Supporting web-only information

Supplemental Materials and Methods

cDNA labelling and microarray hybridization

Total RNA (15 µg) was converted into target cDNA by reverse transcription using the SuperScriptTM Indirect cDNA Labelling System (Invitrogen), following the manufacturer’s instructions. During the labelling phase, amino-modified cDNAs, deriving one from the treated RNA sample and one from the control RNA sample, were coupled to two different monoreactive N-hydroxysuccinimide (NHS)-ester fluorescent dyes (Cy3 and Cy5 mono-reactive dye, GE Healthcare).Labelled cDNAs were then purified, in order to remove any trace of unincorporated dye, their quantity (expressed in pmol) was checked by absorbance measurements from 240 to 750 nm, and their concentrations were calculated by applying the formulas supplied with the labelling kit instruction manual.

Both the pre-hybridization and hybridization reactions were carried out in Corning® hybridization chambers with 120 l of 0.3X SSC to maintain proper internal humidity and salt concentration, immersed in a water bath at 48°C. A 2 hours-pre-hybridization step was performed in a solution containing 5X SSC, 0.1% SDS, 1X Denhardt’s solution and 100 ng ml-1 DNA carrier. The pre-hybridization solution (65 l) was boiled for 5 min and then deposited onto the spotted area of the microarray slide, which was further covered with a cover glass slip. At the end of the pre-hybridization stage, microarray slides were washed with a 0.2X SSC solution and immediately centrifuged for 2 min at 2500 rpm to remove any traces of liquid.

Equal amounts (90-120 pmol) of cDNA-Cy3 and cDNA-Cy5 were gently mixed together in a vial. The cDNA mixture (200 l final) was then precipitated with ammonium acetate (2M final) and 2.5 V of absolute ethanol at -20°C for at least 30 min. The pellet was resuspended in 65 l of hybridization solution (5X SSC, 0.1% SDS, 25% formamide, and 40 ng ml-1 DNA carrier). The obtained solution was first denatured by boiling it for 30 s, dropped above the spotted area of the chip by covering it with a glass cover slip, and further hybridized for 48 h at 48°C in a water bath.

Once hybridization was completed, microarray slides were submitted to a series of washing steps, as follows: 5 min in solution I (1X SSC and 0.2% SDS), 5 min in solution II (0.1X SSC and 0.2% SDS), twice for 5 min in solution III (0.2X SSC) and 5 min in solution IV (0.1X SSC). The slides were finally dried by a brief centrifugation step (2 min at 2500 rpm) and stored at 20-25°C in dark conditions until optical scanning.

Supplemental Table S2 Name, TC number, and sequence of the gene-specific primers designed for qRT-PCR assays. Asterisks indicate primers taken from Choat et al. (2009)

PRIMER DESCRIPTION / CORRESPONDENT TC PROBE ON THE ARRAY / PRIMER SEQUENCE
VvPIP 1;1 FW / TC51776 / 5'-GAGTGGTGCTGGGCGTTGATG-3'
VvPIP 1;1 RV / 5'-CCATCGCCTTTCTCTGTTGTG-3'
VvPIP 1;2. FW* / TC60619 / 5'-TCCTCCATTTTCGTTTCTTC-3'
VvPIP 1;2. RV* / 5'-ATTGTAATAGAAGCAGCCCAG-3'
VvPIP 1;3 FW* / TC55780 / 5'-CCATCGCCTTTCTCTGTTGTG-3'
VvPIP 1;3 RV* / 5'-AGAATACTCAATAATTTACAC-3'
VvPIP 2;1 FW* / TC58240 / 5'-CCATTTTGATACCTTCTTCC-3'
VvPIP 2;1 RV* / 5'-TATCTACAATTTCATGCCCTC-3'
VvPIP 2;4 FW / TC53235 / 5'-CTAGGATCTTTCAGGAGCAA-3'
VvPIP 2;4 RV / 5'-TACTCCTCCACCATTGATGT-3'
VvNAC72 FW / TC60322 / 5'-CGCCCTCCAATCTTCTTCTCT-3'
VvNAC72 RV / 5'-AGCTGTGAAAGCGGGTCAGT-3'
VvNAC2 FW / TC64843 / 5'-CGGGCTGGGTTCTACCTTAT-3'
VvNAC2 RV / 5'-GGATCAAGGGTCTTACCTGCT-3'
VvLEA 14 FW / TC65080 / 5'-CGTACAACGCCAAGGTCTCA-3'
VvLEA 14 RV / 5'-CATCTTCCCCGACGCTATCA-3'
Vvdh11 DEHYDRIN 11 FW / TC54112 / 5'-AACCCGGCGTGCTTCAT-3'
Vvdh11 DEHYDRIN 11 RV / 5'-CATGCCCGGTATCCTCTCTTT-3'
VvSUC-transp (Sucrose transporter) FW / TC51724 / 5'-TGACCCCCTACGTTCAGCTT-3'
VvSUC-transp (Sucrose transporter) RV / 5'-CCAACTACCGGCTGCACAAT-3'
VvGLUC-transp (Glucose-6-phosphate transporter) FW / TC64506 / 5'-TTCCGGTGCCGGTCTACTT-3'
VvGLUC-transp (Glucose-6-phosphate transporter) RV / 5'-GCCCCCATAAACCCAGTCAT-3'
VvSKc (serine trehonine kinase) FW / TC58392 / 5'-AGATGTTTGGTCTTGTGGTGTGA-3'
VvSKc (serine trehonine kinase) RV / 5'-CCCAATGGTCTTCCGGAAAT-3'
VvCAL (Calmodulin) FW / TC54154 / 5'-TGGTCAGAGAAGTGGACTGCAA-3'
VvCAL (Calmodulin) RV / 5'-CAGGTGCTGCTGCTACCAACT-3'
VvRD-22 (dehydration-responsive factor) FW / TC38296 / 5'-TCATCTCCTACCCATCCTTGCT-3'
VvRD-22 (dehydration-responsive factor) RV / 5'-TTGGCATGGGAGTGTTTGG-3'
VvK-transp (Potassium transporter) FW / TC59127 / 5'-GCCGCGTGTGGTGTTAGTATAA-3'
VvK-transp (Potassium transporter) RV / 5'-CGTTCTCTCGGCTACGTCAAG-3'
VvBeta-amylase enzyme FW / TC67979 / 5'-CTAGCAGCTGCCGAAGGAAT-3'
VvBeta-amylase enzyme RV / 5'-CAGCCGCATGAGACCTTGTT-3'
HOUSEKEEPING GENES USED / TC NUMBER (TIGR Release 7) / PRIMER SEQUENCE
VvUbiquitin FW / TC117219 / 5'-TCTGAGGCTTCGTGGTGG TA-3'
VvUbiquitin RV / 5'-AGGCGTGCATAACATTTGCG-3'
VvActin1 FW / TC134791 / 5'-GCCCCTCGTCTGTGACAATG-3'
VvActin1 RV / 5'-CCTTGGCCGACCCACAATA-3'

Captions to supplemental tables in the excel file

Supplemental Table S1 Grouping of GO terms belonging to higher order GO functional categories used to classify probes

Supplemental Table S3 Correlation between quantitative real-time PCR (qRT-PCR) analysis and microarray measurements for the WS-IRR and for the REC-IRR comparisons in Exp. B. Data are expressed as log2 of expression ratios. Not all the transcripts analysed in the WS/IRR comparison were also considered in the REC/IRR comparison and viceversa, thus an empty space is left on the corresponding raw. Pearson correlation coefficients (r) are reported at the end of the table for each set of data

Supplemental Table S4 Complete list of probes that showed significant expression differences after SAM analysis. Only probes that reached the log2>1 threshold of expression ratio, in REC relative to IRR plants in Exp A, and that were not significantly affected by WS treatment, were considered. Probes are grouped according to the respective GO functional category. Petioles were sampled at 2; 5; 8; 11 HAR. For each probe the TC or EST number of the VvGI database (release 5), the best UniProt match with the related annotation, and the log2 of expression ratios in REC plants related to IRR plants are also reported

Supplemental Table S5 Complete list of probes that showed significant expression differences after SAM analysis. Only probes that reached the log2>1 threshold of expression ratio, in REC relative to IRR plants in Exp A, and that were also significantly affected by WS treatment, were reported. Probes are grouped according to the respective GO functional category. Petioles were sampled at 2; 5; 8; 11 HAR. For each probe the TC or EST number of the VvGI database (release 5), the best UniProt match with the related annotation, and the log2 of expression ratios in REC plants related to IRR plants at the different time points, and in WS plants relative to IRR plants (WS), are also reported

Supplemental Table S6 Complete list of probes that showed significant expression differences at the SAM analysis. Only probes that reached the log2>1 threshold of expression ratio, in REC relative to IRR plants in Exp B, and that were not significantly affected by WS treatment, were shown. Probes are grouped according to the respective GO functional category. Petioles were sampled at 8 HAR. For each probe the TC or EST number of the VvGI database (release 5), the best UniProt match with the related annotation, and the log2 of expression ratios in REC plants related to IRR plants are also reported

Supplemental Table S7 Complete list of probes that showed significant expression differences at the SAM analysis. Only probes that reached the log2>1 threshold of expression ratio, in REC relative to IRR plants in Exp B, and that were also significantly affected by WS treatment, were shown. Probes are grouped according to the respective GO functional category. Petioles were sampled at 8 HAR. For each probe the TC or EST number of the VvGI database (release 5), the best UniProt match with the related annotation, and the log2 of expression ratios in REC plants related to IRR plants, and in WS plants relative to IRR plants (WS), are also reported

Supplemental Table S8Complete list of probes that showed significant expression differences at the SAM analysis. Only probes that reached the log2>1 threshold of expression ratio, in REC relative to IRR plants in Exp A, and that were also significantly affected by REC treatment in Exp. B, were shown. Probes are grouped according to the respective GO functional category. Petioles were sampled at 2; 5; 8; 11 HAR in Exp. A and at 8 HAR in Exp.B. For each probe the TC or EST number of the VvGI database (release 5), the best UniProt match with the related annotation, and the log2 of expression ratios in REC plants related to IRR plants, and in WS plants relative to IRR plants (WS), are also reported