ABSTRACT: Experiments were carried out in 2008 on 8-year-old apple trees (cv. Golden Delicious/M9) trained with standard horticultural practices at the experimental farm of the IASMA (Trento, Italy). Populations of fruits with different abscission potentials (abscising fruitlets versus persisting fruitlets), were established as described by Dal Cin et al. (2005, 2007, 2009), Angeli et al. (2002), and other preliminary experiments (unpublished data). Briefly, the abscising population was made up of lateral fruitlets treated with benzylaminopurine (BA) at 200 ppm (commercial name Brancher-Dirado), when fruits had an average size of 13 mm (about 15 days after petal fall, DAPF). The population of central persisting fruitlets was generated by removing all the laterals from each cluster at petal fall, and leaving exclusively the central flower that had been hand-pollinated at full bloom with compatible pollen (cv. Stark Red). Samples of the two populations were collected at defined time points from groups of twenty homogeneous trees randomly distributed in the orchard in four blocks. Fruits were collected and categorised into three classes of size (class 1, smaller fruits; class 2, medium fruits; class 3, bigger fruits), two classes related to the position within the clusters (lateral versus central fruits), and two classes on the base of the treatment (BA-treated versus untreated fruits). Fruits of the intermediate size (class 2 fruits in Figure 1) were not considered for sampling and subsequent molecular analyses, and only fruits of the two more divergent size classes 1 and 3 were kept. This resulted in a combined categorisation of fruits into 4 classes: 1) untreated lateral fruitlets, 2) lateral fruitlets treated with BA, 3) untreated central fruitlets, and 4) central fruitlets treated with BA (Figure 1). Each class was further distinguished into the two size categories 1 (small fruits) and 3 (bigger fruits), for a total of eight experimental theses. Fruitlet shedding and ethylene evolution were monitored throughout the physiological drop from the beginning of the experiments up to 46 DAPF in all fruitlet classes, separately. Seed and cortex (including epidermis) samples were collected from all classes of fruitlets at 0 (T0), 1 (T1), 2 (T2), 4 (T3), 6 (T4), and 8 (T5) days after the BA treatment from control and treated trees, and according to their position within the clusters (central vs lateral) and size (small vs big), as described. The latter parameter was decided at each sampling date based upon the mean cross diameter of the whole population of lateral fruits, calculated over a sample of 100 fruits measured randomly on twenty trees. The small ones had a cross diameter below the mean - s.d. (standard deviation), whereas the big ones had a cross diameter above the mean + s.d. Lateral fruitlets were collected from intact clusters showing a clear hierarchy in terms of fruit size (i.e. with a clearly distinguishable central fruit, bigger than any lateral). Acronyms were ascribed to samples according to the following code: the first letter(s) describes fruitlet position within the cluster and the presence of BA treatment (L=untreated lateral, C=untreated central, LB=treated lateral, CB=treated central), then a digit to describe the size (1=small, 3=big), and finally a digit to described the time point (0=time of the treatment, 1=1 day after treatment, 2=2 days after treatment, 3=4 days after treatment, etc.). For example, LB32 is a lateral fruit, treated with BA, big sized, 2 days after treatment.
For microarray hybridizations, only samples of T0, T2, and T3 were used.
