Genome-Wide Analysis of Translational Dynamics in Chloroplasts

The translation of chloroplast mRNAs is subject to regulation by developmental, environmental and physiological cues.  For example, translation initiation in chloroplasts is modulated by light, redox poise and the assembly status of the gene product, and translation elongation can be influenced by cold stress and membrane targeting of the nascent peptide. Genetic approaches have identified nucleus-encoded proteins that stimulate the translation of specific chloroplast mRNAs, but little is known about the roles of such proteins in linking environmental, developmental, and physiological signals to translational efficiency.  Progress in this area has been limited by the assays that have been available to monitor chloroplast ribosome behavior in vivo: pulse-labeling, polysome, and reporter gene approaches have been informative, but they are labor intensive, have limited resolution, and are not suited to genome-wide explorations. To address this need, we developed a method that reports the abundance and ribosome occupancy of every chloroplast mRNA at ~30-nt resolution for low cost. Our method is similar to a ribosome profiling method developed for cytosolic mRNAs (Ingolia et al, 2009) but substitutes high-resolution tiling microarrays for deep sequencing to map the positions of ribosome “footprints”.  We are using this method to (i) comprehensively analyze the functional repertoire of nucleus-encoded proteins that modulate chloroplast gene expression; (ii) address questions in chloroplast translation that were opaque to prior methods; and (iii) explore how ribosome dynamics influence and are influenced by membrane targeting and assembly of the nascent peptide. Examples of insights obtained in each of these areas will be discussed.