Plants are indispensable for life on earth and represent organisms of extreme biological diversity with unique molecular capabilities 1. Here, we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. It provides initial answers to how many genes exist as proteins (>18,000), where they are expressed, in which approximate quantities (>6 orders of magnitude dynamic range) and to what extent they are phosphorylated (>43,000 sites). We present examples for how the data may be used, for instance, to discover proteins translated from short open reading frames, to uncover sequence motifs involved in protein expression regulation, to identify tissue-specific protein complexes or phosphorylation-mediated signaling events to name a few. Interactive access to this unique resource for the plant community is provided via ProteomicsDB and ATHENA which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interplay. Main The plant model organism Arabidopsis thaliana (AT) has revolutionized our understanding of plant biology and influenced many other areas of the life sciences 1. Knowledge derived from Arabidopsis has also provided mechanistic understanding of important agronomic traits in crop species 2. The Arabidopsis genome was sequenced 20 years ago and hundreds of natural variants have since been analyzed at the genome and epigenome level 3,4. In contrast, the Arabidopsis proteome as the main executer of most biological processes is far less comprehensively characterized. To address this gap, we used state-of-the-art mass spectrometry and RNA sequencing (RNA-seq) to provide the first integrated proteomic, phosphoproteomic and transcriptomic atlas of Arabidopsis. Illustrated by selected examples, we show how this rich molecular resource can be used to explore the function of single proteins or entire pathways across multiple omics levels. Multi-omics atlas of Arabidopsis We generated an expression atlas covering, on average, 17,603 ± 1,317 transcripts, 14,430 ± 911 proteins and 14,689 ± 2,509 phosphorylation sites (p-sites) per tissue, using a reproducible biochemical and analytical approach (Fig. 1a,b; Extended Data Fig. 1a-c; Supplementary Data 1,2). In total, the protein expression data covers 18,210 of the 27,655 protein-coding genes (66%) annotated in Araport11 5. This is a substantial increase compared to the percentage of genes with protein level evidence reported in UniProt (27%) 6 and more than double the number of proteins identified in an earlier tissue proteome analysis 7 (Fig. 1c, Extended Data Fig. 1d-f). In addition, we report tissue-resolved quantitative evidence for a total of 43,903 p-sites making this study the most comprehensive single Arabidopsis phosphoproteome published to date (Fig. 1c). 47% of the expressed proteome was found to be phosphorylated in at least one instance, confirming earlier analyses of individual

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ProteomicsDB (https://www.ProteomicsDB.org) started as a protein-centric in-memory database for the exploration of large collections of quantitative mass spectrometry-based proteomics data. The data types and contents grew over time to include RNA-Seq expression data, drug-target interactions and cell line viability data. In this manuscript, we summarize new developments since the previous update that was published in Nucleic Acids Research in 2017. Over the past two years, we have enriched the data content by additional datasets and extended the platform to support protein turnover data. Another important new addition is that ProteomicsDB now supports the storage and visualization of data collected from other organisms, exemplified by Arabidopsis thaliana. Due to the generic design of ProteomicsDB, all analytical features available for the original human resource seamlessly transfer to other organisms. Furthermore, we introduce a new service in ProteomicsDB which allows users to upload their own expression datasets and analyze them alongside with data stored in ProteomicsDB. Initially, users will be able to make use of this feature in the interactive heat map functionality as well as the drug sensitivity prediction, but ultimately will be able to use all analytical features of ProteomicsDB in this way.

Integrated analysis of genomes, transcriptomes, proteomes and drug responses of cancer cell lines (CCLs) is an emerging approach to uncover molecular mechanisms of drug action. We extend this paradigm to measuring proteome activity landscapes by acquiring and integrating quantitative data for 10,000 proteins and 55,000 phosphorylation sites (p-sites) from 125 CCLs. These data are used to contextualize proteins and p-sites and predict drug sensitivity. For example, we find that Progesterone Receptor (PGR) phosphorylation is associated with sensitivity to drugs modulating estrogen signaling such as Raloxifene. We also demonstrate that Adenylate kinase isoenzyme 1 (AK1) inactivates antimetabolites like Cytarabine. Consequently, high AK1 levels correlate with poor survival of Cytarabine-treated acute myeloid leukemia patients, qualifying AK1 as a patient stratification marker and possibly as a drug target. We provide an interactive web application termed ATLANTiC ( http://atlantic.proteomics.wzw.tum.de ), which enables the community to explore the thousands of novel functional associations generated by this work.

The evolutionarily conserved 8-kD protein NEDD8 (NEURAL PRECURSOR CELL EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED8) belongs to the family of ubiquitin-like modifiers. Like ubiquitin, NEDD8 is conjugated to and deconjugated from target proteins. Many targets and functions of ubiquitylation have been described; by contrast, few targets of NEDD8 have been identified. In plants as well as in non-plant organisms, the cullin subunits of cullin-RING E3 ligases are NEDD8 conjugates with a demonstrated functional role for the NEDD8 modification. The existence of other non-cullin NEDD8 targets has generally been questioned. NEDD8 is translated as a precursor protein and proteolytic processing exposes a C-terminal glycine required for NEDD8 conjugation. In animals and yeast, DENEDDYLASE1 (DEN1) processes NEDD8. Here, we show that mutants of a DEN1 homolog from Arabidopsis thaliana have no detectable defects in NEDD8 processing but do accumulate a broad range of NEDD8 conjugates; this provides direct evidence for the existence of non-cullin NEDD8 conjugates. We further identify AUXIN RESISTANT1 (AXR1), a subunit of the heterodimeric NEDD8 E1 activating enzyme, as a NEDD8-modified protein in den1 mutants and wild type and provide evidence that AXR1 function may be compromised in the absence of DEN1 activity. Thus, in plants, neddylation may serve as a regulatory mechanism for cullin and non-cullin proteins.