Mechanism of Light Signaling in Controlling Chloroplast Biogenesis

Phytochromes are red and far-red light receptors that initiate photomorphogenesis by reprogramming both nuclear and plastidial genomes. Early light signaling events include translocation of photoactivated phytochromes from the cytoplasm to subnuclear photobodies and phytochrome-mediated degradation of a group of transcription factors, PHYTOCHROME INTERACTING FACTORs (PIFs). The degradation of PIFs not only inhibits the elongation of hypocotyl but also promotes chloroplast development by activating photosynthetic genes. However, the mechanisms by which phytochrome signaling initiates chloroplast development remain elusive. The main challenge in determining these mechanisms has been that previous genetic screens have been unable to distinguish mutants involved in plastidial gene regulation from mutants of essential components of chloroplast functionality (Chen et al., 2010; Chen and Chory, 2011). We have previously reported a new phytochrome signaling component, HEMERA (HMR), which is a transcriptional coactivator required for both phytochrome signaling and chloroplast development. The hmr mutant has a combination of long-hypocotyl and albino phenotypes, representing the founding member of a new class of photomorphogenetic mutants that has been overlooked by previous genetic screens (Chen et al., 2010; Chen and Chory, 2011). We hypothesized that these tall-and-albino mutants define uncharacterized components of phytochrome signaling required for chloroplast development. To investigate this hypothesis, we conducted a forward genetic screen for tall-and-albino mutants, which identified a novel phytochrome signaling component named Regulator-for-Chloroplast-Biogenesis-by-Light (RCBL). To determine the evolutionary history of RCBL, I acquired the homologous sequences of RCBL and its paralog, REGULATOR-FOR-CHLOROPLAST-BIOGENESIS (RCB), from the available genomes and transcriptomes of a wide range of land plants. Phylogenetic analyses of these sequences demonstrate that RCBL and RCB diverged after the emergence of seed plants, but their mutant phenotypes show they are not functionally redundant. Characterization of rcbl mutants show that RCBL is required for both red and far-red light signaling, and it acts genetically downstream of phytochrome A and B. Similar to HMR, RCBL is also essential for photobody assembly, PIF1 and PIF3 degradation, and the expression of PIF-dependent light-responsive genes. Knocking out four PIFs (pifq, pif1/pif3/pif4/pif5) in the rcbl mutant background largely rescued the elongated hypocotyl phenotype of rcbl, indicating that the phytochrome-mediated phenotype of rcbl is dependent on PIFs. In chloroplasts, RCBL is required for transcription of plastid-encoded photosynthesis genes. However, this defective chloroplast phenotype of rcbl cannot be rescued by knocking out PIFs, suggesting RCBL plays a PIF-independent role in chloroplast development.

Since RCBL is involved in both phy signal transduction and chloroplast biogenesis, I examined whether RCBL is dual-localized to the nucleus and chloroplasts. Fluorescently-tagged RCBL shows dual-localization to chloroplasts and nuclei in both tobacco and Arabidopsis. Additionally, RCBL protein can be detected in protein fractions isolated from nuclei and plastids. I therefore conclude that RCBL is a dual-localized protein, which suggests that RCBL might be directly involved in both nuclear and plastidial events of photomorphogenesis.

Further investigation of the structure of RCBL revealed that the C-terminus of RCBL contains a domain similar to E. coli thioredoxin but without the canonical catalytic CxxC motif. Biochemical analyses confirmed that RCBL lacks thioredoxin reductase activity. Instead, in vitro experiments suggest that RCBL directly interacts with RCB through its C-terminal thioredoxin-like domain.

Taken together, this study revealed a previously uncharacterized early phytochrome signaling component which plays a critical role in chloroplast development, and demonstrated a mechanistic link between the nucleus and plastids during the initiation of photomorphogenesis.