Evolutionary Refinement of Mitochondrial and Plastid Targeting Sequences Coincides with the Late Diversification of Land Plants

Plastids and mitochondria are key to plant survival and adaptation. The evolutionary progress of land plants (embryophytes) witnessed gene and genome duplications, and the expansion of organelle-localized proteins. To deal with the increase of nuclear-encoded proteins, targeting to and import by the mitochondrion and plastid are known to have adapted in multiple ways. It included the addition of entirely new import channels and lineage-specific import receptors. Through comparative genomics and experimental biology, we uncover further changes in the organelle import machineries. Their evolution likely served to enhance the rate of protein import and improve its physiological regulation, e.g. via interactions between the import channel and respiratory complex. On the cargo side, nuclear-encoded N-terminal targeting sequences of mitochondrial targeting peptide (TP) and plastidal (pTPs) proteins have diverged in their charge via a preference for phosphorylatable amino acids (AA) (adding negative charges after phosphorylation) and an avoidance of positive charges in the pTPs, which is most evident in eudicots. Using Chlamydomonas and Marchantia, we experimentally underscore that the evolved TP divergence prevents mis-sorting between mitochondria and plastids. In accordance with the increase in phosphorylatable AA in the pTPs, we pinpoint the embryophytic origin of a membrane-anchored phosphatase, PAP2, which is associated with targeting sequence processing. On the whole, we propose a revised model for the evolution of plant organelle protein import from algae to angiosperms, which facilitated the flourishing of this lineage on land.