*3.3. Functional Subsets within the Core Module*

Because of the high number of genes encoding proteins with unknown or unassigned functions, we curated the functional annotation of each gene in the identified core module manually using current databases and literature and generated an up-dated list that covers detailed information on function, potential interaction partners and intracellular localization (Supplemental Table S1). By this means we uncovered a number of interesting novel targets for RB signals not yet described (see description of functional subsets below).

In total 84 of the 152 genes encode proteins that were predicted to be localized with low, medium or high probability in plastids. The plastid localization of 67 of them was experimentally confirmed by proteomic approaches according to AT\_CHLORO. Roughly 45–55% of all genes in the module encode components that have predicted cellular destinations other than plastids (i.e., cytosol, Golgi apparatus, nucleus, endoplasmic reticulum, plasma membrane and mitochondria). Since prediction is not 100% precise and dual localization may also occur the results must be taken with care. Nevertheless, the data suggest that RB signals affect also other parts of the cell besides plastids. We noted that 80% of the genes with enhanced expression upon blocked chloroplast biogenesis belong to the group of genes encoding non-plastid localized components. Vice versa, 94% of the genes encoding plastid localized components displayed a low expression upon blocked chloroplast biogenesis indicating that the genes within the identified core module are dominated by two opposing expression modes that are largely associated with the potential localization of the affected gene products. In order to test whether or not the opposing expression modes are also associated to specific functions we analyzed the available information in a number of databases and ordered the genes of the core module into functional subsets with at minimum four genes (Figure 2A–F) and analyzed the corresponding expression profiles (see below for more details). 23 genes remained uncategorized and are given in the supplement (Supplemental Figure S2)
