Studying Translation in Arabidopsis Chloroplasts

Abstract

Chloroplasts as descendents of a cyanobacterial endosymbiont have retained, during evolution, their own genome together with the gene expression machinery, including the translation apparatus. Therefore, chloroplast protein synthesis is not only a key process in organello biogenesis and maintenance, but it also represents the major regulatory step in chloroplast gene expression. In fact, several independent evidences have shown that the accumulation of template messengers is not limiting in the expression of chloroplast genes. On the contrary, translation regulatory processes based on selection of translatable mRNA by either nucleus-encoded activation factors or sensors of the assembly status of chloroplast multiprotein complexes have been reported. Additionally, we have shown that organelle translation rate triggers an organelle-to-nucleus signaling cascade aimed to modulate nuclear gene expression according to the organelle’s needs. Therefore, the study of chloroplast translation appears to be essential for the comprehension of several aspects of chloroplast activity. Here, we describe the in vivo pulse-chase and the polysome isolation approaches. Taken together, the two methods allow one to assess rates of protein synthesis and degradation as well as defects during the initial steps of protein synthesis.