Changes in organellar gene expression (OGE) trigger retrograde signaling. The molecular dissection of OGE-dependent retrograde signaling based on analyses of mutants with altered OGE is complicated by compensatory responses that mask the primary signaling defect and by secondary effects that influence other retrograde signaling pathways. Therefore, to identify the earliest effects of altered OGE on nuclear transcript accumulation, we have induced OGE defects in adult plants by ethanol-dependent repression of PRORS1, which encodes a prolyl–tRNA synthetase located in chloroplasts and mitochondria. After 32h of PRORS1 repression, the translational capacity of chloroplasts was reduced, and this effect subsequently intensified, while basic photosynthetic parameters were still unchanged at 51h. Analysis of changes in whole-genome transcriptomes during exposure to ethanol revealed that induced PRORS1 silencing affects the expression of 1020 genes in all. Some of these encode photosynthesis-related proteins, including several down-regulated light-harvesting chlorophyll a/b binding (LHC) proteins. Interestingly, genes for presumptive endoplasmic reticulum proteins are transiently up-regulated. Furthermore, several NAC-domain-containing proteins are among the transcription factors regulated. Candidate cis-acting elements which may coordinate the transcriptional co-regulation of genes sets include both G-box variants and sequence motifs with no similarity to known plant cis-elements.