wine quality. A new extraction method for the recovery of flavonoids, e.g. anthocyanins, flavonols and flavan-3-ols from grape skins was developed. The optimization of solid-liquid extraction of flavonoids was conducted, with respect to the type of the organic solvent and its percentage in the extraction solvent as well as the extraction temperature and extraction time, using response surface methodology. Optimal conditions were obtained by using extraction solvent composed from acetonitrile:water:formic acid (20:79:1; v/v/v), at an extraction temperature of 50 °C, an extraction time of 1 h in a single-step extraction and with a solid-to-solvent ratio of 1:80 g mL(-1) (125 mg of grape skin powder and 10 mL of extraction solvent). The new optimal extraction method is inexpensive, simple, fast, accurate and selective for the recovery of simple flavonoids.

Application of pectinases for recovery of grape seeds phenolics.

Grape marc seeds contain large amounts of different polyphenolic compounds so they can be used for the recovery of these classes of compounds. A new green extraction method for the recovery of phenols from the grape seeds was developed. To provide a high quality extract sourced from natural product by using enzymes as alternative solvents and spending less energy, it is impeccable to call this method "green". Furthermore, this method was optimized by finding out which conditions provide the best possible results and achieving the maximum recovery of polyphenols from grape seeds. The optimization of the enzyme-assisted extraction of phenols was conducted using the commercially available oenological enzyme preparations with respect to the enzyme dosage, temperature, extraction time, pH value and enzyme preparation by applying the response surface methodology. Optimal conditions were determined using the enzyme preparation Lallzyme EX-V, at the extraction temperature of 48 °C, extraction time of 2 h and 43 min, pH 3.5 and enzyme dosage of 20.00 mg g-1. The new optimized extraction method is less expensive, simple, fast, precise and selective for the recovery of simple phenols (monomeric and dimeric form) and since it is based on the environmentally friendly extraction solvent it may provide a valuable alternative to the conventional extraction methods. The obtained extracts can be used for the application in pharmaceutical, food and cosmetic industry.

Multi-response optimisation of ultrasound-assisted extraction for recovery of flavonoids from red grape skins using response surface methodology.

INTRODUCTION: For the characterisation of grape cultivars, the profile and content of flavonoids are important because these compounds impact grape and wine quality. To determine the correct profile and content of flavonoids, the use of robust, sensitive and reliable methods is necessary. OBJECTIVE: The object of this research is to develop a new ultrasound-assisted extraction (UAE) method for the recovery of flavonoids from grape skins using response surface methodology. METHOD: Optimisation of UAE was performed using a complementary study combining a Box-Behnken experimental design with qualitative analysis by high-performance liquid chromatography. RESULTS: Optimal extraction conditions were obtained using the extraction solvent composed of acetonitrile:water:formic acid (26:73:1, v/v/v) at an extraction temperature of 50 °C, an extraction time of 15 min in a single-extraction step and with a solid-to-solvent ratio of 1:80 g/mL. The calculated relative standard deviations for the optimal extraction method were very low, measuring less than 5%. CONCLUSIONS: This study demonstrates that numerous factors have strong effects on the extraction efficiency, including the type of organic modifier and its percentage in the extraction solvent, the number of extraction steps, the solid-to-solvent ratio, the extraction time and temperature and, finally, the particular nature of analyte and their position within the grape skin cell.