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Abstract We developed a resource, the Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/), to solve central questions about the Arabidopsis thaliana proteome, such as the significance of protein splice forms and post-translational modifications (PTMs), or simply to obtain reliable information about specific proteins. PeptideAtlas is based on published mass spectrometry (MS) data collected through ProteomeXchange and reanalyzed through a uniform processing and metadata annotation pipeline. All matched MS-derived peptide data are linked to spectral, technical, and biological metadata. Nearly 40 million out of ∼143 million MS/MS (tandem MS) spectra were matched to the reference genome Araport11, identifying ∼0.5 million unique peptides and 17,858 uniquely identified proteins (only isoform per gene) at the highest confidence level (false discovery rate 0.0004; 2 non-nested peptides ≥9 amino acid each), assigned canonical proteins, and 3,543 lower-confidence proteins. Physicochemical protein properties were evaluated for targeted identification of unobserved proteins. Additional proteins and isoforms currently not in Araport11 were identified that were generated from pseudogenes, alternative start, stops, and/or splice variants, and small Open Reading Frames; these features should be considered when updating the Arabidopsis genome. Phosphorylation can be inspected through a sophisticated PTM viewer. PeptideAtlas is integrated with community resources including TAIR, tracks in JBrowse, PPDB, and UniProtKB. Subsequent PeptideAtlas builds will incorporate millions more MS/MS data. 

Summary A network of peptidases governs proteostasis in plant chloroplasts and mitochondria. This study reveals strong genetic and functional interactions in Arabidopsis between the chloroplast stromal CLP chaperone‐protease system and the PREP1,2 peptidases, which are dually localized to chloroplast stroma and the mitochondrial matrix.Higher order mutants defective in CLP or PREP proteins were generated and analyzed by quantitative proteomics and N‐terminal proteomics (terminal amine isotopic labeling of substrates (TAILS)).Strong synergistic interactions were observed between the CLP protease system (clpr1‐2,clpr2‐1,clpc1‐1,clpt1,clpt2)and both PREP homologs (prep1,prep2) resulting in embryo lethality or growth and developmental phenotypes. Synergistic interactions were observed even when only one of the PREP proteins was lacking, suggesting that PREP1 and PREP2 have divergent substrates. Proteome phenotypes were driven by the loss of CLP protease capacity, with little impact from the PREP peptidases. Chloroplast N‐terminal proteomesshowed that many nuclear encoded chloroplast proteins have alternatively processed N‐termini inprep1prep2,clpt1clpt2andprep1prep2clpt1clpt2.Loss of chloroplast protease capacity interferes with stromal processing peptidase (SPP) activity due to folding stress and low levels of accumulated cleaved cTP fragments. PREP1,2 proteolysis of cleaved cTPs is complemented by unknown proteases. A model for CLP and PREP activity within a hierarchical chloroplast proteolysis network is proposed. 

Summary Eukaryotic cells represent an intricate collaboration between multiple genomes, even down to the level of multi‐subunit complexes in mitochondria and plastids. One such complex in plants is the caseinolytic protease (Clp), which plays an essential role in plastid protein turnover. The proteolytic core of Clp comprises subunits from one plastid‐encoded gene (clpP1) and multiple nuclear genes. TheclpP1gene is highly conserved across most green plants, but it is by far the fastest evolving plastid‐encoded gene in some angiosperms. To better understand these extreme and mysterious patterns of divergence, we investigated the history ofclpP1molecular evolution across green plants by extracting sequences from 988 published plastid genomes. We find thatclpP1has undergone remarkably frequent bouts of accelerated sequence evolution and architectural changes (e.g. a loss of introns andRNA‐editing sites) within seed plants. AlthoughclpP1is often assumed to be a pseudogene in such cases, multiple lines of evidence suggest that this is rarely true. We applied comparative native gel electrophoresis of chloroplast protein complexes followed by protein mass spectrometry in two species within the angiosperm genusSilene, which has highly elevated and heterogeneous rates ofclpP1evolution. We confirmed thatclpP1is expressed as a stable protein and forms oligomeric complexes with the nuclear‐encoded Clp subunits, even in one of the most divergentSilenespecies. Additionally, there is a tight correlation between amino acid substitution rates inclpP1and the nuclear‐encoded Clp subunits across a broad sampling of angiosperms, suggesting continuing selection on interactions within this complex. 

With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them.