Semi-Targeted Profiling of the Lipidome Changes Induced by Erysiphe Necator in Disease-Resistant and Vitis vinifera L. Varieties.

The ascomycete Erysiphe necator is a serious pathogen in viticulture. Despite the fact that some grapevine genotypes exhibit mono-locus or pyramided resistance to this fungus, the lipidomics basis of these genotypes' defense mechanisms remains unknown. Lipid molecules have critical functions in plant defenses, acting as structural barriers in the cell wall that limit pathogen access or as signaling molecules after stress responses that may regulate innate plant immunity. To unravel and better understand their involvement in plant defense, we used a novel approach of ultra-high performance liquid chromatography (UHPLC)-MS/MS to study how E. necator infection changes the lipid profile of genotypes with different sources of resistance, including BC4 (Run1), "Kishmish vatkhana" (Ren1), F26P92 (Ren3; Ren9), and "Teroldego" (a susceptible genotype), at 0, 24, and 48 hpi. The lipidome alterations were most visible at 24 hpi for BC4 and F26P92, and at 48 hpi for "Kishmish vatkhana". Among the most abundant lipids in grapevine leaves were the extra-plastidial lipids: glycerophosphocholine (PCs), glycerophosphoethanolamine (PEs) and the signaling lipids: glycerophosphates (Pas) and glycerophosphoinositols (PIs), followed by the plastid lipids: glycerophosphoglycerols (PGs), monogalactosyldiacylglycerols (MGDGs), and digalactosyldiacylglycerols (DGDGs) and, in lower amounts lyso-glycerophosphocholines (LPCs), lyso-glycerophosphoglycerols (LPGs), lyso-glycerophosphoinositols (LPIs), and lyso-glycerophosphoethanolamine (LPEs). Furthermore, the three resistant genotypes had the most prevalent down-accumulated lipid classes, while the susceptible genotype had the most prevalent up-accumulated lipid classes.

Aroma Investigation of New and Standard Apple Varieties Grown at Two Altitudes Using Gas Chromatography-Mass Spectrometry Combined with Sensory Analysis.

The aromatic profile of apples constitutes important information for the characterization and description of local products. Apple flavor is determined by perception in mouth and aroma; while the first is mainly defined by sugars and organic acids, aroma is a complex mixture of many volatile organic compounds (VOCs) whose composition is often specific to the variety. Headspace-solid phase microextraction gas chromatography coupled with mass spectrometry (HS-SPME-GC-MS) allows for the detection of detailed information of volatile constituents. In this study, eleven apple varieties (Braeburn, Fuji, Gala, Golden Delicious, Coop 39-Crimson Crisp®, Dalinette-Choupette®, Fujion, CIV323-Isaaq®, Coop43-Juliet®, SQ159-Natyra®, UEB32642-Opal®) grown in two pedoclimatic locations at different altitudes in South Tyrol (Italy) (ca. 225 m and ca. 650 m a.s.l.) were investigated. Thirty-eight VOCs were identified and combined with sensory analysis results (from 11 trained panelist) to characterize the aroma of new and standard apple varieties with a special focus on pedoclimatic location differences. The study shows strong diversification of the varieties based on their VOC profiles and sensory attributes, as expected. Moreover, investigating how the pedoclimatic location at different altitudes can influence the apple aroma profile, we identified twelve VOCs involved in these differences and provided a deeper investigation on how different altitudes can influence the apple aroma composition and perceptions combining the analytical and sensory parameters.

Volatile Profile of Mead Fermenting Blossom Honey and Honeydew Honey with or without Ribes nigrum.

Mead is a not very diffused alcoholic beverage and is obtained by fermentation of honey and water. Despite its very long tradition, little information is available on the relation between the ingredient used during fermentation and the aromatic characteristics of the fermented beverage outcome. In order to provide further information, multi-floral blossom honey and a forest honeydew honey with and without the addition of black currant during fermentation were used to prepare four different honey wines to be compared for their volatile organic compound content. Fermentation was monitored, and the total phenolic content (Folin-Ciocalteu), volatile organic compounds (HS-SPME-GC-MS), together with a sensory evaluation on the overall quality (44 nontrained panelists) were measured for all products at the end of fermentation. A higher total phenolic content resulted in honeydew honey meads, as well as the correspondent honey wine prepared with black currant. A total of 46 volatile organic compounds for pre-fermentation samples and 62 for post-fermentation samples were identified belonging to higher alcohols, organic acids, esters, and terpenes. The sensory analysis showed that the difference in meads made from blossom honey and honeydew honey was perceptible by the panelists with a general greater appreciation for the traditional blossom honey mead. These results demonstrated the influences of different components in meads, in particular, the influence of honey quality. However, further studies are needed to establish the relationship between the chemical profile and mead flavor perception.

Apple fruit superficial scald resistance mediated by ethylene inhibition is associated with diverse metabolic processes.

Fruits stored at low temperature can exhibit different types of chilling injury. In apple, one of the most serious physiological disorders is superficial scald, which is characterized by discoloration and brown necrotic patches on the fruit exocarp. Although this phenomenon is widely ascribed to the oxidation of α-farnesene, its physiology is not yet fully understood. To elucidate the mechanism of superficial scald development and possible means of prevention, we performed an integrated metabolite screen, including an analysis of volatiles, phenols and lipids, together with a large-scale transcriptome study. We also determined that prevention of superficial scald, through the use of an ethylene action inhibitor, is associated with the triggering of cold acclimation-related processes. Specifically, the inhibition of ethylene perception stimulated the production of antioxidant compounds to scavenge reactive oxygen species, the synthesis of fatty acids to stabilize plastid and vacuole membranes against cold temperature, and the accumulation of the sorbitol, which can act as a cryoprotectant. The pattern of sorbitol accumulation was consistent with the expression profile of a sorbitol 6-phosphate dehydrogenase, MdS6PDH, the overexpression of which in transgenic Arabidopsis thaliana plants confirmed its involvement in the cold acclimation and freezing tolerance.

Untargeted lipidomic profiling of grapes highlights the importance of modified lipid species beyond the traditional compound classes.

The aim of this paper is to provide a detailed characterisation of grape lipidome. To achieve this objective, it starts by describing a pipeline implemented in R software to allow the semi-automatic annotation of the detected lipid species. It also provides an extensive description of the different properties of each molecule (such as retention time dependencies, mass accuracy, adduct formation and fragmentation patterns), which allowed the annotations to be made more accurately. Most annotated lipids in the grape samples were (lyso)glycerophospholipids and glycerolipids, although a few free fatty acids, hydroxyceramides and sitosterol esters were also observed. The proposed pipeline also allowed the identification of a series of methylated glycerophosphates never previously observed in grapes. The current results highlight the importance of expanding chemical analyses beyond the classical lipid categories.

Secondary and primary metabolites reveal putative resistance-associated biomarkers against Erysiphe necator in resistant grapevine genotypes.

Numerous fungicide applications are required to control Erysiphe necator, the causative agent of powdery mildew. This increased demand for cultivars with strong and long-lasting field resistance to diseases and pests. In comparison to the susceptible cultivar 'Teroldego', the current study provides information on some promising disease-resistant varieties (mono-locus) carrying one E. necator-resistant locus: BC4 and 'Kishmish vatkana', as well as resistant genotypes carrying several E. necator resistant loci (pyramided): 'Bianca', F26P92, F13P71, and NY42. A clear picture of the metabolites' alterations in response to the pathogen is shown by profiling the main and secondary metabolism: primary compounds and lipids; volatile organic compounds and phenolic compounds at 0, 12, and 48 hours after pathogen inoculation. We identified several compounds whose metabolic modulation indicated that resistant plants initiate defense upon pathogen inoculation, which, while similar to the susceptible genotype in some cases, did not imply that the plants were not resistant, but rather that their resistance was modulated at different percentages of metabolite accumulation and with different effect sizes. As a result, we discovered ten up-accumulated metabolites that distinguished resistant from susceptible varieties in response to powdery mildew inoculation, three of which have already been proposed as resistance biomarkers due to their role in activating the plant defense response.

Mono-Locus and Pyramided Resistant Grapevine Cultivars Reveal Early Putative Biomarkers Upon Artificial Inoculation With Plasmopara viticola.

One of the most economically important grapevine diseases is Downy mildew (DM) caused by the oomycete Plasmopara viticola. A strategy to reduce the use of fungicides to compensate for the high susceptibility of V. vinifera is the selection of grapevine varieties showing pathogen-specific resistance. We applied a metabolomics approach to evaluate the metabolic modulation in mono-locus resistant genotypes carrying one locus associated with P. viticola resistance (Rpv) (BC4- Rpv1, Bianca- Rpv3-1, F12P160- Rpv12, Solaris- Rpv10), as well as in pyramided resistant genotypes carrying more than one Rpv (F12P60- Rpv3-1; Rpv12 and F12P127- Rpv3-1, Rpv3-3; Rpv10) taking as a reference the susceptible genotype Pinot Noir. In order to understand if different sources of resistance are associated with different degrees of resistance and, implicitly, with different responses to the pathogen, we considered the most important classes of plant metabolite primary compounds, lipids, phenols and volatile organic compounds at 0, 12, 48, and 96 h post-artificial inoculation (hpi). We identified 264 modulated compounds; among these, 22 metabolites were found accumulated in significant quantities in the resistant cultivars compared to Pinot Noir. In mono-locus genotypes, the highest modulation of the metabolites was noticed at 48 and 96 hpi, except for Solaris, that showed a behavior similar to the pyramided genotypes in which the changes started to occur as early as 12 hpi. Bianca, Solaris and F12P60 showed the highest number of interesting compounds accumulated after the artificial infection and with a putative effect against the pathogen. In contrast, Pinot Noir showed a less effective defense response in containing DM growth.

Grape Lipidomics: An Extensive Profiling thorough UHPLC-MS/MS Method.

Lipids play many essential roles in living organisms, which accounts for the great diversity of these amphiphilic molecules within the individual lipid classes, while their composition depends on intrinsic and extrinsic factors. Recent developments in mass spectrometric methods have significantly contributed to the widespread application of the liquid chromatography-mass spectrometry (LC-MS) approach to the analysis of plant lipids. However, only a few investigators have studied the extensive composition of grape lipids. The present work describes the development of an ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method that includes 8098 MRM; the method has been validated using a reference sample of grapes at maturity with a successful analysis and semi-quantification of 412 compounds. The aforementioned method was subsequently applied also to the analysis of the lipid profile variation during the Ribolla Gialla cv. grape maturation process. The partial least squares (PLS) regression model fitted to our experimental data showed that a higher proportion of certain glycerophospholipids (i.e., glycerophosphoethanolamines, PE and glycerophosphoglycerols, PG) and of some hydrolysates from those groups (i.e., lyso-glycerophosphocholines, LPC and lyso-glycerophosphoethanolamines, LPE) can be positively associated with the increasing °Brix rate, while a negative association was found for ceramides (CER) and galactolipids digalactosyldiacylglycerols (DGDG). The validated method has proven to be robust and informative for profiling grape lipids, with the possibility of application to other studies and matrices.

Two-omics data revealed commonalities and differences between Rpv12- and Rpv3-mediated resistance in grapevine.

Plasmopara viticola is the causal agent of grapevine downy mildew (DM). DM resistant varieties deploy effector-triggered immunity (ETI) to inhibit pathogen growth, which is activated by major resistance loci, the most common of which are Rpv3 and Rpv12. We previously showed that a quick metabolome response lies behind the ETI conferred by Rpv3 TIR-NB-LRR genes. Here we used a grape variety operating Rpv12-mediated ETI, which is conferred by an independent locus containing CC-NB-LRR genes, to investigate the defence response using GC/MS, UPLC, UHPLC and RNA-Seq analyses. Eighty-eight metabolites showed significantly different concentration and 432 genes showed differential expression between inoculated resistant leaves and controls. Most metabolite changes in sugars, fatty acids and phenols were similar in timing and direction to those observed in Rpv3-mediated ETI but some of them were stronger or more persistent. Activators, elicitors and signal transducers for the formation of reactive oxygen species were early observed in samples undergoing Rpv12-mediated ETI and were paralleled and followed by the upregulation of genes belonging to ontology categories associated with salicylic acid signalling, signal transduction, WRKY transcription factors and synthesis of PR-1, PR-2, PR-5 pathogenesis-related proteins.

Lipid, phenol and carotenoid changes in 'Bianca' grapevine leaves after mechanical wounding: a case study.

Metabolic changes can occur in plants in response to abiotic stress. Extensive use of leaf discs (mechanical wounding) in studies regarding the effect on the biochemical response of the grapevine to different types of biotic stress makes it necessary to understand metabolic perturbation after injury. In this study, we investigate how mechanical wounding can affect the metabolism of grapevine leaf tissue using Bianca variety as case study. Two sizes of leaf discs (1.1 and 2.8 cm in diameter) were excised from leaves, and phenol, lipid and carotenoid perturbation were investigated 0, 6, 12, 24, 48, 96 and 120 h post cutting. In our study, we found an accumulation of molecules belonging to stilbenoid and stilbene classes such as trans-resveratrol, trans-piceide, Z-miyabenol C, E-cis-miyabenol C and ampelopsin D + quadrangularin A after abiotic stress. The increase in fatty acids such as linoleic acid, linolenic acid and oleic + cis-vaccenic acid during the first 12 h after injury, followed by a return to basal level, allowed us to surmise their role in response to abiotic stress, in particular to mechanical wounding in grapevine leaves. Different-sized discs caused a different response to the tissue, with a higher accumulation in 1.1-cm-diameter discs, especially of phenol compounds. The results of this work can be used to better understand metabolic changes due to biotic stress, having previous knowledge about the perturbation caused by abiotic stress.

Identification of Biomarkers for Defense Response to Plasmopara viticola in a Resistant Grape Variety.

Downy mildew (Plasmopara viticola) is one of the most destructive diseases of the cultivated species Vitis vinifera. The use of resistant varieties, originally derived from backcrosses of North American Vitis spp., is a promising solution to reduce disease damage in the vineyards. To shed light on the type and the timing of pathogen-triggered resistance, this work aimed at discovering biomarkers for the defense response in the resistant variety Bianca, using leaf discs after inoculation with a suspension of P. viticola. We investigated primary and secondary metabolism at 12, 24, 48, and 96 h post-inoculation (hpi). We used methods of identification and quantification for lipids (LC-MS/MS), phenols (LC-MS/MS), primary compounds (GC-MS), and semi-quantification for volatile compounds (GC-MS). We were able to identify and quantify or semi-quantify 176 metabolites, among which 53 were modulated in response to pathogen infection. The earliest changes occurred in primary metabolism at 24-48 hpi and involved lipid compounds, specifically unsaturated fatty acid and ceramide; amino acids, in particular proline; and some acids and sugars. At 48 hpi, we also found changes in volatile compounds and accumulation of benzaldehyde, a promoter of salicylic acid-mediated defense. Secondary metabolism was strongly induced only at later stages. The classes of compounds that increased at 96 hpi included phenylpropanoids, flavonols, stilbenes, and stilbenoids. Among stilbenoids we found an accumulation of ampelopsin H + vaticanol C, pallidol, ampelopsin D + quadrangularin A, Z-miyabenol C, and α-viniferin in inoculated samples. Some of these compounds are known as phytoalexins, while others are novel biomarkers for the defense response in Bianca. This work highlighted some important aspects of the host response to P. viticola in a commercial variety under controlled conditions, providing biomarkers for a better understanding of the mechanism of plant defense and a potential application in field studies of resistant varieties.

A rapid LC-MS/MS method for quantitative profiling of fatty acids, sterols, glycerolipids, glycerophospholipids and sphingolipids in grapes.

The abundance of lipids in plants is influenced by genotype and phenotype. Despite being a very important class of plant metabolites, knowledge of grape lipids is still very limited to date, with the exception of those located in seeds. Few investigations of grape lipids have shown that their profile depends on grape maturity, the variety and their location in the berry. Recent advances in liquid chromatography coupled to mass spectrometry have paved the way for faster analysis of lipids with minimal sample preparation. Here we describe a validation method for the extraction, identification and quantification of different classes of grape lipids: fatty acids, sterols, glycerolipids, glycerophospholipids and sphingolipids using liquid chromatographic electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The method was validated for 33 lipids, with linearity range (R(2)=0.95-1.00), LOQ (0.003-14.88 ng mL(-1)) and intraday and interday repeatability being evaluated for each lipid. The lipid profiling method developed was successfully applied to the analysis of 18 grape samples (10 red grape and 8 white grape varieties) from 4 different genetic groups: Vitis vinifera, Vitis non-vinifera, Muscat and hybrid; 33 lipids were identified and quantified. This method, which can be easily expanded to include further compounds and other plant tissues, is the starting point for analysis of the lipid profile in different grape tissues, an essential goal for better understanding the role of lipids in grape physiology.