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MapMan4: A Refined Protein Classification and Annotation Framework Applicable to Multi-Omics Data Analysis

Genome sequences fromover 200 plant species have already been published, with this number expected to increase rapidly due to advances in sequencing technologies. Once a new genome has been assembled and the genes identified, the functional annotation of their putative translational products, proteins, using ontologies is of key importance as it places the sequencing data in a biological context. Furthermore, to keep pace with rapid production of genome sequences, this functional annotation process must be fully automated. Here we present a redesigned and significantly enhanced MapMan4 framework, together with a revised version of the associated online Mercator annotation tool. Compared with the original MapMan, the new ontology has been expanded almost threefold and enforces stricter assignment rules. This framework was then incorporated into Mercator4, which has been upgraded to reflect current knowledge across the land plant group, providing protein annotations for all embryophytes with a comparably high quality. The annotation process has been optimized to allow a plant genome to be annotated in a matter of minutes. The output results continue to be compatible with the established MapMan desktop application.


INTRODUCTION
Plant sciences have seen a dramatic increase in the use of highthroughput omics platforms, driven by recent technological improvements. Among the quantitative omics technologies, transcriptomics can be seen as mature with continually decreasing costs as protocols are improved (Tzfadia et al., 2018), metabolomics is widely adopted , and proteomics offers great potential (Vanderschuren et al., 2013). Application of omics technologies to epigenetics data is allowing great strides, for example in understanding the role of plant DNA methylation . Since these technologies are complementary, their combined use is uncovering novel key players in plant metabolism, signaling and regulation, and driving functional pathway elucidation. This is especially the case for coupled transcriptomics and metabolite data, for example, in secondary metabolism (Fernie and Tohge, 2017;Wisecaver et al., 2017) and for transcriptomics in combination with chromatin immunoprecipitation sequencing to study transcriptional regulation (e.g., Ezer et al., 2017). Genomics analyses are greatly aided by pathway and process databases that capture existing knowledge and allow visualizations of individual or combined datasets. Crossspecies genomics analyses are also playing an increasing role While initially focused on metabolic processes, the MapMan framework rapidly evolved to include regulatory processes such as transcription factors and signaling pathways as well as biotic and abiotic stress responses. The ontology was exploited as the foundation for the MapMan application, which allows quantitative omics data to be visualized on functional pathways (Thimm et al., 2004). It also allows users to investigate enriched pathways and to functionally explore differentially expressed genes and accumulated metabolites.
Although MapMan was originally developed for use with the model species A. thaliana, it was later adapted to other species by similarity transfer and manual curation (Ling et al., 2013). This proved to be infeasible as a long-term approach, due to the rapid increase in the number of species for which genomics data were available. The tool Mercator was therefore developed to allow automatic annotation of plant protein sequences with MapMan terms (Lohse et al., 2014). Mercator relied on sequence similarity and, when appropriate, protein domains from InterPro (Mulder and Apweiler, 2007) and CDD (Marchler-Bauer et al., 2013) that had been manually assigned to the bins. However, this approach resulted in many annotations to abstract levels, partly due to the absence of appropriate bins for the new species.
In terms of describing proteins, the most commonly used framework is the Gene Ontology (GO), which is widely used across all life forms. The GO framework defines terms and their relationship to each other as a means to formalize protein description (Gene Ontology Consortium, 2014). The terms are partitioned in three specific categories (named ''Biological Process,'' ''Molecular Function,'' and ''Cellular Component''). These GO terms are arranged as a directed acyclic graph (DAG), where a child term may have more than a single parent. Generally, a single protein can expect to be annotated with a multitude of GO terms originating from each category. While GO is beneficial for a rich annotation, it can pose difficulties when it is used to visualize omics data because the multiple annotations lead to a strong redundancy (Jantzen et al., 2011).
The Kyoto Encyclopedia of Genes and Genomes (KEGG) ontology is a collection of databases covering many different aspects of biology (Kanehisa et al., 2017). Of these, the KEGG Orthology (KO) database is the closest equivalent to the MapMan framework, using a similar hierarchical structure of protein function terms. KO encompasses genes from both eukaryotes and prokaryotes, and while it was traditionally focused on metabolism, it has been expanded to include a wider range of biological processes. Other frameworks such as PlantCyc (Schl€ apfer et al., 2017) and Plant Reactome (Naithani et al., 2017) focus primarily on metabolic processes, and, while highly detailed within their area of focus, do not cover a broad range of biological processes and thus cannot easily be compared with MapMan.
We have now completely redesigned the MapMan framework and developed a more powerful Mercator pipeline to sustainably annotate the proteome of any land plant. Here we present the first stable release of the new MapMan4 framework and the improved online tool Mercator4, and showcase their application in determining gene loss in parasitic plants.

MapMan4: A Novel Biological Context-Based Framework
The MapMan4 ontology represents a comprehensive set of common biological processes and incorporates genetic information from a wide variety of plant species. The core design principles from the original MapMan (Thimm et al., 2004) have been retained, such as the simple tree structure with each toplevel category representing a main biological concept with each sublevel becoming increasingly specialized. In MapMan4, proteins are only classified into leaf node categories ( Figure 1, Lopez-Obando et al., 2015), thus ensuring that all assignments receive precise protein descriptions. In contrast to the original MapMan framework, assigning proteins to top-level or intermediate nodes is no longer possible. The total number of bin categories has been almost tripled, with currently 4147 leaf nodes and 1340 branch nodes, compared with 1550 leaf nodes and 341 branch nodes in MapMan v.3. This increase provides a finer granularity that enables users to perform more precise analyses at the biological level.
Currently, the MapMan4 ontology comprises 27 functional toplevel categories representing a diverse range of biological processes (Table 1). In principle, these 27 top-level bins should contain only proteins that have a strong biological context, e.g., the well-defined function of a protein within a pathway. However, this strict approach results in the classification of around only one-third of plant proteins, due to limited plant biological knowledge.
Therefore, the criteria were broadened, in specific cases, to also accept proteins that had weaker biological contexts. One example are transcription factors, which are simply classified using their canonical transcription factor family as context. MapMan4 currently distinguishes 91 transcription factor families. These families were designed primarily using PlantTFDB (Jin et al., 2017) and PlnTFDB (Pé rez-Rodríguez et al., 2010) as a guide, but in some cases sequence comparison suggested additional subdivision. As an example, for the HD-ZIP family from PlantTFDB, this has been divided into HD-ZIP I/II transcription factor, HD-ZIP III transcription factor, and HD-ZIP IV transcription factor in MapMan4, following the structure from Ariel et al. (2007). Comparison of the MapMan4 transcription factor bins (BIN-15.7 and BIN-15.8) against the iTAK (Zheng et al., 2016) transcription factor classification for A. thaliana revealed 1688 common transcription factor genes, 256 genes in MapMan4 but not in iTAK, and 79 genes in iTAK but not in MapMan4, indicating substantial agreement between the annotations. Mutual Information between the specific classes assigned by iTAK and MapMan4 to these shared 1688 genes is 3.499, very close the maximum possible value, given the entropy of each classification (3.589 for iTAK, 3.665 for MapMan4), thus indicating almost complete agreement of the specific class of each transcription factor gene.
Other cases where limited functional context is available are the large enzyme families, which are currently gathered into Bin-50. This category includes proteins that are known to belong to enzyme families, but information pertaining to their specific function may not have been ascertained. This category follows the Enzyme Commission structure to the second level, and currently contains 50 categories applicable to plants. In compliance with the original MapMan v.3 framework, proteins that have not been classified are assigned to Bin-35. This bin is further subdivided into Bin-35.1 (not assigned.annotated) and Bin-35.2 (not assigned.not annotated), depending whether they can or cannot be assigned Swiss-Prot based annotations (for details see Methods).

Comparison with Kyoto Encyclopedia of Genes and Genomes
The KO uses a hierarchical structure of protein function terms similar to MapMan4 (see Introduction). However, MapMan4 focuses exclusively on the plant kingdom, and thus includes plant-specific processes at a finer level of granularity. The plant focus of MapMan4 is more apparent in some biological processes than others. In metabolism, there is a considerable similarity, with, e.g., hexokinases, which in KO are under the hierarchy ''Metabolism.Carbohydrate metabolism.Glycolysis/Gluconeogenesis.HK; hexokinase'' (ko0844), in MapMan4 as ''Carbohydrate metabolism.sucrose metabolism.degradation.hexokinase'' (BIN-3.1.4.3). In other areas, there is a substantially different organization, with important plant-specific processes, such as Cell wall and Nutrient uptake, which are top-level categories in MapMan4 distributed in unrelated parts of the KO hierarchy, based on, for example, the substrates involved.
The KO structure uses up to four levels, which results in very high branching factors, particularly between the third and fourth levels. In contrast, the MapMan4 framework currently uses up to eight levels, and can allocate hierarchy levels as needed to the biological process (e.g., Protein degradation.peptide  tagging.Ubiquitin (UBQ)-anchor addition (ubiquitylation)), the particular step in the process (UBQ-ligase E3 activities), the protein complex grouping (Cullin-based ubiquitylation complexes), the specific protein complex (SKP1-CUL1-FBX (SCF) E3 ligase complexes), and specific protein component within the complex (F-BOX substrate adaptor components.SKP component).

Comparison with Gene Ontology
The GO consists of a DAG of annotation terms, from three categories, Cellular Component, Molecular Function, and Biological Process (see Introduction). Furthermore, it is common in GO that a single protein will be annotated with multiple terms from each GO category, e.g., the A. thaliana Hexokinase 1 protein (HXK1, At4g29130) is annotated (on www.arabidopsis.org) with multiple Biological Processes including hexose catabolic process, glycolytic process, cellular glucose homeostasis, and likewise multiple Molecular Functions including hexokinase activity, ATP binding, and zinc ion binding. Since the various terms capture different aspects of the protein, the terms cannot easily be inferred from each other, and thus GO terms should be considered as a group rather than individually. As previously noted in the Introduction, the DAG-based structure and the assignment of multiple terms per protein makes the visualization and interpretation of the GO annotations more complex than those from MapMan4 or KEGG, where annotation with a single term is often possible.

Automatic Annotation of Plant Proteomes
The Mercator4 Table 1). When considering all splice forms, the average protein classification rate was found to be 43.51% for dicots, 39.42% for monocots, and lower for other species (33.83%), reflecting the high diversity within the algae. Annotation rates were notably higher, with 64.65%, 58.5%, and 46.88% for dicots, monocots, and other species, respectively. Repeating this analysis using only the longest splice isoform of each gene resulted in a 1%-2% drop in the rate of bin assignment and annotation. These figures compare favorably with the annotation state of most sequenced plant species (Rhee and Mutwil, 2014).
For the model plant A. thaliana, the Mercator4 protein classification rate is currently at about 47% ( Figure 2 and Supplemental Table 1). This compares favorably with a KEGG annotation using the KAAS pipeline (Moriya et al., 2007), which covered 32%. It is, however, lower than the rate of 64% achieved by the GO framework (having at least one GO term assigned to Molecular Function or Biological Process) . However, as previously noted, assignment of a single GO term is not generally equivalent to a MapMan4 classification, and this annotation is the result of a large-scale community effort rather than an automated annotation pipeline.

Web Annotation Interface
To facilitate use of MapMan4 for any plant proteome or transcriptome, we have made available the corresponding Mercator4 pipeline for automated plant protein annotation (Figure 3, https://www. plabipd.de/portal/web/guest/mercator4). This web-based user interface allows the user to submit a text file (FASTA format) of either nucleotide or protein sequences. The user can provide an optional job name that will be used to name the result file. Multiple MapMan4 Refined Protein Annotation jobs can be simultaneously submitted and monitored. Users have the option of providing an email address that will be notified when their jobs are complete.
Once a job has finished, a summary of the protein categorization is provided along with a bar chart. This bar chart shows how many of the leaf categories belonging to each top-level bin contain at least one protein. In cases where a complete proteome was submitted, this chart can immediately suggest whether some biological pathways are missing. However, this insight relies on the completeness of the submitted proteome because underpopulated categories could also be an indication of an incomplete proteome. Table 1 depicts these values for A. thaliana, tomato, wheat, and corn, and demonstrates that these values are usually above 90% for well-conserved processes.
For a more detailed view of the protein classification, the user can launch the Mercator4 Tree Viewer (Figure 4). This visualization shows the number of proteins assigned to each bin, displayed on the MapMan4 hierarchical tree. The tree structure can also be used to compare multiple proteomes. To support this, a selection of reference proteomes is provided for comparison purposes. A download option is provided that creates a tabdelimited text file for all jobs and for the selected reference proteomes. The data can easily be loaded into a variety of statistical programs to allow a more detailed analysis or to perform comparison between proteomes.
Protein classification and annotation is performed on an HPC cluster that was recently upgraded. Further speed enhancements were achieved by reducing the number of sequence comparison tools and reference databases used during the bin assignment and annotation. The post-processing code was also optimized, resulting in a dramatic reduction in disk I/O. These hardware and software enhancements have resulted in speed improvements such that a typical diploid plant proteome (30 000 proteins) can be processed within a few minutes.

Legacy Versions
Access to the original Mercator v.3 will continue to be supported, and a separate tool (Legacy Mercator4) is available to support legacy versions of Mercator4 (https://www.plabipd.de/portal/ legacy-mercator4). Providing access to legacy Mercator4 versions ensures that any analyses carried out will remain reproducible in the future. However, given that users could potentially run a job against a variety of versions, the Mercator4 tree viewer is not supported in the legacy version.

Visualization in the MapMan Desktop Application
Given the extensive redesign of the MapMan framework for this release, it was necessary to create new MapMan4 pathway diagrams to reflect these changes and provide compatibility with the MapMan desktop application. A series of new pathway diagrams have been released that enable the visualization of transcripts using the MapMan desktop application. These include, for example, new kinase families and nitrogen uptake. The new pathway diagrams are available via the MapMan website (https://mapman. gabipd.org/mapmanstore).
As data analyses are often performed online nowadays, we have developed a feature-reduced web version of the MapMan desktop application. This allows visualization of functional responses based on client web browser technology and can easily integrate into functional genomics analysis platforms. As all rendering and business logic is performed on the client side Fifty-seven plant proteomes from Ensembl Plants v41 categorized by the Mercator4 annotation. The diagram distinguishes between ''classified'' proteins (assignable to a MapMan4 Bin-1/27 and Bin-50) and ''annotated'' proteins (assignable to a MapMan4 Bin-1/27, Bin-50, or a Swiss-Prot protein entry). The rate is given as proportion of the total number of proteins.
(i.e., on the web browser), and this component can be integrated into simple analysis platforms or static websites. Alternatively, the demo website can be downloaded and used as an embedded MapMan desktop application (https://usadellab. github.io/MapManJS/ultramicro.html).

Performance Measures and Comparison with Other Annotation Frameworks
Gene Network-Based Assessment of MapMan4 Protein Functional Annotation A significant challenge with evaluating the effectiveness of protein functional annotation is the lack of large evaluation datasets that are independent of the protein sequence. One strategy to elucidate such sequence-independent information is by using ''guilt-by-association'' approaches based on expression information (Di Salle et al., 2017). It has previously been reported by Klie and Nikoloski (2012) that the MapMan v.3 ontology outperforms GO in this area, providing a higher annotation rate of unknown proteins based on, for example, a simple k-nearest neighbor approach. As the same design principles were adhered to in the redesigned and expanded MapMan4 ontology, we expected it to perform even better in the analysis of biological networks.
We compared the performance of MapMan4 and the original MapMan v.3 release. Using a similar approach to that described by Klie and Nikoloski (2012), we calculated simple gene networks based on Pearson and Spearman correlation for the model plant A. thaliana, based on data downloaded from GeneCAT (Mutwil et al., 2008). For each gene pair, we assessed where both corresponding protein members had strong context bin assignments and how many of these pairs shared at least one MapMan top-level category. As can be seen in Figure 5, the new MapMan4 framework consistently outperformed the old MapMan v.3 annotation in terms of precision, regardless of whether the gene networks were constructed using Pearson or Spearman correlation thresholds.

Assessment of MapMan4 Using the Gene Ontology Framework
To bridge and compare the MapMan4 annotation with the corresponding GO annotation, 572 412 reference protein sequences from the public Swiss-Prot database were annotated by Mercator4 (using 3989 distinct MapMan4 leaf categories). On average, 143 reference proteins were assigned to each MapMan4 bin. The GO terms for each individual protein of a MapMan4 category were extracted, proteins without GO annotations discarded, and the GO terms shared by all proteins assigned to the MapMan4 bin. On average, 41 GO terms were assigned to each bin. Less than 1% of the MapMan4 bins were assigned no GO term at all. This was either because there was a single reference protein without GO annotations assigned to the bin (0.1%) or because the reference proteins did not share any GO terms (0.75%).
Approximately 34% of the MapMan4 bins showed a nonunique pattern of GO terms (i.e., the GO term collection was shared with at least one other bin). In some cases, these shared GO term patterns are the result of the same protein function existing in different biological contexts. In other cases, they were caused by the fine granularity of the MapMan4 bins-for example, the individual ribosomal proteins are categorized into many specifiable MapMan4 bins that all share the same GO term pattern. Sometimes, the shared term patterns may have been a result of the stringent filtering requiring a GO term to be shared by all proteins before getting included. This therefore resulted in an enrichment of the higher-level, more generic GO terms that would be expected to be present in a number of bins.
As an assessment of specificity or detail of the protein function description formed by the overall GO annotation of a bin, we computed the depth of each GO term. The depth of a given GO term was measured as the minimum number of edges separating the root node of the GO graph from the respective GO term. The relationship between the depth of a GO term and its information content was previously analyzed by Klie and Nikoloski (2012) using GO annotations from the A. thaliana proteome. They found that a depth value of 6 corresponds to an information content of 90% of its maximum content, when terms from the GO categories ''Biological Process'' and ''Cellular Component'' are assessed. The depth value of the MapMan4 bins (compound GO annotation) was calculated to Mercator4 assigns MapMan4 categories to protein sequences provided by the user. The resulting categorization can be visualized online by the Mercator4 tree viewer (Figure 4) or downloaded as a tab-delimited text file. On a local computer, the file together with a corresponding expression data file can be loaded into the MapMan desktop application to visualize the expression for each MapMan4 category. be 6.6 on average, thus suggesting a very detailed description of the protein function.
To assess the quality of Mercator4-inferred GO terms, we used the Oryza sativa proteins, from UniProt/trEMBL, as a test dataset. To avoid bias, 3467 proteins that overlapped with Swiss-Prot were removed. As a gold standard for the annotation, we downloaded the latest O. sativa GO term annotation (http://geneontology.org/ gene-associations/gene_association.gramene_oryza.gz) and retained only those annotations that had experimental evidence or were made by a human expert curator. For reasons of completeness, the remaining GO annotations were extended with their respective ancestral terms as obtainable from the directed GO graph. This resulted in a gold standard of 19 629 distinct GO annotations. Due to removing electronically inferred annotations, 68 572 proteins were left unannotated and were also removed. In total, 616 proteins from the original dataset of 72 655 remained.
Mercator4 was used to classify the test proteins, and GO terms were inferred from these assignments. An independent GO term assignment was also performed using InterProScan. Merca-tor4 and InterProScan inferences were compared with the gold standard annotation, and resulted in a Matthews Correlation Coefficient (MCC) of 0.16 for both Mercator4 and InterProScan (with Mercator4 yielding a slightly higher value), indicating that GO terms inferred are indeed meaningful and not biased.
Strikingly, when the MapMan4 categories were mapped onto GO terms using only a subset of 41 898 Swiss-Prot plant proteins, the GO annotation results were similar to the results obtained using all Swiss-Prot references (see above). This suggests that the MapMan4 framework is not only able to annotate protein functions present in plants, but also to assign these functions to conserved non-plant proteins. However, protein functions not found in plants are beyond the scope of the MapMan4 framework.

Usage Example: Detecting Gene Losses in Cuscuta Species
Data mining for genome changes should be promising approach when analyzing gene loss in plants with a reduced gene content requirement such as parasitic plants. In a test case, we used the Screenshot of the Mercator4 tree viewer that compares annotations from user jobs and-this example-from the reference species Arabidopsis thaliana. Each entry (colored circles within the tree) has the number of proteins assigned to a certain MapMan4 category, displayed with the option to see the protein names by hovering over the protein number.
Transcription in plastids is mediated by two different RNA polymerases, a nucleus-encoded RNA polymerase, and a plastidencoded RNA polymerase (PEP) complex (Yu et al., 2014;Pfannschmidt et al., 2015). The PEP is predominantly responsible for transcribing genes involved in photosynthesis (Yagi and Shiina, 2014) and consists of the conserved plastidencoded core subunits rpoA, rpoB, rpoC1, and rpoC2. All the core subunits have been reported as missing in the plastid genomes of C. gronovii (Funk et al., 2007) and C. obtusiflora (McNeal et al., 2007). The Mercator4 analysis with visualization of Bin-15.9 (RNA biosynthesis.organelle machineries.RNA polymerase activities) confirms the lack of the PEP core subunits ( Figure 6B) in these Cuscuta species. In comparison, the plastid genome of the related autotrophic I. batatas contains the genes for the PEP subunits ( Figure 6B). In addition, Mercator4 reveals that the nuclear genomes of C. campestris and C. australis have experienced major losses of the PAP/pTAC PEP-associated co-factors ( Figure 6A). Moreover, the putative regulatory co-factors PrdA and Prin2, as well as the six Sigma-like factors required for the initiation of the plastidial transcription by PEP, are lacking. Interestingly, the transcriptionally active plastid chromosome proteins pTAC9 and pTAC17 were identified by Mercator4 in both Cuscuta proteomes ( Figure 6A).
It has been shown that many plastidial RNA editing sites were abolished in Cuscuta species (Tillich and Krause, 2010). The visualization of Bin-16.10.2 (RNA processing.organelle machineries.RNA editing) confirms major losses of plastidial RNA editing factors in Cuscuta species. In accordance with this reduction in editing sites, 11 of the 15 editing factors found in tomato and I. nil are lacking in C. campestris and C. australis ( Figure 6C).

Comparison Using Other Ontologies and Automated Annotation Tools
To determine whether the biological insights described above, regarding the PEP complex and plastidial RNA editing factors, could potentially be discovered with other annotation pipelines and frameworks, we reannotated the 27 PEP core subunits and associated factors and 32 plastidial RNA editing factors from S. lycopersicum, using both the previous Mercator v.3 release and the KAAS pipeline (Moriya et al., 2007).
Mercator4 had assigned these 59 proteins to 42 different bins, illustrating the fine granularity of the MapMan4 framework structure. Furthermore, these categories were coherently structured under two branch nodes, representing the PEP complex components and RNA editing factors, respectively, making the loss of these mechanisms readily apparent by comparing the gene counts across species.
In contrast, Mercator v.3 could annotate 33 of the 59 proteins. Of these, 17 proteins were classified in broadly appropriate bins (RNA.transcription, RNA.regulation of transcription, RNA.RNA binding) while 16 others were assigned to unrelated bins. Loss of the PEP core subunits, which are assigned to RNA.transcription, would however be difficult to notice as this bin is quite general, and thus many other proteins are assigned to the same category. Likewise, any signal from the loss of PEPassociated components and plastidial RNA editing factors would be difficult to discern, due to the low annotation rate and large number of other proteins in the relevant categories.
Annotation using the KAAS pipeline could assign only 11 of the 59 proteins. Of these, the four PEP core subunits were correctly assigned to the PEP RNA polymerase KO groups, while others were assigned to spliceosome, signal transduction, and chaperones KO groups. Loss of the PEP core subunits would be clear within the RNA polymerase KO groups; however, the related loss of PEP-associated components and loss of plastidial RNA editing factors would likely be missed.

DISCUSSION
The rapidly expanding number of available plant genome sequences (https://www.plabipd.de/plant_genomes_pa.ep) offers an opportunity for unraveling protein function through comparative gene regulatory network analysis (Ferrari et al., 2018). In addition, it widens the application of transcriptomics and proteomics tools (Sheth and Thaker, 2014) and plant genome scale metabolic prediction (De Oliveira Dal'molin and Nielsen,  2013) to phylogenetically remote plants. Genomics and systems biology share common ground (Conesa and Mortazavi, 2014) and a draft genome is often the start for additional downstream analyses (Cuevas et al., 2016). However, protein sequences need to be put into a functional biological context to enable meaningful genome comparison within and between species. The demand for functional annotation is already visible in the use of the original Mercator tool, which has processed more than 12 000 datasets (i.e., genomes and transcriptomes) since its release (Lohse et al., 2014). While there is likely to be considerable redundancy between these datasets with regard to species, a recent study (Rai et al., 2017) came to the conclusion that almost 1000 different plant species have been studied by RNA-sequencing technology. This is likely to be an underestimate as it was based merely on publicly available data extracted from the NCBI Sequence Read Archive (https://www. ncbi.nlm.nih.gov/sra).
An increasing number of studies are coupling metabolite and transcriptome profiling data. This has been done in model plants for some years (Gibon et al., 2006;Satou et al., 2014). A rapidly expanding area is secondary metabolism in medicinal or ornamental plants, with the goal of discovering novel pathway members and gaining insights into pathway regulation (Polturak et al., 2018;Scossa et al., 2018).
Thus, the need for functional annotation and tools to analyze and interpret multi-omics data for model and in particular nonmodel plant species is increasing . This is especially necessary to bridge between omics data and research questions about physiology (Do Amaral and Souza, 2017). Mercator was initially developed for MapMan against this background (Lohse et al., 2014), but its extension was restricted due to the underlying design of MapMan. Thus, we have redesigned the MapMan framework and have adapted Mercator4 to reflect these changes. While the current version of Mercator4 does not yet capture as many protein sequences as the original version (Supplemental Table 2), the new classifications are more specific.
The increased specificity together with the design goals offer several advantages. (1) Statistical representation analyses such as an over-representation analysis is straightforward and can be restricted to any level on the hierarchical bin tree. Although the previous version of MapMan v.3 already performed reasonably well for protein functional annotation (Klie and Nikoloski, 2012), performance has been substantially improved by a complete redesign of the framework. (2) The new MapMan4 framework is fully deterministic and can be applied to any land plant proteome to allow a consistent comparison between proteomes. Finally, (3) the annotation process performed by the online tool Mercator4 takes only minutes to annotate a complete plant proteome.
To demonstrate the usefulness of the MapMan4 framework, we analyzed the deduced proteomes of two Cuscuta species. For survival, these holoparasitic plants strictly rely on a nutrient supply from their host plants, which has relieved them from an autotrophic lifestyle. In line with this evolutionary step, the plastid and nuclear genomes have experienced extensive losses in genes related to photosynthesis and other processes not needed for a parasitic lifestyle (Krause, 2008;Vogel et al., 2018). With the new MapMan4 framework, substantial protein losses in categories containing components of the PEP complex and protein factors involved in plastidial RNA editing are detectable in detail and can be visualized by the Mercator4 tree viewer ( Figure 6). The Mercator4 annotation enables a quick and reliable survey of which proteins within a certain biological context are available or not in a given plant proteome.

Design of the MapMan4 Framework
The MapMan4 hierarchical category structure was designed based on published experimental evidence and textbook knowledge (Figure 1). This also provided initial reference proteins that were used as seeds to find orthologs in high-quality annotated plant genomes across the plant kingdom. Determining whether a protein is a true ortholog involved manual examination of multiple sequence alignments from many plant species. The curated set of orthologous proteins identified for each MapMan4 bin was used to create one or more bin-specific Hidden Markov Models (HMMs), which can identify orthologous proteins from additional species.
The main functional bins in MapMan4 were designed to contain proteins that have a strong biological context, e.g., the well-defined function of a protein within a pathway. However, it was necessary to relax this criterion for transcription factors, which are simply classified using their canonical transcription factor family as context. Another case where limited functional context is available are the large enzyme families, which were currently gathered in Bin-50. This category includes proteins that are known to belong to enzyme families, but information pertaining to their specific function may not have been ascertained.

Automatic Annotation of Plant Proteomes
Each protein to be classified is tested against the bin-specific HMMs, using hmmscan from the HMMER3 software package (Eddy, 2011). If the provided sequences are nucleotides, a 6-frame translation of the sequence is generated for testing against the HMMs.
Many proteins that are assigned to the subcategories under the 27 main categories, based on their biological role, will also have assignment to categories under Bin-50 on the basis of their enzymatic activity. In these cases, assignments to the 27 main categories are considered to have a higher priority, and the redundant enzymatic activity bin assignment is filtered out.
The remaining unclassified proteins are subsequently compared with protein sequences contained in the Swiss-Prot database (The UniProt Consortium, 2018) using BLASTP. Positive matches (using a BLAST bit score >80) inherit the annotation from the matching Swiss-Prot hit and move to Bin-35.1 (not assigned.annotated). This provides an annotation for a number of proteins that were not assigned to a functional MapMan4 bin. Proteins that remain unannotated are placed in Bin-35.2 (not assigned.not annotated).

Mercator4 Implementation
The Mercator4 annotation web tool was implemented using the Java Portlet technology on a Liferay Portal (https://www.liferay.com). The front-end website is written in HTML with interactive components provided using the Javascript library D3 (https://d3js.org MapMan4 Refined Protein Annotation the jobs has been written in Java. The Distributed Resource Management Application API (DRMAA) is used to submit and monitor the jobs. The results are evaluated and collated before generation of the output file, which is presented to the user for download.

Proteome Annotation
All available plant proteomes were downloaded from Ensembl Plants (release 41, ftp://ftp.ensemblgenomes.org/pub/plants/release-41). Genomes that contained multiple splice variants were further processed to remove all but the longest form. Before being processed by Mercator4, each FASTA-formatted file was validated using the Mercator FASTA Validator, with records shorter than 5 amino acids or longer than 25000 amino acids removed (https://www.plabipd.de/portal/web/guest/mercatorfasta-validator). The validated files were submitted to the online Mercator4 annotation tool (https://www.plabipd.de/portal/mercator4).

Analysis of Concordant Bin Pairs in Correlation Networks
Within the expression dataset for A. thaliana (file ExpMatAra.exp, available at http://aranet.mpimp-golm.mpg.de/download.html), all ambiguous gene code assignments were deleted. In addition, for genes measured by multiple probes only one was retained. The resulting gene expression matrix was loaded into R and a correlation network was calculated using the R cor (correlation between matrices) function both for Spearman and Pearson correlation. The resulting matrix was then inspected for correlation values between genes with correlation thresholds of 0.7-0.966 in steps of 0.033 and only pairs where both proteins had an assignment in Bin-1 to 27 for MapMan4, and in Bin-1 to 34 (without Bin-26) for MapMan v.3. The pair was counted as concordant if at least one MapMan top-level bin assignment between the two proteins was shared and discordant otherwise. Code and data are available from https://github.com/ usadellab/MapMan-Mercator-4.

Web-Available MapMan Application
To allow the integration of the MapMan application into web services, we have ported some basic MapMan application components into Javascript. In addition, we have implemented code for data visualization that relies on Javascript D3 (https://d3js.org) and allows quick rendering of simple data formats. An over-representation analysis is also included based on Javascript D3 and relies on a Fisher's exact test that uses the Lanczos approximation to compute the gamma function (Lanczos, 1964). Values and code were translated from the GNU Scientific Library. All components were written to be maximally portable and light, therefore pathway files, for example, need to comply with the formatting as provided by MapMan to allow simpler parsing. The code in addition to a working version is available from https://github.com/usadellab/ usadellab.github.io.

MapMan4 Comparison with Gene Ontology
The complete Swiss-Prot protein sequence dataset was annotated by Mercator4 to find sets of reference proteins with each set representing a MapMan4 leaf category. The subsequent GO annotation for each reference protein also includes ancestral GO terms, i.e., terms found in all paths leading from the GO root term to the respective descendant term. Reference proteins without any GO annotation were removed from the sets. The collected GO terms for a reference protein form what we call the compound GO annotation of the protein. Finally, a MapMan4 category was annotated by the GO terms shared by all reference proteins of the category. To evaluate how detailed the GO annotation of a MapMan4 category describes a protein function, we measured the depth of each GO term appearing in the bin. The depth is defined as the minimum number of edges leading from the root of the GO graph to the respective GO term.
We assessed the quality of protein function predictions obtained using Mercator4 and InterProScan (Quevillon et al., 2005). Performance was assessed on a gold standard of rice proteins that were not present in Swiss-Prot and that had manually curated GO annotations on the GO website. As performance measures we used the MCC (Matthews, 1975;Powers, 2011). Before calculating these measures, all GO term predictions were extended to include related ancestral terms. All material, code, documentation, and results are available as R-package (MapMan2GO, http://github.com/usadellab/MapMan2GO).

SUPPLEMENTAL INFORMATION
Supplemental Information is available at Molecular Plant Online.