Plastid transit peptides-where do they come from and where do they all belong? Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution.

Subcellular relocalization of proteins determines an organism's metabolic repertoire and thereby its survival in unique evolutionary niches. In plants, the plastid and its various morphotypes import a large and varied number of nuclear-encoded proteins to orchestrate vital biochemical reactions in a spatiotemporal context. Recent comparative genomics analysis and high-throughput shotgun proteomics data indicate that there are a large number of plastid-targeted proteins that are either semi-conserved or non-conserved across different lineages. This implies that homologs are differentially targeted across different species, which is feasible only if proteins have gained or lost plastid targeting peptides during evolution. In this study, a broad, multi-genome analysis of 15 phylogenetically diverse genera and in-depth analyses of pangenomes from Arabidopsis and Brachypodium were performed to address the question of how proteins acquire or lose plastid targeting peptides. The analysis revealed that random insertions or deletions were the dominant mechanism by which novel transit peptides are gained by proteins. While gene duplication was not a strict requirement for the acquisition of novel subcellular targeting, 40% of novel plastid-targeted genes were found to be most closely related to a sequence within the same genome, and of these, 30.5% resulted from alternative transcription or translation initiation sites. Interestingly, analysis of the distribution of amino acids in the transit peptides of known and predicted chloroplast-targeted proteins revealed monocot and eudicot-specific preferences in residue distribution.