Three evolutionary scenarios for the origin of endomembrane system-mediated protein targeting to higher plant plastids. The phagotrophic ancestor of the kingdom Archaeplastida, including glaucophytes, red algae, and green plants, regularly fed on cyanobacteria, from which genes migrated to the host nucleus via endosymbiotic gene transfer (A). When these endosymbionts evolved into primary plastids, they made use of both cyanobacteria- and host-derived genes present in the host nucleus. According to the ‘relic’ hypothesis for endomembrane system (ES)-mediated plastid protein targeting , all such proteins were targeted to the new primary plastid via the endoplasmic reticulum and/or Golgi apparatus (B1). In a later evolutionary stage, this co-translational pathway was replaced by a post-translational route involving Toc and Tic translocons for most plastid-targeted proteins (C). The hypothesis implies that proteins currently imported into higher plant plastids via the ES, such as αAmy3, αAmy7, CAH1, NPP1, are relics of ancestral ES-mediated protein trafficking to the primary plastid. Two alternative scenarios (B2 and B3) conflict with the ‘relic’ hypothesis; they postulate that the Toc and Tic translocons evolved very early in the primary endosymbiosis. In one (B2), a limited subset of host-derived proteins, previously targeted via the ES to different compartments within the host cell, exploited their pre-existing signal peptides to reach the primary plastid. Alternatively (B3), host-derived proteins carrying signal peptides were directed to primary plastids much later, well after the initial primary endosymbiosis, and possibly only in some higher plant lineages (C). Thickness of the colored arrows is proportional to the presumed or known commonality of a given pathway: ES (pink) or Toc-Tic translocons (orange). Stacked thylakoids only evolved in the green primary plastid lineage.