*3.8. Transcription*

Special interest in retrograde signaling is paid to the identification of nuclear localized transcription factors that mediate the corresponding gene expression responses (Figure 2E). An often discussed prime candidate, ABI4 (ABA insensitive 4), has been recently shown to be likely not involved in retrograde signaling [47]. Our meta-analysis identified nine genes encoding other potential candidates that exhibit diverse expression patterns. One highly interesting candidate is GLK1 that exhibits the "photosynthesis-type" transcript accumulation. It was shown to be a major activator of PhANG expression and chloroplast biogenesis [48] and is also down-regulated at protein level in *pap8-1*, an albino mutant related to *pap7-1* [49]. The low expression of GLK1 in response to blocked chloroplast biogenesis likely accounts for a large part of the expression profiles observed here. We noticed that the partner regulator GLK2 did not appear in our gene list suggesting that it is regulated differently. GLK1 may potentially work together with another candidate, COL7, a transcription factor involved in light signaling that interacts with HY5 (Supplemental Table S1). The gene for the MYB-domain containing transcription factor MYB29 shares the "photosynthesis-type" expression pattern. It is known to be a major activator of the biosynthesis of glucosinolates, secondary metabolites involved in responses to biotic and abiotic stresses [50]. This observation aligns well with the expression patterns of MAM1 and BGL28 (encoding two enzymes required for glucosinolate biosynthesis) (Supplemental Figure S2 and Table S1). MYB29 is also involved in retrograde control from mitochondria regulating the expression of alternative oxidase 1a providing an interesting link between the two organelles [51]. Genes for three transcription factors RVE2, CGA1 and CRF6 (Reveille 2, Cytokinin-responsive GATA factor 1 and Cytokinin response factor 6) exhibit enhanced expression upon block of chloroplast biogenesis. Reveille 2, also a MYB-domain containing factor, is known to promote primary dormancy, but is repressed under illumination and imbibition by PhyB [52]. Its opposite expression in the NF sample may be an ABA-mediated effect as with SEN1 (see above). CGA1 and CRF6 are both implicated in cytokinin signaling. Cytokinin activates chloroplast biogenesis by inducing multiple target genes, among them HY5, CGA1 and CRF6. CGA1 is also known to act in an additive manner to GLK1 and both together represent a major regulatory hub of chloroplast biogenesis with GLKs likely acting down-stream of GNCs [53,54]. A recent study revealed that *pap* mutants overproduce cytokinins [55]. We regard it as likely that blocking of chloroplast biogenesis by LIN or NF induce similar responses. In sum, we identified a set of key transcription factors that is most likely involved in the mediation of RB signals during early chloroplast development and provides interesting candidates for future research in this context. Apart from directly affecting the expression of these key transcription regulators, RB signals appear to affect also other components involved in transcription or RNA metabolism both in plastid and in nucleus. A number of recent studies have identified numerous connections between retrograde signals and RNA metabolism in nucleus and plastid pointing to a potentially important regulatory level [56–59].

## *3.9. Proteins and Stress*

Our meta-analysis was focused on the implications of RB signals on transcript accumulation and cannot account for potential effects on other gene expression levels. However, we observed significant effects of RB signals on genes encoding components involved in protein folding, proteostasis and stress responses, which imply that blocking chloroplast biogenesis affects also these functions. Recent studies revealed a major impact of plastid signals on the unfolded protein response in plastids, endoplasmic reticulum and cytosol as well as on protein accumulation [60–62]. This includes the action of heat shock proteins and likely other functionally related proteins such as immunophilins. The GLK1 transcription factor has been recently shown to be controlled at the protein level by RB signals [63]

providing an important example. This aspect of retrograde signaling certainly will likely expand a lot in future research.

## *3.10. Lipids and Hormones*

Lipids are major constituents of membranes and are absolutely essential for the buildup of chloroplast. Although blocking of chloroplast biogenesis results in plastids without thylakoid formation we did not find any impact on genes encoding proteins involved in plastid lipid metabolism, but only for non-plastid components. The corresponding gene expression profiles are very complex and difficult to interpret and more analyses will be necessary to understand and to explain the RB impact on them. We also found an impact of RB signals on multiple genes involved in hormone biosynthesis and/or signaling. We identified already in other functional subsets a number of candidate genes that are associated with synthesis and/or the action of several hormones (see above). ABA appears to be an interceptive signal in a few cases (Supplemental Figure S3), but likely is not a major contributor to RB signaling in chloroplast biogenesis since NF treatment results in the same expression pattern as in the LIN and *pap7-1* profiles.

Interestingly, we found a strong impact on two genes encoding key enzymes of allene oxide biosynthesis (lipoxygenase and allene oxide synthase) that produce the precursor for peroxisomal jasmonic acid production, the oxophytodienoic acid (OPDA) [64]. A distinct role of OPDA in retrograde signaling has been not yet reported, but the molecule represents a likely candidate for a metabolite signal since it leaves the plastid for further metabolic processing. The impact on the allene oxide pathway probably is connected to the missing allene oxide precursor molecule linolenic acid that usually originates from the thylakoid lipids and which are not present in the arrested plastid [64]. A number of other genes encoding components involved in or connected to plant defense to biotic and abiotic stressors or peroxisome biogenesis were identified in this study (see above) supporting the view that a block of chloroplast biogenesis generates a situation of severe stress that is not only based on the missing photosynthetic function, but also caused by a dysregulation in the build-up of the plant defense system. Besides, most other genes identified in the "Hormones" subset encode proteins with non-plastidial locations demonstrating the broad impact of RB signals on the hormone-signaling network.
