Following pre-treatment, 16 rooted-leaves of K51-40, 140 Ruggeri and Cabernet Sauvignon were subjected to nutrient solution only (control) or to 50 mM Cl– (Na+: Ca2+: Mg2+ = 6:1:1) in nutrient solution for 4 days. At harvest, the rooted-leaves of each genotype were washed in de-ionised water, blotted dry with paper towel, weighed, then separated into lamina, petiole and roots. Fresh weights of all plant parts were also obtained. Separate root samples were taken for RNA extraction.
Growth protocol
Pot-grown grapevines of K51-40 (Vitis champinii X Vitis riparia), 140 Ruggeri (Vitis berlandieri X Vitis rupestris) and Cabernet Sauvignon (Vits vinifera) were established from cuttings and maintained in a glasshouse as described previously (Gong et al., 2011). Grapevines were watered daily with tap water that contained 3.2 mM Cl–. Vines were given fortnightly applications via fertigation of Megamix Plus (Rutec Pty. Ltd., Tamworth, New South Wales) and Iron Sodium EDTA (concentration 42.6 g/L), 25 mLs per 250 L. Typical analysis of this solution was macro elements in mM: K, Na, Ca, Mg and Cl, respectively, 1.3, 2.4, 1.5, 1.1 and 2.7, and micro elements in μM: Fe, B, respectively, 25.2 and 3. Mature leaves were excised from grapevines and the petioles were dipped in a hormone gel containing 1.5 g.l-1 IBA (Clonex, Growth Technology, O’Connor, Western Australia) and then placed in propagation trays containing vermiculite and perlite at a 1:1 ratio (Australian Vermiculite and Perlite Pty Ltd, Campbellfield, Victoria, Australia). The propagation trays (33 × 28 cm x 5.5 cm deep) were placed on heat beds (30 ºC) (Heat ’n’ Grow by Thermofilm, Springvale, Victoria, Australia) in a mini-shadehouse within the glasshouse. To maintain high humidity, the mini-shadehouse was covered with 90 % shadecloth and the leaves were misted with fine sprays for 1 min every hour during the day from 7 am to 7 pm. After approximately 3 weeks, rooted-leaves were transferred to aerated hydroponics tanks containing modified Hoagland Solution (Hoagland & Arnon, 1950) containing the following nutrients (mM) for a two-week pre-treatment period: KNO3, 1.0; Ca(NO3)2·4H2O, 1.0; MgSO4·7H2O, 0.4; KH2PO4, 0.2; H3BO3, 4.6×10-2; MnCl2·4H2O, 9.1×10-3; ZnSO4·7H2O, 7.6×10-4; CuSO4·5H2O, 3.2×10-4; Na2MoO4·2H2O, 2.4×10-4; EDTA-Fe-Na, 7.1×10-2 (pH 6.5).
Extracted molecule
total RNA
Extraction protocol
Frozen roots were ground to a fine powder in liquid nitrogen using a mortar and pestle. RNA was extracted using the Spectrum Plant Total RNA Kit (Sigma, St. Louis, Missouri, USA) following the manufacturer’s recommended protocol. RNA was DNase I treated with Turbo DNA-free (Life Technologies, Carlsbad, California, USA) for 1 hour at 37 oC to remove any contaminating genomic DNA. RNA was then precipitated at minus 80 oC overnight in 5 volumes of 100 % ethanol (v/v) and 1/10 volumes of 3 M NaOAC. After ethanol precipitation, RNA was resuspended in nuclease free water and analysed on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Only RNA samples with 260/280 and 260/230 absorbance ratios greater than 1.8 were used. RNA integrity was measured on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, California, USA).
Label
cy3
Label protocol
Single colour labelling was performed at the Ramaciotti Centre for Gene Function Analysis (University of New South Wales, Australia) using reagents, kits and protocols as recommended by Agilent.
Hybridization protocol
Hybridisations were performed at the Ramaciotti Centre for Gene Function Analysis (University of New South Wales, Australia) using custom 8 x 60K gene expression microarrays from Agilent, and using methods recommended by Agilent.
Scan protocol
Image scanning was performed at the Ramaciotti Centre for Gene Function Analysis (University of New South Wales, Australia) using methods as recommended by Agilent.
Data processing
Scanned images were analysed with Feature Extraction Software 10.7.3 (Agilent Technologies, Santa Clara, California, USA) and the Cy3 median signal intensities for each spot on the arrays were imported into R for further processing. The data was log(2) transformed and quantile normalized. Since the microarray hybridizations were performed at different dates we observed batch effects which we corrected for with the ComBat package (Johnson et al., 2007). Differentially expressed genes were identified using the Linear Model for Microarray Data (LIMMA) package (Smyth, 2004), and the Benjamini and Hochberg correction method was applied to account for multiple testing (Benjamini & Hochberg, 1995).
