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annotation was tentatively performed by comparing experimental tandem mass spectra with MS-DIAL LC-MS reference libraries “ESI(+)-MS/MS from authentic standards (16,481 unique compounds)” and “ESI(-)-MS/MS from authentic standards (9,033 unique compounds)”. Unknown interesting compounds were analysed with MS-FINDER 3.5, an open access structure-elucidation program that allows the annotation of unknown compounds by predicting their elemental formula and by comparing the experimental tandem mass spectra with metabolome databases and an in-silico mass spectral fragmentation (http://prime.psc.riken.jp/compms/msfinder/main.html). When standards were available, retention times and tandem mass spectra of compounds present in samples were compared with the one of the standards injected under the same conditions.&nbsp;</p>MetaboLightsPublicLiquid Chromatography MS - negative - reverse phaseLiquid Chromatography MS - positive - reverse phase<p>Leaf extracts (2 µL) were analysed in random order with an Ultimate 3000 UHPLC chromatograph coupled with a Q-Exactive™ hybrid quadrupole-Orbitrap™ mass spectrometer (Thermo Fisher Scientific). An Acclaim mixed-mode WCX-1, 3 µm, 120 Å, 2.1 x 150 mm (Thermo Fisher Scientific) column thermostated at 20 °C was used. Eluents were ultrapure water and acetonitrile, both acidified with 0.1% of formic acid, at a flow rate of 0.2 mL/min. The gradient was 100% water for 3 min, linear increasing to 100% acetonitrile in 15 min, 100% constant acetonitrile for 4 min and then 100% water in 1 min, equilibration for 7 min. </p>Decoding heat-shock responses in Vitis vinifera L. by metabolic, VOC and physiological profiling: towards the identification of volatile and metabolic biomarkers.Silvia PettenuzzoUniversity of PadovaleafSolventmass spectrometry assay<p>For the experiment conducted in controlled conditions, the 2nd, 5th and 8th leaf of each biological replicate were collected as control samples before the start of the heat shock, while the 4th, 7th and 10th leaf were collected as stress samples at the end of the 3 h of heat shock.</p><p>Leaf sampling in the field during hot days was performed for each genotype on the 6th leaf on the three shoots between 5.30 and 8.00 in the morning as control, while the 7th leaf was sampled during heat stress between 13.00 and 16.00. Leaves were immediately frozen in liquid nitrogen and subsequently stored at -80 °C.</p><p>For each treatment, leaves collected for each genotype were pulled together and grinded in liquid nitrogen.</p><p>Frozen leaf powder (200 mg) was extracted with 1 mL of a methanol:ultrapure water (70:30) solution containing phenylalanine-d5 added as surrogate IS with a concentration of 200 µg/L. The extraction mixture was sonicated for 15 min and then centrifuged for 10 min at 14000 rpm. The supernatant was then lyophilized and resuspended in 200 µL of ultrapure water with 0.1% of formic acid. Quality Control (QC) samples were prepared from a mixture of frozen leaf powder of all the genotypes under analysis at control and stress and extracted with the same protocol.</p>SolventVitis viniferahttps://www.ebi.ac.uk/metabolights/MTBLS13195Silvia Pettenuzzo. Università degli Studi di Padova. Via Marzolo, 1. silvia.pettenuzzo@unipd.it.Maria Grando. Università di Trento. stella.grando@unitn.it.Sara Bogialli. Università degli Studi di Padova. sara.bogialli@unipd.it.Pietro Franceschi. Fondazione Edmund Mach. pietro.franceschi@fmach.it.Emanuela Betta. Fondazione Edmund Mach. emanuela.betta@fmach.it.Franco Biasioli. Fondazione Edmund Mach. franco.biasioli@fmach.it.Luca Cappellin. Università degli Studi di Padova. luca.cappellin@unipd.it.Laura Costantini. Fondazione Edmund Mach. laura.costantini@fmach.it.Marco Roverso. Università degli Studi di Padova. Via Marzolo, 1. marco.roverso@unipd.it.Michele Faralli. Università di Trento. michele.faralli@unitn.it.<p>Raw data were converted to ABF format (Reyfics Analysis Base File Converter) and processed with MS-DIAL 4.9. Parameters for data extraction were selected on the basis of the chromatographic performances of the above-mentioned equipment. The feature’s areas were extracted from MS-DIAL, after alignment, identification and normalization by internal standard (phenylalanine-d5, relative standard deviation (RSD%)= 2-20%) and QCs (using the LOESS normalization). Data were further normalized by subtracting at each feature the mean of the genotype to reduce the variability related to varietal abundance and highlight differences related to the application of the heat shock.&nbsp;</p><p><br></p><p>Multivariate analysis, statistical analysis and graphical representation were performed using R software environment 4.2.1 (https://www.r-project.org/). For Principal Component Analysis (PCA) the following packages were used: “ggfortify” (Horikoshi and Tang 2016), “ggplot2” (Wickham 2016). Correlations were calculated with the package ‘tidyverse’ (Wickham et al. 2019). Due to the non-normal distribution of data, Spearman correlations were calculated and T-test and One-Way ANOVA Test (ANOVA) were performed on logarithm transformed data with the packages ‘rstatix’ (Kassambara 2023) and ‘devtools’ (Wickham at al. 2022). Effect sizes were calculated and plotted with packages ‘effectsize’ and ‘effsize’ (Ben-Shachar et al. 2020). Graphical representations were performed with the package ‘ggplot2’. Gene Ontology analysis for metabolomics was performed using INCHIKEY codes of identified metabolites on the IDSL.GOA website.</p>TreatmentExperimentsilvia.pettenuzzo@unipd.it<p><strong>Plant Materials:</strong></p><p>The study was conducted on selected genotypes of a mapping population obtained from the cross ‘Rhine Riesling’ x ‘Cabernet Sauvignon’ (RRxCS). The cross was made in 2005 and the seedlings were planted in the field on their own roots in 2008. Plants were grown at the Giaroni experimental field of Edmund Mach Foundation (San Michele all’Adige, Trentino Alto Adige, Italy, 46°18′N, 11°13′E) and trained according to the Guyot system.</p><p>Woody shoots of parental lines and RRxCS offspring were collected in January 2023 and cut in smaller pieces containing 3 nodes. Only the apical bud was left, the other two were cut and covered with grafting wax (Fitobalsam, Zapi garden). Cuttings were grown in a climatic chamber at 25 °C with 24 h of light until bud burst and root appearance. Stem cuttings were then transferred in a 13 x 9 x 9 cm (1 L) plastic container filled with peat moss and grown in greenhouse conditions. Vines were irrigated and fertilized according to the greenhouse management and treated against powdery mildew to avoid plant infection. After about two months of growth, cuttings with 12-15 leaves were selected to be studied in a growth chamber. Cuttings of different genotypes were prepared with the same procedure at three different times (January-February-March) to perform the study on plants with the same age.</p><p><br></p><p><strong>Experimental design:</strong></p><p>Stem cuttings of each genotype were acclimated for 5 days in a controlled environment (CLIMACELL-E-707-c, MMM Group, Munich, Germany) with 70 % ± 5 % of relative humidity (RH). Temperatures were set at 25/20 °C day/night, with a photoperiod of 16 h under a white fluorescent light at 100 µmol m-2 s-1 plus a red/blue LED light at 600 µmol m-2 s-1. Plants were irrigated to full pot capacity on alternate days during the acclimatation period and after the stress application. Chamber temperature and humidity were monitored with a data logger during experiments. After the acclimatation period, plants were subjected to a sudden heat shock (HS). The temperature of the chamber was raised to 40 or 43 °C in 30 min and maintained for 3 h (from 11:00 to 14:00). After the heat shock, the temperature was set back to 25 °C for recovery. For each genotype two identical but distinct experiments were performed with different biological replicates due to analysis incompatibility. In particular, one experiment (N= 5 biological replicates) was performed to sample the volatile organic compounds emitted by plants with CLSA, while physiological measurements and leaf sampling for metabolomic analysis were performed in a second experiment (N= 6 biological replicates). Measurement of physiological parameters, VOCs and leaves sampled before the stress application were considered as control. VOC collection with CLSA technique was then performed during 3 h of heat shock, while leaves were sampled immediately after the end of the treatment. Physiological parameters were measured during stress application, at the end of the temperature ramp and after 1, 2 and 3 h of heat shock, and during recovery step (1-3 h after the end of the heat shock and after 24 h). To confirm results obtained in controlled conditions, leaves of selected genotypes of the population were sampled in the field to perform metabolomic analysis in two different seasons (2022 and 2023).&nbsp;</p>MetabolomicsMetabolomicsPhysiological responseGrapevineHeat StressVolatile Organic CompoundsMetabolomicsPhysiological responseGrapevineHeat StressVolatile Organic Compounds<p>Mass spectrometric conditions were: electrospray ionization (ESI) in both positive (+) and negative (-) mode, data dependent acquisition with resolution 35,000 in full MS and 17,500 in MS/MS, AGC target 1·106 in full MS and 1·105 in MS/MS, max injection time of 100 ms, m/z range 70-1000, isolation window 2.0 m/z, collision gas nitrogen, normalized collision energy 25 eV. Data dependent settings were: Minimum AGC target 9·103 and dynamic exclusion 20s. The capillary voltage was 4.0 kV (+)/ 2.8 kV (-), capillary temperature 300 °C (+)/280 °C (-), and auxiliary gas was nitrogen at 20 psi. The calibration was performed with a standard solution (Pierce LTQ Velos ESI positive/negative ion calibration solution, Thermo Fisher Scientific) and the software Xcalibur 3.1 was used for instrument control (Thermo Fisher Scientific).</p>Benzoic acidNaringeninRiboflavinRUTOSIDE (rutin)CitrusinProcyanidin B1Protocatechuic acid6,7-Dihydroxycoumaringamma-Glutamyl-cysteineINDOLEisomaltulosePhloretinSpermidine4-Guanidinobutyric acidConiferyl aldehydeKynurenic acidL-HistidineTyraminegamma-GlutamylleucinePyridoxalTrigonelline5-O-methylvisammiosidePeonidin-3-O-glucosideEsculinThreonineMethionineGlucose-1-phosphateKaempferol 3-glucuronideconiferinProcyanidin B2Syringic acidIndole-3-carboxyaldehydeCyanidin-3-O-alpha-arabinosideCysteinylglycinehyperosideL-kynurenineIsorhamnetin 3-galactosidePhenylacetaldehydeGlutamic acidSalicyclic acidsyringinPyruvic acidN-AcetylargininePaeonolL-Pipecolic acidSalidrosideTarginineGentisic acidKaempferol-3-O-rhamnosidePyroglutamic acidsn-Glycero-3-phosphocholineLuteolinquercetin 3-O-glucuronideN-Acetylglutamic acidPYRIDOXINEPhenylalanineKaempferolmyricetin-3-O-hexosideNordihydroguaiaretic acidDL-NorleucineTryptophanPHENETHYLAMINEAdenosine MonophosphateCaffeic acidFerulic acidS-Adenosyl-methionineCholineL-ARGININEP-Coumaric acidUracilPantothenic acidIndolelactic acidepicatechin gallateGuanosineLEVODOPAl-ascorbic acidCHLOROGENIC ACIDAbscisic acidL-5-OxoprolineGlutathione (reduced)TyrosinePruninGallic acid6-Methylcoumarincuminyl alcoholSpermineHypoxanthine2'-DeoxyadenosineProcyanidin CProlineBenzyl glucopyranosideEpicatechinO-Acetyl-L-homoserineResveratrolAspartic acidLoliolideAdenosineAdenine5'-S-MethylthioadenosinePhosphocholineIsoquercitrinQuercetin 3-O-malonylglucosidetaxifolinsinapic acidBETA-INDOLEACETIC ACIDQuercetin-3,4'-O-di-beta-glucosideGlutaminefalseDecoding heat-shock responses in Vitis vinifera L. by metabolic, VOC and physiological profiling: towards the identification of volatile and metabolic biomarkers<p>Grapevine (Vitis spp.) is a crop of major economic importance in Europe. Rising temperatures and extreme weather events reduce yield and alter berry composition, affecting wine quality. Despite advances in vineyard management, mechanisms underlying grapevine thermotolerance remain poorly understood. To fill this gap, we combined physiological, volatile organic compound profiling and untargeted metabolomics. Selected genotypes from a ‘Rhine Riesling’ x ‘Cabernet Sauvignon’ progeny were exposed to a 3 h heat shock at 40 and 43 °C under controlled conditions. At 43 °C genotypes were heat stressed, with significant chlorophyll fluorescence decrease and modulation of leaf transpiration and metabolism. VOC analysis highlighted 6-methyl-5-hepten-2-one as a candidate heat stress marker, as well as benzenic compounds (i.e 2-phenyl-2-propanol). Untargeted metabolite profiling revealed several candidate heat-stress biomarkers; notably, the hypothesised 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate (MEP-cPP) and guanosine accumulated preferentially in susceptible genotypes. Both markers were validated in the field, providing candidate molecular tools for breeding heat-tolerant grapevines.</p>2026-06-262025-10-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:15584CHEBI:15671CHEBI:18026CHEBI:24673CHEBI:28635CHEBI:30831CHEBI:17069CHEBI:15864CHEBI:16630CHEBI:28908CHEBI:172661CHEBI:29749CHEBI:30959CHEBI:4853CHEBI:16258CHEBI:16761CHEBI:16619CHEBI:18148CHEBI:18367CHEBI:16778CHEBI:29073CHEBI:16971CHEBI:16433CHEBI:3293CHEBI:16112CHEBI:18135CHEBI:45712CHEBI:16947CHEBI:32374CHEBI:15937CHEBI:3978CHEBI:22660CHEBI:27956CHEBI:80441CHEBI:48095CHEBI:28860CHEBI:90CHEBI:70255CHEBI:4806CHEBI:139458CHEBI:17620CHEBI:86388CHEBI:16238CHEBI:18012CHEBI:30778CHEBI:68433CHEBI:16015CHEBI:18050CHEBI:82966CHEBI:17859CHEBI:16856CHEBI:57925CHEBI:33508CHEBI:16750CHEBI:44747CHEBI:16831CHEBI:67486CHEBI:17596CHEBI:6052CHEBI:139417CHEBI:30838CHEBI:191842CHEBI:189575CHEBI:30200CHEBI:16010CHEBI:177059CHEBI:27432CHEBI:80845CHEBI:167535CHEBI:68986CHEBI:18300CHEBI:30796CHEBI:17369CHEBI:18152CHEBI:17533CHEBI:21626CHEBI:17846CHEBI:28327CHEBI:28658CHEBI:17295CHEBI:17276CHEBI:8016CHEBI:17289CHEBI:18021CHEBI:30764CHEBI:8056CHEBI:8317CHEBI:75633CHEBI:132142CHEBI:75643CHEBI:16243CHEBI:66395CHEBI:32080CHEBI:36124CHEBI:9009CHEBI:16914CHEBI:16119CHEBI:18428CHEBI:15741CHEBI:17992CHEBI:192639CHEBI:26984CHEBI:18283CHEBI:16828CHEBI:18186CHEBI:17775CHEBI:16704CHEBI:16650CHEBI:18066CHEBI:17712CHEBI:18107CHEBI:16708CHEBI:18237CHEBI:60961CHEBI:58196CHEBI:17009CHEBI:28788CHEBI:16236CHEBI:28456CHEBI:16467CHEBI:17053CHEBI:16551CHEBI:64701CHEBI:28737CHEBI:28838CHEBI:15956CHEBI:28839CHEBI:28840CHEBI:28842CHEBI:28843CHEBI:28844CHEBI:28845CHEBI:28846CHEBI:28848CHEBI:28847CHEBI:16199CHEBI:17924CHEBI:58195CHEBI:25057CHEBI:28849CHEBI:28850CHEBI:15827CHEBI:16737CHEBI:16157CHEBI:28851CHEBI:28852CHEBI:28853CHEBI:28854CHEBI:28855CHEBI:28784CHEBI:28856CHEBI:28857CHEBI:16473CHEBI:16954CHEBI:16336CHEBI:25314CHEBI:17052CHEBI:28762CHEBI:28858CHEBI:16429CHEBI:16643CHEBI:28859CHEBI:28861CHEBI:28862CHEBI:28863CHEBI:15953CHEBI:28864CHEBI:28865CHEBI:28866CHEBI:16849CHEBI:28867CHEBI:28868CHEBI:28869CHEBI:28870CHEBI:10567CHEBI:28871CHEBI:16239CHEBI:16237CHEBI:15361CHEBI:28872CHEBI:28873CHEBI:28874CHEBI:5280807CHEBI:16679CHEBI:28875CHEBI:28876CHEBI:16469CHEBI:16470CHEBI:15949CHEBI:10548CHEBI:28877CHEBI:28878CHEBI:16857CHEBI:5589CHEBI:16846CHEBI:28879CHEBI:16835CHEBI:16951CHEBI:16414CHEBI:28880