cultivar: Cabernet Sauvignon treatment: 0 µM ABA + 0.05% adjuvant + 0.002% ethanol, Control year: 2011 location: University of Nevada, Reno, USA tissue: Leaf
Treatment protocol
A 10 µM ABA spray was made by first dissolving ABA ([+]-ABA, A.G. Scientific, Inc., http://www.agscientific.com) to 500 mM in 100% ethanol and then diluting to 10 µM in water containing 0.05% adjuvant (Latron-B, DOW AgroSciences LLC, http://www.dowagro.com). Control solution spray was distilled-deionized water containing 0.05% adjuvant and 0.002% ethanol. Shoot tips, berry clusters, and cell culture were sprayed with the 10 µM ABA treatment until running off. Roots were treated with 10 µM ABA in the aeroponic system by adding the ABA to the nutrient solution in the nebulizer for 2 hours; roots and leaves of root-treated vines were harvested. Samples were quickly rinsed and rapidly frozen in liquid nitrogen before storage at -80°C. Three independent experimental (and biological) replicates were harvested to compare between the ABA-treated and untreated samples.
Growth protocol
Shoot tips and berries samples were collected at the University of California, Davis in 2010; cell culture samples were sampled at Oregon State University in 2010; roots and leaf samples were collected at the University of Nevada, Reno in 2011. Own-rooted vines of Vitis vinifera (L.) cv. Cabernet Sauvignon were used for the shoot tips and berries (before véraison) assays at UC Davis. These vines were grown from dormancy in 4-L tree pots filled with 1/3 peat, 1/3 sand, 1/3 redwood compost, with 2.4 kg m-2 dolomite lime in a greenhouse (30/20 ºC ± 3 ºC; 40/70% ± 10% RH; and natural light with a daily maximum of 1200 µmol m-2 s-1 PAR). The vines were pruned to two shoots, and the shoots were vertically trained to ~1.5 m. Pots were drip irrigated four times a day (at 06.00, 09.00, 14.00, and 18.00) for 4 min at 7.57 L h-1 (2 L d-1) with dilute nutrient solution (90 ppm calcium, 24 ppm magnesium, 124 ppm potassium, 6 ppm nitrogen as ammonium, 96 ppm nitrogen as nitrate, 26 ppm phosphate, 16 ppm sulfate, 1.6 ppm iron, 0.27 ppm 195 manganese, 0.16 ppm copper, 0.12 ppm zinc, 0.26 ppm boron, and 0.016 ppm molybdenum) at pH 5.5 to 6.0. Cell suspension cultures (CS4) at Oregon State University were maintained under continuous fluorescent light (~68 μmol m-2 s-1) at 25°C on an orbital shaker (120 rpm). Suspension cultures were subcultured weekly in 250 mL Erlenmeyer flasks containing 50 mL of cell suspension in B5 medium supplemented with 20 g L-1 sucrose, 250 mg L-1 casein hydrolysate, 0.5 mg L-1 1-naphtalene-acetic acid and 0.12 mg L-1 benzylaminopurine, by inoculating the cells at a 1/5 (v/v) ratio into a fresh medium. For experimental purposes, 7-day-old cell suspensions were inoculated into a fresh medium (3:7) and cultured for 3 days before treatment. Young vines propagated from leaf cuttings were grown in an aeroponic system in a greenhouse at UNR. The rooted cuttings from Cabernet Sauvignon were grown in a growth chamber for 2 to 3 weeks before being carefully transferred to the aeroponic system. Each container (43.2 cm (L) x 27.9 cm (W) x 20.3 cm (H)) had its own aeroponic nebulizer with a fogger head size of 3.8 cm diameter x 4.4 cm height for each experimental replicate (3 containers for control and 3 containers for ABA treatment). There were small holes in the lid of each container large enough for the rooted plant to pass through. Gibeaut’s solution [78] was used to provide the macronutrients and micronutrients to the vines in the aeroponic mist. The pH of the solution was maintained at 6.0. Root and leaf samples were grown for 3 months in this system before treatment.
Extracted molecule
total RNA
Extraction protocol
Samples were quickly rinsed and rapidly frozen in liquid nitrogen before storage at -80°C. Three independent experimental (and biological) replicates were harvested to compare between the ABA-treated and untreated samples. Each biological replicate was ground in a frozen mortar and pestle. Total RNA was isolated using a cetyltrimethylammonium bromide (CTAB) based method and RNeasy plant mini kit (Quiagen) following the manufacture’s protocol. The total RNA was treated with RNAse-free DNAse I (Qiagen) to eliminate any genomic DNA contamination and then quantified by using a Nanodrop spectrophotometer (Thermo Scientific NanoDrop 2000c). An aliquot of each RNA sample was also analyzed using an Agilent 2100 Bioanalyzer using RNA LabChip® assays according to the manufacturer's instructions.
Label
Cy3
Label protocol
cDNA synthesis and labeling reactions were performed according to the NimbleGen Arrays User's Guide (V 3.2).
Hybridization protocol
Labeled cDNA was hybridized to a NimbleGen microarray 090818 Vitis exp HX12 (Roche, NimbleGen Inc., Madison, WI, USA), which contains probes targeted to 29,549 grapevine genes predicted from the V1 annotation of the 12x grapevine genome (http://srs.ebi.ac.uk/), and 19,091 random probes as negative controls.
Scan protocol
Each microarray was scanned using an Axon GenePix 4400A (Molecular Devices, Sunnyvale, CA, USA) at 532 nm (Cy3 absorption peak) and GenePix Pro7 software (Molecular Devices) according to the manufacturer’s instructions.
Description
Leaf_Control-rep1 (LC1) Leaf_Control-rep2 (LC2) Leaf_Control-rep3 (LC3) Young vines propagated from leaf cuttings were grown in an aeroponic system in a greenhouse at UNR. The rooted cuttings from Cabernet Sauvignon were grown in a growth chamber for 2 to 3 weeks before being carefully transferred to the aeroponic system. Each container (43.2 cm (L) x 27.9 cm (W) x 20.3 cm (H)) had its own aeroponic nebulizer with a fogger head size of 3.8 cm diameter x 4.4 cm height for each experimental replicate (3 containers for control and 3 containers for ABA treatment). There were small holes in the lid of each container large enough for the rooted plant to pass through. Gibeaut’s solution [78] was used to provide the macronutrients and micronutrients to the vines in the aeroponic mist. The pH of the solution was maintained at 6.0. Root and leaf samples were grown for 3 months in this system before treatment. Roots were treated with a control solution spray of distilled-deionized water containing 0.05% adjuvant (Latron-B, DOW AgroSciences LLC, http://www.dowagro.com) and 0.002% ethanol added to the nutrient solution in the nebulizer for 2 hours; roots and leaves of root-treated vines were harvested.
Data processing
Images were analyzed using NimbleScan v2.5 software (Roche), which produces pair files containing the raw signal intensity data for each probe and calls files with normalized expression data derived from the average of the intensities of the four probes for each gene. Background correction and standard RMA normalization were selected.
