Background:

Plants as sessile organisms cannot run when challenged by environmental stress, including temperature stress. Instead, they have developed a plethora of sophisticated physiological and molecular reactions to maintain homeostasis. How important phase separation is for plants is just beginning to emerge. We found that the chloroplast RNA binding protein CP29A is capable of phase separation in vivo and in vitro. Phase separation is induced by low temperatures, is fully reversible after return to ambient temperature and depends on the presence of CP29A’s prion-like domain (PLD). Disruption of CP29A or deletion of the PLD leads to leaf bleaching and compromised photosynthetic activity in the cold (Fig. 1). eCLIP and RBNS experiments identified multiple chloroplast RNA targets of CP29A, including the rbcL mRNA. Mutants of CP29A show a strong reduction of rbcL mRNA in the cold. NMR analyses of recombinant CP29A during cooling unraveled a pronounced structural shift with in the PLD, indicative of a strong reduction in protein mobility resulting from molecular interactions in the condensed phase (Fig. 2). Moreover, NMR analysis of CP29As two individual RRM motifs identified surprising differences in their ability to bind RNA. Encouraged by our findings with CP29A, we asked whether other chloroplast RBPs are also capable of LLPS. Using a combination of in silico predictions, in vivo transient expression analysis and in vitro LLPS assays, we present evidence for a handful of additional chloroplast RNA binding proteins capable of LLPS.