Plastid to nucleus signals have been shown to regulate core photosynthetic genes, in both C3 and C4 species. However, the regulatory mechanisms of this signalling are not well understood. C4 photosynthesis has evolved from the ancestral C3 state in over sixty lineages of plants. Although commonly described as the most remarkable example of convergent evolution known to biology, more recent analysis indicates that the C4 pathway is also underpinned by parallel evolution. For example, orthologous genes from separate C4 lineages have repeatedly been recruited into the C4 pathway such that they are co-regulated with existing genes of C3 photosynthesis. For decades, the mechanisms allowing co-regulation of C4 and C3 photosynthesis genes have been unclear, but it was recently shown that in C3 Arabidopsis thaliana C4 orthologues can be controlled by plastid-to-nucleus signalling (Burgess et al. 2016). This strongly implies that evolution has re-enforced existing regulatory networks that operate in the C3 state to control expression of C4 genes. Currently, the extent to which this phenomenon is true in additional C3 lineages is not known. The work presented here aimed to understand in more detail which genes are under plastid control in a eudicotyledon and monocotyledon model and identify the factors involved.

To investigate this, Arabidopsis thaliana and Oryza sativa were subjected to inhibitors of chloroplast development and differential gene expression analysis performed by mRNA sequencing. Comparisons between illuminated and dark-grown plants for each treatment enabled the complementary roles of plastid-to-nucleus signalling and light regulation to be identified. Over 20,000 genes were detected for each sample, with over 1000 genes differentially expressed in lincomycin-treated plants compared to controls, and over 3500 genes differentially expressed when plants were treated with norflurazon. Furthermore, over half of the C4 orthologues in these two C3 species were regulated by both light and the chloroplast. Genes whose transcript abundance was significantly affected by chloroplast inhibition were scanned for known motif binding sites. A number of putative transcription factors were identified that bind to motifs found in both A. thaliana and rice chloroplast responsive genes. An additional method for identifying potential cis-regulatory elements involved in plastid to nucleus signalling is ATAC-seq. A trial has allowed this method, including the analysis pipeline, to be optimised such that in the future it could be employed to create an atlas of transcription factor binding sites involved in plastid to nucleus signalling.