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

## TL;DR

This study explores how plant cells evolved better ways to transport proteins into mitochondria and plastids, helping land plants thrive.

## Contribution

The study reveals evolutionary changes in targeting sequences and import mechanisms that prevent mis-sorting in land plants.

## Key findings

- N-terminal targeting sequences in eudicots evolved to avoid positive charges and favor phosphorylatable amino acids.
- Experimental evidence shows evolved targeting peptides prevent mis-sorting between mitochondria and plastids.
- A phosphatase, PAP2, is linked to targeting sequence processing and originated in embryophytes.

## Abstract

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.