**4. Conclusions**

The core module responsive to RB signals identified in this study is different from retrograde controlled gene modules identified in earlier studies [65–67], most likely because these studies included conditions in which also retrograde operational (RO) signals are active (i.e., in the presence of fully developed chloroplasts). All three conditions analyzed in this study did not include the action of RO signals. The molecular targets causing the arrest in chloroplast biogenesis were different in the three conditions (cf. introduction), but the affected processes are not independent from each other and are interlinked by negative feedback loops affecting the generation of components of the transcriptional (rpo subunits) and translational (ribosomal components) machineries (Figure 3). Because of these connections and the high similarity in the three expression profiles we regard it as very likely that all three conditions generate signals that feed into the same signaling pathway(s). This signaling pathway targets mainly genes for photosynthesis or processes coupled to photosynthesis. Typically such genes are up-regulated by illumination and RB signals appear to intercept this light-dependent activation. A much smaller group of genes displayed the opposite expression profile indicating that RB signals can be of positive or negative effect. This master expression switch accounts for most genes in the core module. Only a few genes displayed more complex patterns that suggest the involvement of additional regulatory signals in their expression (such as ABA mediated signals). It must be noted that our meta-analysis describes only gene expression changes. Thus, it cannot draw conclusions on the positive or negative nature of RB signals and cannot distinguish between missing activation or active repression (or vice versa) [21,68]. It also

cannot explain whether the listed genes are direct or indirect targets. We regard it as likely that most genes in this module are controlled by just a few regulators primarily targeted by transcription factors responding to the RB signal(s) (Figure 2E). This would explain the homogeneous expression profiles of so many different genes. Novel prime candidates for the mediation of the RB signal(s) towards such primary regulators are dually localized PEPassociated proteins (PAPs) that have been shown to be essential for chloroplast biogenesis and formation of late photobodies during early steps of photomorphogenesis [49,69,70]. They belong to a group of proteins that appear to move via the plastid towards the nucleus representing genuine retrograde signals [71]. The core module identified here provides novel insights into the targets of RB signals and may serve as base for more detailed working models in future studies. This will include the determination of the accumulation of the corresponding proteins.

**Figure 3.** Working hypothesis for the action of retrograde biogenic signals. Oval on top represents a plastid that is arrested in its development either genetically (*pap7-1* mutant) or chemically (LIN or NF). The molecular inhibitory effects are connected by negative feedback loops resulting in a uniform retrograde biogenic (RB) signal in all three cases. Major target is the group of photosynthesis and photosynthesis-associated genes that normally are activated during chloroplast biogenesis by light and cytokinins with GNC and GLK transcription factors as major regulatory hubs. RB signals intercept into this activation resulting in low expression of these genes. RB signals may act also oppositely by promoting the expression of a small set of genes, many of them are involved in or related to defence responses to biotic and abiotic stresses. Largely not understood is the action of RB signals on genes with mixed expression. Likely additional regulatory factors may play a role such as the suppression of abscisic acid (ABA) formation upon NF treatment. Arrows represent conceptual positive influences, bars represent conceptual negative influences.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2223-774 7/10/2/296/s1, Figure S1: Comparison of RB-affected genes with direct HY5 target genes, Figure S2: Genes in the core module encoding proteins not related to one of the major functional groups, Figure S3: Genes with opposite expression profiles in the LIN and NF data sets, Table S1: Analysis of 152 genes responsive to retrograde biogenic signals.

**Author Contributions:** Conceptualization, T.P.; Formal analysis, B.G., C.C., T.P.; Data curation, C.C., T.P.; Writing, B.G., C.C., T.P.; Supervision, T.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Original data sources are described in the Methods section.

**Conflicts of Interest:** The authors declare no conflict of interest.
