The B vitamins and the cofactors derived from them are essential for life. B vitamin synthesis in plants is consequently as crucial to plants themselves as it is to humans and animals, whose B vitamin nutrition depends largely on plants. The synthesis and salvage pathways for the seven plant B vitamins are now broadly known, but certain enzymes and many transporters have yet to be identified, and the subcellular locations of various reactions are unclear. Although very substantial, what is not known about plant B vitamin pathways is regrettably difficult to discern from the literature or from biochemical pathway databases. Nor do databases accurately represent all that is known about B vitamin pathways-above all their compartmentation-because the facts are scattered throughout the literature, and thus hard to piece together. These problems (i) deter discoveries because newcomers to B vitamins cannot see which mysteries still need solving; and (ii) impede metabolic reconstruction and modelling of B vitamin pathways because genes for reactions or transport steps are missing. This review therefore takes a fresh approach to capture current knowledge of B vitamin pathways in plants. The synthesis pathways, key salvage routes, and their subcellular compartmentation are surveyed in depth, and encoded in the SEED database (http://pubseed.theseed.org/seedviewer.cgi?page=PlantGateway) for Arabidopsis and maize. The review itself and the encoded pathways specifically identify enigmatic or missing reactions, enzymes, and transporters. The SEED-encoded B vitamin pathway collection is a publicly available, expertly curated, one-stop resource for metabolic reconstruction and modeling.

Many common metabolites are intrinsically unstable and reactive, and hence prone to chemical (i.e. non-enzymatic) damage in vivo Although this fact is widely recognized, the purely chemical side-reactions of metabolic intermediates can be surprisingly hard to track down in the literature and are often treated in an unprioritized case-by-case way. Moreover, spontaneous chemical side-reactions tend to be overshadowed today by side-reactions mediated by promiscuous ('sloppy') enzymes even though chemical damage to metabolites may be even more prevalent than damage from enzyme sloppiness, has similar outcomes, and is held in check by similar biochemical repair or pre-emption mechanisms. To address these limitations and imbalances, here we draw together and systematically integrate information from the (bio)chemical literature, from cheminformatics, and from genome-scale metabolic models to objectively define a 'Top 30' list of damage-prone metabolites. A foundational part of this process was to derive general reaction rules for the damage chemistries involved. The criteria for a 'Top 30' metabolite included predicted chemical reactivity, essentiality, and occurrence in diverse organisms. We also explain how the damage chemistry reaction rules ('operators') are implemented in the Chemical-Damage-MINE (CD-MINE) database (minedatabase.mcs.anl.gov/#/top30) to provide a predictive tool for many additional potential metabolite damage products. Lastly, we illustrate how defining a 'Top 30' list can drive genomics-enabled discovery of the enzymes of previously unrecognized damage-control systems, and how applying chemical damage reaction rules can help identify previously unknown peaks in metabolomics profiles.

The increasing number of sequenced plant genomes is placing new demands on the methods applied to analyze, annotate, and model these genomes. Today's annotation pipelines result in inconsistent gene assignments that complicate comparative analyses and prevent efficient construction of metabolic models. To overcome these problems, we have developed the PlantSEED, an integrated, metabolism-centric database to support subsystems-based annotation and metabolic model reconstruction for plant genomes. PlantSEED combines SEED subsystems technology, first developed for microbial genomes, with refined protein families and biochemical data to assign fully consistent functional annotations to orthologous genes, particularly those encoding primary metabolic pathways. Seamless integration with its parent, the prokaryotic SEED database, makes PlantSEED a unique environment for crosskingdom comparative analysis of plant and bacterial genomes. The consistent annotations imposed by PlantSEED permit rapid reconstruction and modeling of primary metabolism for all plant genomes in the database. This feature opens the unique possibility of modelbased assessment of the completeness and accuracy of gene annotation and thus allows computational identification of genes and pathways that are restricted to certain genomes or need better curation. We demonstrate the PlantSEED system by producing consistent annotations for 10 reference genomes. We also produce a functioning metabolic model for each genome, gapfilling to identify missing annotations and proposing gene candidates for missing annotations. Models are built around an extended biomass composition representing the most comprehensive published to date. To our knowledge, our models are the first to be published for seven of the genomes analyzed.systems biology | computational biochemistry | plant metabolism | plant genomics

There is a growing demand for genome-scale metabolic reconstructions for plants, fueled by the need to understand the metabolic basis of crop yield and by progress in genome and transcriptome sequencing. Methods are also required to enable the interpretation of plant transcriptome data to study how cellular metabolic activity varies under different growth conditions or even within different organs, tissues, and developmental stages. Such methods depend extensively on the accuracy with which genes have been mapped to the biochemical reactions in the plant metabolic pathways. Errors in these mappings lead to metabolic reconstructions with an inflated number of reactions and possible generation of unreliable metabolic phenotype predictions. Here we introduce a new evidence-based genome-scale metabolic reconstruction of maize, with significant improvements in the quality of the gene-reaction associations included within our model. We also present a new approach for applying our model to predict active metabolic genes based on transcriptome data. This method includes a minimal set of reactions associated with low expression genes to enable activity of a maximum number of reactions associated with high expression genes. We apply this method to construct an organ-specific model for the maize leaf, and tissue specific models for maize embryo and endosperm cells. We validate our models using fluxomics data for the endosperm and embryo, demonstrating an improved capacity of our models to fit the available fluxomics data. All models are publicly available via the DOE Systems Biology Knowledgebase and PlantSEED, and our new method is generally applicable for analysis transcript profiles from any plant, paving the way for further in silico studies with a wide variety of plant genomes.

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide. In Escherichia coli and other bacteria, sequential deamination and reduction steps in riboflavin biosynthesis are catalyzed by RibD, a bifunctional protein with distinct pyrimidine deaminase and reductase domains. Plants have two diverged RibD homologs, PyrD and PyrR; PyrR proteins have an extra carboxyl-terminal domain (COG3236) of unknown function. Arabidopsis (Arabidopsis thaliana) PyrD (encoded by At4g20960) is known to be a monofunctional pyrimidine deaminase, but no pyrimidine reductase has been identified. Bioinformatic analyses indicated that plant PyrR proteins have a catalytically competent reductase domain but lack essential zinc-binding residues in the deaminase domain, and that the Arabidopsis PyrR gene (At3g47390) is coexpressed with riboflavin synthesis genes. These observations imply that PyrR is a pyrimidine reductase without deaminase activity. Consistent with this inference, Arabidopsis or maize (Zea mays) PyrR (At3g47390 or GRMZM2G090068) restored riboflavin prototrophy to an E. coli ribD deletant strain when coexpressed with the corresponding PyrD protein (At4g20960 or GRMZM2G320099) but not when expressed alone; the COG3236 domain was unnecessary for complementing activity. Furthermore, recombinant maize PyrR mediated NAD(P)H-dependent pyrimidine reduction in vitro. Import assays with pea (Pisum sativum) chloroplasts showed that PyrR and PyrD are taken up and proteolytically processed. Ablation of the maize PyrR gene caused early seed lethality. These data argue that PyrR is the missing plant pyrimidine reductase, that it is plastid localized, and that it is essential. The role of the COG3236 domain remains mysterious; no evidence was obtained for the possibility that it catalyzes the dephosphorylation that follows pyrimidine reduction.