The plastid is a defining structure of photosynthetic eukaryotes and houses many plant-specific processes, including the light reactions, carbon fixation, pigment synthesis, and other primary metabolic processes. Identifying proteins associated with catalytic, structural, and regulatory functions that are unique to plastid-containing organisms is necessary to fully define the scope of plant biochemistry. Here, we performed phylogenomics on 20 genomes to compile a new inventory of 597 nucleus-encoded proteins conserved in plants and green algae but not in non-photosynthetic organisms. 286 of these proteins are of known function, whereas 311 are not characterized. This inventory was validated as applicable and relevant to diverse photosynthetic eukaryotes using an additional eight genomes from distantly related plants (including Micromonas, Selaginella, and soybean). Manual curation of the known proteins in the inventory established its importance to plastid biochemistry. To predict functions for the 52% of proteins of unknown function, we used sequence motifs, subcellular localization, co-expression analysis, and RNA abundance data. We demonstrate that 18% of the proteins in the inventory have functions outside the plastid and/or beyond green tissues. Although 32% of proteins in the inventory have homologs in all cyanobacteria, unexpectedly, 30% are eukaryote-specific. Finally, 8% of the proteins of unknown function share no similarity to any characterized protein and are plant lineage-specific. We present this annotated inventory of 597 proteins as a resource for functional analyses of plant-specific biochemistry.The plastid is an organelle in plants and algae that evolved from a photosynthetic cyanobacterium after it was engulfed by an ancestral eukaryotic cell over 1.5 billion years ago (1, 2). How the endosymbiont became integral to host cell functions and evolved into a plastid is still under debate (3), but functions localized to the present day plastid depend on both plastid-and nucleus-encoded proteins. The latter are synthesized in the cytoplasm and imported into the organelle by a specific multiprotein complex composed of the translocon of the outer and inner chloroplast envelope membrane (TOC 4 and TIC, respectively) proteins (4, 5). Over 2000 proteins are estimated to be located in the plastid with the vast majority (Ͼ90%) encoded by genes in the nucleus (6 -9). Many of the nucleus-encoded proteins that function within plastids are conserved among photosynthetic organisms. These conserved proteins function in processes such as the capture and utilization of excitation energy, carbohydrate metabolism, and the synthesis of key cellular metabolites (such as lipids, isoprenoids, pigments, and amino acids). Interestingly, however, many plastid-localized proteins have not yet been assigned a specific biochemical function.The increasing availability of sequence information from diverse organisms has allowed the application of comparative genomics, or phylogenomics, to discover proteins specific to bacteria (...