Supplementary Data 1

cDNA microarray preparation, hybridization and analysis

Most cDNA libraries were generated from subtractive hybridization according to the Clontech PCR select cDNA subtraction kit manual (Clontech, Palo Alto, USA), using stress-treated versus non-treated wild-type plants. Collections involving 6,848 cDNA clones from 15 cDNA subtractive libraries, including various developmental stages, CBF1 transgenic plants, and stress treatments such as chilling, heavy metal, heat treatment, phosphate starvation, and pathogen infection, and 5,600 EST clones that were selected from 4 root EST libraries (cLEX, cLEY, cLEW and cLEZ, Tomato Expression Database, http://ted.bti.cornell.edu/) were printed on glass slides for hybridization (Liu et al. 2006). PCR products of each cDNA clone from the libraries were resuspended in 50% DMSO and then printed on glass slides (CMT-GAPSTM coated slides, Corning, NY, USA) using microarray machine PixSys 4500 (Cartesian Technologies, Irvine, CA, USA).

Total RNA was extracted from normal growth and stress-treated wild-type plants (Trizol, Invitrogen, Carlsbad, CA, USA), then purified polyA+ RNA were obtained using Qiagen mRNA purification kit (Qiagen, Valencia, CA, USA). One mg polyA+ RNA was reverse transcribed into cDNA in a volume of 40 ml containing 1 mg of oligo dT (20 mer), 10 mM DTT, 500 mM each of dATP, dCTP, dGTP, and dTTP and 300 units MMLV reverse transcriptase in 1X Superscript first-strand buffer (Invitrogen). After incubation for 1 h at 42°C, 0.5 units RNaseH were added and the reaction incubated for 0.5 h at 37°C. The reaction samples were purified on Microcon YM-30 (Millipore, Bedford, MA, USA) according to the supplied manual, and divided into two samples for the second-strand cDNA synthesis with individual Cy3 and Cy5 labeling. The labeling reaction was carried out in a volume of 40 ml containing 3 mg random primer (Invitrogen) with 25 mM each dATP, dCTP, and dGTP, 9 mM dTTP, 25 mM Cy3-dUTP (or 25 mM Cy5-dUTP) and 10 units of Klenow fragment (New England BioLabs, Ipswich, MA, USA) in 1X Klenow buffer. After incubation for 3 h at 37°C, the reaction products were combined and purified using the Qiagen PCR purification kit. The purified probes containing 20 mg human Cot-1 DNA (Invitrogen), 20 mg poly dA, 50% formamide, 0.1% SDS, and 5X SSC were denatured for 3 min at 100°C, and then allowed to cool for 20 min at room temperature. The probe samples were dropped onto the cover slip, and then placed onto the center of entire array surface.

The hybridized slides were placed in a hybridization chamber and submerged into a 42°C water bath for 16 h. After hybridization, slides were washed with 2X SSC and 0.1% SDS for 5 min at 42°C, 0.1X SSC and 0.1% SDS for 10 min at room temperature, 0.1X SSC for 5 min at room temperature, and finally rinsed with water. Slides were dried by centrifugation at 1,000 g for 5 min and then scanned with a scanning laser microscope (GenePix 4000B, Axon Instruments, Foster, CA, USA).

We used GenePix Pro4.0 (Axon Instruments, Union City, CA, USA) to record image intensity and GeneSpring 7.3.1 (Silicon Genetics, San Carlos, CA, USA) software to perform data analysis.

RACE (Rapid Amplification of cDNA Ends) and RT-PCR

One mg mRNA extracted from leaves of salt-treated wild-type plants was used to perform RACE according to the supplied user manual (Clontech, Palo Alto, CA, USA). The obtained PCR products were then cloned into the pGEMT easy vector (Promega, Madison, WI, USA) and sequenced. To amplify the 5’ and 3’ fragments, the RACE primers used were reverse specific primer (5`-TTCTATGCAT GATGCCATCGAAGGGG-3`; located at +1517 to +1493 in 3’-UTR, ATG as +1) and forward specific primer (5`-AAAGGCGATGCTTAAGACGGACACAAGC-3`; located at +1304 to +1331 in the coding sequence) that were designed from the EST clone LEPSR08G09 with an interval of 214 bp. DNA sequence was then determined by an ABI PRISM 373 automatic DNA sequencing system. Following sequence alignment, we designed primers (5`- ATGGGGAGTA ATTATCATTT CAAGAAC-3`) and (5`- TTACCATGGA CCAGTTTGTG TC-3`) to isolate an open reading frame of SlAREB by RT-PCR.