*3.2. Identification of a Core-Module of Genes Controlled by Biogenic Signals*

The chosen gene sets contained 3004 genes for the LIN set, 1957 genes for the *pap7-1* set and 1128 genes for the NF set. Comparison of the three conditions revealed a common gene module of 152 genes being significantly regulated in all three conditions (Figure 1A). The highest bivalent similarity was found for the NF set that shared 51.1% of its genes with the LIN set and 21.5% with the *pap7-1* set. The *pap7-1* set shared 25.8% of its genes with the NF set and 12.4% with the LIN set while the LIN set shared 19.2% of its genes with the NF set and 16.8% with the *pap7-1* set (Figure 1B). These results strongly suggest that each of the three different blocks in chloroplast biogenesis induce largely their own specific responses in nuclear gene expression. These differences in expression are likely attributed to the different sites of the respective blocks and/or the slightly different experimental set-ups and laboratory conditions. Nonetheless, there apparently exists also a common and robust element in all three conditions giving rise to the shared group of 152 genes. Therefore, we regard this group as a highly trustful core module of genes being influenced in their expression when chloroplast biogenesis is blocked, thus representing genuine target genes regulated (directly or indirectly) by retrograde biogenic (RB) signals.

An initial functional annotation of the 152 genes based on the bin definition in Map-Man [31] was performed in order to understand the regulatory implications of the biogenic control. The largest group comprised 38 genes encoding proteins of unassigned, unknown or hypothetical function (Figure 1C) strongly suggesting that many aspects of chloroplast biogenesis at the molecular level are not understood yet. The second largest group contained 18 genes encoding components implicated in "Photosynthesis" followed by groups involved in Protein" (15), "RNA" (11), "Redox" (10), "Transport" (9), "Hormone metabolism" (6), "Stress" (5) and 14 further groups represented by one to four genes.

The expression behavior of the 152 genes was highly similar in the three analyzed conditions with approximately one third (47) of genes being stronger expressed than in unaffected wild-type (WT) controls and two third (105) of genes exhibiting a weaker expression (Supplemental Table S1). In control conditions detecting the influence of light (WT-light versus WT-dark) most of the genes displaying a weaker expression upon the block in chloroplast biogenesis exhibited an opposite response with a clear induction upon illumination (and vice versa). This opposite expression pattern corresponds to recent reports where this pattern was interpreted in a way that biogenic signals turn light signals from positive into negative stimuli [32]. We, therefore, compared the core module to a set of recently identified direct HY5 target genes [33]. HY5 is one of the central regulators in photomorphogenesis and expected to control or co-regulate a large part of the light regulated genes [34] during seedling photomorphogenesis and chloroplast biogenesis. However, we identified only 5 of the 152 genes in the set of HY5-dependent genes (Supplemental Figure S1) indicating that the core module is likely not directly regulated by the classical light-dependent signaling pathways.

Except for a few condition-specific expressed genes (see below), the general profiles of genes affected by LIN and NF treatments were found to be highly similar in strength and direction of expression change. The expression profiles in the *pap7-1* mutant were comparable to both LIN and NF profile concerning the direction of expression change; however, the degree of variation was weaker in most cases. Pharmaceutical inhibition of chloroplast development by LIN or NF treatments, thus, appears to have a stronger impact on nuclear gene expression than the genetic block in the *pap7-1* mutant, although the overall plant phenotype in all three conditions was largely the same. This suggests that the inhibitor treatments (i) induce stronger inhibitory effects in the plastid than the genetic block and/or (ii) the respectively affected plastid processes contribute to biogenic retrograde signaling with different strength. This suggest that the albino phenotype *per se* is likely not the cause of the changes in nuclear gene expression arguing for a specific molecular signaling pathway.

**Figure 1.** Comparison of target gene modules identified in three experimentally different approaches blocking chloroplast biogenesis. (**A**) Venn diagram describing the overlap in target genes of biogenic signals identified by norflurazon (NF) treatment, 24 h lincomycin (LIN) treatment and by genetic inactivation in *pap7-1* mutants. In all studies significantly regulated genes were defined by an expression change of at least log2 fold change ≥ 1 and a p-value ≤ 0.05. (**B**) Percentage of shared genes between the treatments. Since the sizes of the affected gene groups in the three conditions are different, the overlap is given for each of the three binoms (indicated on bottom) separately. (**C**) Functional annotation of genes shared between all three conditions. The categorization followed the Bin system of MapMan. Gene groups with a minimum of at least three genes are mentioned. A complete list of all genes is given in Supplemental Table S1.
