Diagram of water deficit experiment conducted with ‘Royal Gala’. A total of twenty-five trees were selected for the experiment. Five trees were sampled (leaves, bark and roots) at the beginning of the experiment (T = 0), after one week acclimation in the growth chamber. The controls, water deficit and recovery treatments are indicated in the boxes. Black arrows indicate how the experiment was conducted in time. Subtractions are indicated next to the red arrows showing the direction of subtraction, forward or reverse. For example, the forward subtraction between T1E and T1C (AAF) involves T1E cDNA as the tester and ten times concentrated T1C cDNA as the driver. Genes isolated from this subtraction represent those whose levels are upregulated in response to two weeks of water deficit treatment. In the reverse subtraction (AAR), T1C is the tester and T1E (ten times concentrated) is the driver. The reverse subtraction identifies genes downregulated in response to water deficit. The two controls (T1C and T2C) were subtracted to account for differences in gene abundance as a consequence of age (three weeks vs four weeks). This subtraction resulted in only a few sequences.