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3 Material and Methods
3.1 Berry sampling
Grape berries from three wine varieties – Alvarinho, Arinto and Padeiro de Basto -, were collected in 2012 season in two different Portuguese ampelographic collections located in North - Demarcated Region of Vinho Verde (Estação Vitivinícola Amândio Galhano - EVAG) and South – Estremadura Region (Instituto Nacional de Investigação Agrária e Veternária - INIAV). From each variety three clusters from tree different vines were collected at different phenological stages: green pea, veraison and mature at 18 ºBrix. Each sample was stored separately and carried in a thermal luggage. Grape berries were ground with mortar and pestle in liquid nitrogen. The powder was stored at -80 ºC for posterior use.
Grape berries from Cabernet Souvignon and Touriga Nacional clean and infected with the Grapevine Leaf Roll virus (GLRa-V3) were also collected in 2012 season in the ampelographic collection of Instituto Nacional de Investigação Agrária e Veternária (INIAV). Three clusters from each clean and infected variety at the mature stage were sampled. Each sample was stored separately and carried in a thermal luggage. Part of the samples were ground with mortar and pestle in liquid nitrogen. The powder was stored at -80 ºC for posterior use.
In a different approach, grape berries from twenty one grape varieties, five white (Malvasia Fina Perrum, Vital, Antão Vaz, Airén) and sixteen red (Cinsaut, Castelão, Moreto Trincadeira, Jaén, Aragonês Padeiro, Corropio, Tinto Cão, Tinta Miuda, Touriga Nacional, Alfrocheiro, Merlot, Alvarelhão, Alicante Bouschet, and Borraçal) were collected in 2011 season in the ampelographic collection of the Instituto Nacional de Investigação Agrária e Veternária (INIAV) to study in berry tissues genotype-dependent phenolics and proline content and antioxidant capacity. For each variety, three clusters at the mature stage were collected from three different vines. Each sample was stored separately and carried in a thermal luggage. The samples were ground with mortar and pestle in liquid nitrogen. The powder was stored at -80 ºC for posterior use.
3.2 Sample preparation for metabolomic analysis
To provide detailed information on the grape berry metabolome in vines from two different regions the above-mentioned powder was lyophilized for six days. Metabolite extraction from the lyophilized samples and analysis by GC-TOF-MS were carried out at UC
Material and Methods
Davis Genome Center Metabolomics Laboratory, as described by Fiehn et al. (2008). After metabolite extraction and derivatization, samples were injected in split-less mode with a cold injection system (Gerstel, Germany) and analyzed by GC (Agilent 6890, San Jose, USA) using a Rtx 5Sil MS column (30 m x 0.25 mm, 0.25 µm film thickness) and an integrated guard column (Restek, Bellefonte, USA). The GC was connected to a Leco Pegasus IV TOFMS spectrometer controlled with Leco ChromaTOF software v.2.32 (Leco, St. Joseph, USA). Peak detection and mass spectra deconvolution were performed with Leco Chroma- TOF software v.2.25. GC-MS chromatograms were processed as described by Fiehn et al. (2008). Further analysis after deconvolution was made using the semi-automated workflow of the UC Davis Genome Center Metabolomics Laboratory (Fiehn et al., 2005). Metabolite data were normalized using the dry weight (DW) of the lyophilized samples. For all experimental conditions, three independent runs were performed in all metabolomic analysis.
3.3 Quantification of free amino acids
Whole grape berries at mature stage from North and South regions were ground in liquid nitrogen and lyophilized for six days. Extraction was performed by adding 25 ml of milli-Q H2O to 1 g of grape berry powder and quantification of natural free amino acids
(excluding tryptophan) was performed in a Biochrom 30 Aminoacid Analyser with a weak acidic cation exchange resin acting as stationary phase (200 x 4.6 mm column) and a number of weak acidic Li-citrate buffers acting as mobile phase. Stepwise pH, temperature and salt concentration gradients were applied. Detection after post column derivatization with Ninhydrin (135°C) at 570 or 440 nm was performed (Ansynth Service, B.V.). For tryptophan quantification, the sample solutions were diluted in milli-Q H2O (1:10) and analysis was performed using a Beckman System Gold HPLC equipped with an Allsphere C8, 250 x 4.6 mm (stationary phase) and using a phosphate buffer/MeOH gradient (mobile phase). Detection was performed by fluorimetry, with emission wavelength set at 340 nm and excitation wavelength set at 280 nm (Ansynth Service, B.V.).
Material and Methods
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Triton X-100. The homogenates were then centrifuged at 18,000 xg for 20 min at 4 ºC and the supernatants were kept on ice. The total protein content was determined spectrophotometrically by the Bradford method (Bradford, 1976), with bovine serum albumin (BSA) as the standard.
3.5 Enzymatic activities
Malate dehydrogenase activity
The extracts for MDH activity were obtained as described above. MDH activity assay was performed at 30ºC as described by Taureilles-Saurel (1995a) with some modifications. The reaction medium contained 50 mM of Trisaminometano (Tris-HCl) (pH 8), 1.7 mM NaHCO3, 1.3 mM MgSO4, 1.7 EDTA, 0.3 mM NADH and3 mM of oxaloacetic acid in a
final volume of 1 mL. MDH activity was determined spectrophotometrically at 340 nm by measuring the rate of NADH-dependent reduction of oxaloacetate to malate. The reaction was started by the addition of 0.05 mL of extract after equilibrium.
NADP+-dependent malic enzyme activity
The extracts for NADP+ malic enzyme activity were obtained as described above. NADP+ malic enzyme activity assay was performed at 30 ºC, in a total volume of 1 ml. The reaction mixture contained enzyme extract, 100 mM Tris-HCl (pH 7.4), 0.3 mM NADP+, and
30 mM of malic acid (pH 7.4) to ensure the Vmax of the enzyme (different concentrations of
malic acid were used for kinetics studies). The reduction of NADP+ was evaluated spectrophotometrically at 334 nm using a double beam spectrophotometer. One of the cells contained the reaction mixture and the blank was placed in the other. All reactions were initiated by the addition of 0.05 mL of protein extract.
Mannitol dehydrogenase (MTD) activity
The extracts for MTD activity were obtained as described above in the presence of 1% (w/w) of PVPP. MTD activity was assayed at 37 ºC, in a total volume of 1 ml. The reaction mixture contained enzyme extract, 300 mM Bis-Tris propane (pH 9.0), 1mM NAD+, and 200 mM of D-mannitol to ensure the Vmax of the enzyme (Conde et al, 2007b). MTD activity was
determined by measuring the rate of mannitol-dependent conversion of NAD+ to NADH spectrophotometrically at 340 nm. All reactions were initiated by the addition of mannitol.
Material and Methods