Plant Materials

Tomato (Solanum pennellii) seeds of accession numbers LP1940 (S p) and LP2901 (Aco-1 mutants) were obtained from the true-breeding monogenic stocks maintained by the Tomato

Genetics Stock Center (University of California, Davis). Tomato (Solanum lycopersicum) L. cv Moneymaker seeds were obtained from Meyer Beck (Berlin). The seeds were germinated on Murashige and Skoog media (Murashige and Skoog, 1962) containing 2% (w/v) Suc and were grown in a growth chamber (250 µmol photons m–2s–1, 22°C) under a 16-h-light/8-h-dark regime before transfer into the greenhouse, where they were grown in parallel with a minimum of 250 µmol photons m–2s–1, 22°C under a 16-h/8-h day light. Additionally transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the mitochondrial malate dehydrogenase gene in the antisense orientation (mMDH plants) were growing in parallel. Experiments were carried out on mature fully expanded source leaves from 6- to 8- week-old plants.

RNA isolation

For each plant condition, total RNA from three independent biological replicates was isolated using Trisol (BiRad).

Glass slide microarray

Tomato glass slide were provide by The Center for Gene Expression Profiling (CGEP) at the

Boyce Thompson Institute (BTI), Cornell University, the Geneva Agricultural Experiment

Station, and the USDA Federal Plant & Nutrition Laboratory.

Description of the tomato array Clones spotted on the tomato array, were selected at random from cDNA libraries isolated from a range of tissues including leaf, root, fruit, and flowers. The sequences present on the array were selected in order to cover genes that are present in a broad range of metabolic and developmental processes. The gene fragments are printed in 32 sub-grids, in a 21 by 20 spot arrangement per sub-grid. Complete meta-grid size is 13,440 spots per array. A partly annotated gene id file with EST identifier and sequence information was provided for the arrays (for details of the spotting scheme see Tom1 GeneID File 2-03.xls file). Further annotation of this file was carried out to provide gene identities and putative functions for

ESTs described on the TIGR (http://www.tigr.org/tdb/tgi/lgi/) website.

Fluorescent probe preparation and microarray hybridisation

50µg of total RNA was reverse transcribed to synthesise either Cy3 or Cy5 labelled cDNA probes. Total RNA was mixed with 1.25g Oligo d(T) primer (Invitrogen) and denatured at

650C for 5 mins. A master mix containing 8l 5x first strand buffer, 4l low C dNTP mix

(25 mmol each of dGTP, dATP, dTTP and 10 mmol dCTP), 25µmol Cy5 or Cy3 dCTP

(Amersham), 4l 0.1M DTT and 40u RNase out (Invitrogen) was added. Each sample was heated to 420C and then 2l (400u) superscript II reverse transcriptase (Invitrogen) was added. The reaction was incubated for 2 hours at 420C and stopped by the addition of 5l of

0.5M EDTA. Template RNA was hydrolysed by the addition of 10 l of 1M NaOH and incubating at 650C for 30mins. The reaction was neutralized by the addition of 25l of 1M

Tris (pH 8.0). Labeled cDNA was then precipitated by the addition of 8l of 3M NaAc (pH

5.2) and 200l Ethanol. Following incubation at -200C for 2 hours, cDNA was pelleted by centrifugation at 12 000g for 30 mins at 40C. Pellets were allowed to dry, re-suspended in

15l hybridisation solution (0.1% SDS, 25% Formamide, 5 x SSC) and the Cy5 and Cy3 labelled probes were combined to a final volume of 30 l. Microarrays were pre-hybridised by immersing in pre-hybridisation solution (0.1% (w/v)

SDS, 25% (v/v) Formamide, 5 x SSC, 1% (w/v) BSA) for 90 mins at 420C. Slides were rinsed

in ddH2O and air-dried. For hybridisation 2l of liquid block solution (Amersham) was added to the purified, combined Cy3 and Cy5 labeled probes and these were denatured at 950C for

5mins. Probe solution was added to the spotted surface of the slide and hybridisation carried out under a hybri-slip (SIGMA) in a humidified hybridisation cassette (Telechem

International, USA) for 16 hours at 420C. Following hybridisation slides were washed for 5 mins at 420C in 2 x SSC, 0.1% (w/v) SDS, 10 mins at room temperature in 0.1 x SSC, 0.1%

(w/v) SDS and 4 mins at room temperature in 0.1 x SSC.

Micro array scanning and data analysis Microarrays were scanned using a BAS-1800 II, Fuiji and acquisition software according to the manufacturer’s instructions. Briefly, slides were scanned in Cy3 and Cy5 channels separately at a range of photomultiplier gain settings. After scanning images were analysed in

Genepix Pro v. 4.1 software and the raw data collected and imported into Microsoft Excel for further analysis. To identify genes of interest whose expression levels change the ratio of each feature (635nm/532nm) was calculated. Background fluorescence values were automatically calculated by the Genepix program and subtracted from all feature intensities prior to ratio calculation. It was determined that the array data should exclude all features that did not show a median pixel intensity of 2.5 fold greater than the overall mean slide background intensity

In order to account for differences in array-specific effects and to be able to average the results from three replicate arrays (dye-swap replicates were used), normalization between the

Cy3 and Cy5 channels was further achieved by calculating the ratio for each spot based on the

Cy3 and Cy5 fluorescence of the spot in relation to the total Cy3 and Cy5 fluorescence of the whole slide. Clones were described as differentially expressed in the tested situation while differences between average tested values and average references values were two fold