Océane Frelin

Plant B Vitamin Pathways and their Compartmentation: a Guide for the Perplexed

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.

High-throughput comparison, functional annotation, and metabolic modeling of plant genomes using the PlantSEED resource.

Significance Genes must be annotated with their correct functions if genome data are to support hypothesis building and metabolic engineering. PlantSEED was developed to streamline the process of annotating plant genome sequences, to construct metabolic models based on genome annotations automatically, and to use models to test the annotation of these sequences, allowing the detection of gaps and errors in gene annotations and the prediction of new functions. PlantSEED is designed to grow in an iterative manner by including new plant genome sequences, new annotations harvested from the literature, and improved biochemical data, all of which are integrated in a consistent manner into the PlantSEED genomes and metabolic models. 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 cross-kingdom 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 model-based 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.

A cross-kingdom Nudix enzyme that pre-empts damage in thiamin metabolism

Genes specifying the thiamin monophosphate phosphatase and adenylated thiazole diphosphatase steps in fungal and plant thiamin biosynthesis remain unknown, as do genes for ThDP (thiamin diphosphate) hydrolysis in thiamin metabolism. A distinctive Nudix domain fused to Tnr3 (thiamin diphosphokinase) in Schizosaccharomyces pombe was evaluated as a candidate for these functions. Comparative genomic analysis predicted a role in thiamin metabolism, not biosynthesis, because free-standing homologues of this Nudix domain occur not only in fungi and plants, but also in proteobacteria (whose thiamin biosynthesis pathway has no adenylated thiazole or thiamin monophosphate hydrolysis steps) and animals (which do not make thiamin). Supporting this prediction, recombinant Tnr3 and its Saccharomyces cerevisiae, Arabidopsis and maize Nudix homologues lacked thiamin monophosphate phosphatase activity, but were active against ThDP, and up to 60-fold more active against diphosphates of the toxic thiamin degradation products oxy- and oxo-thiamin. Deleting the S. cerevisiae Nudix gene (YJR142W) lowered oxythiamin resistance, overexpressing it raised resistance, and expressing its plant or bacterial counterparts restored resistance to the YJR142W deletant. By converting the diphosphates of damaged forms of thiamin into monophosphates, the Tnr3 Nudix domain and its homologues can pre-empt the misincorporation of damaged diphosphates into ThDP-dependent enzymes, and the resulting toxicity.

Improved evidence-based genome-scale metabolic models for maize leaf, embryo, and endosperm.

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.

Identification of Mitochondrial Coenzyme A Transporters from Maize and Arabidopsis

Coenzyme A made in the cytosol is imported into plant mitochondria by twin transporters from the mitochondrial carrier family that are cognates of coenzyme A transporters of animals and yeast and can functionally replace the yeast transporter. Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5Δ mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes.

A 5-formyltetrahydrofolate cycloligase paralog from all domains of life: comparative genomic and experimental evidence for a cryptic role in thiamin metabolism

A paralog (here termed COG0212) of the ATP-dependent folate salvage enzyme 5-formyltetrahydrofolate cycloligase (5-FCL) occurs in all domains of life and, although typically annotated as 5-FCL in pro- and eukaryotic genomes, is of unknown function. COG0212 is similar in overall structure to 5-FCL, particularly in the substrate binding region, and has distant similarity to other kinases. The Arabidopsis thaliana COG0212 protein was shown to be targeted to chloroplasts and to be required for embryo viability. Comparative genomic analysis revealed that a high proportion (19%) of archaeal and bacterial COG0212 genes are clustered on the chromosome with various genes implicated in thiamin metabolism or transport but showed no such association between COG0212 and folate metabolism. Consistent with the bioinformatic evidence for a role in thiamin metabolism, ablating COG0212 in the archaeon Haloferax volcanii caused accumulation of thiamin monophosphate. Biochemical and functional complementation tests of several known and hypothetical thiamin-related activities (involving thiamin, its breakdown products, and their phosphates) were, however, negative. Also consistent with the bioinformatic evidence, the COG0212 proteins from A. thaliana and prokaryote sources lacked 5-FCL activity in vitro and did not complement the growth defect or the characteristic 5-formyltetrahydrofolate accumulation of a 5-FCL-deficient (ΔygfA) Escherichia coli strain. We therefore propose (a) that COG0212 has an unrecognized yet sometimes crucial role in thiamin metabolism, most probably in salvage or detoxification, and (b) that is not a 5-FCL and should no longer be so annotated.

Evidence that glutamine transaminase and omega-amidase potentially act in tandem to close the methionine salvage cycle in bacteria and plants.

S-Adenosylmethionine is converted enzymatically and non-enzymatically to methylthioadenosine, which is recycled to methionine (Met) via a salvage pathway. In plants and bacteria, enzymes for all steps in this pathway are known except the last: transamination of α-ketomethylthiobutyrate to give Met. In mammals, glutamine transaminase K (GTK) and ω-amidase (ω-Am) are thought to act in tandem to execute this step, with GTK forming α-ketoglutaramate, which ω-Am hydrolyzes. Comparative genomics indicated that GTK and ω-Am could function likewise in plants and bacteria because genes encoding GTK and ω-Am homologs (i) co-express with the Met salvage gene 5-methylthioribose kinase in Arabidopsis, and (ii) cluster on the chromosome with each other and with Met salvage genes in diverse bacteria. Consistent with this possibility, tomato, maize, and Bacillus subtilis GTK and ω-Am homologs had the predicted activities: GTK was specific for glutamine as amino donor and strongly preferred α-ketomethylthiobutyrate as amino acceptor, and ω-Am strongly preferred α-ketoglutaramate. Also consistent with this possibility, plant GTK and ω-Am were localized to the cytosol, where the Met salvage pathway resides, as well as to organelles. This multiple targeting was shown to result from use of alternative start codons. In B. subtilis, ablating GTK or ω-Am had a modest but significant inhibitory effect on growth on 5-methylthioribose as sole sulfur source. Collectively, these data indicate that while GTK, coupled with ω-Am, is positioned to support significant Met salvage flux in plants and bacteria, it can probably be replaced by other aminotransferases.

Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway

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.

Experimental and Metabolic Modeling Evidence for a Folate-Cleaving Side-Activity of Ketopantoate Hydroxymethyltransferase (PanB)

Tetrahydrofolate (THF) and its one-carbon derivatives, collectively termed folates, are essential cofactors, but are inherently unstable. While it is clear that chemical oxidation can cleave folates or damage their pterin precursors, very little is known about enzymatic damage to these molecules or about whether the folate biosynthesis pathway responds adaptively to damage to its end-products. The presence of a duplication of the gene encoding the folate biosynthesis enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (FolK) in many sequenced bacterial genomes combined with a strong chromosomal clustering of the folK gene with panB, encoding the 5,10-methylene-THF-dependent enzyme ketopantoate hydroxymethyltransferase, led us to infer that PanB has a side activity that cleaves 5,10-methylene-THF, yielding a pterin product that is recycled by FolK. Genetic and metabolic analyses of Escherichia coli strains showed that overexpression of PanB leads to accumulation of the likely folate cleavage product 6-hydroxymethylpterin and other pterins in cells and medium, and—unexpectedly—to a 46% increase in total folate content. In silico modeling of the folate biosynthesis pathway showed that these observations are consistent with the in vivo cleavage of 5,10-methylene-THF by a side-activity of PanB, with FolK-mediated recycling of the pterin cleavage product, and with regulation of folate biosynthesis by folates or their damage products.

Drought stress in Pinus taeda L. induces coordinated transcript accumulation of genes involved in the homogentisate pathway

Phenylalanine is a central amino acid in plants and is the precursor of many key secondary metabolites such as lignin, phenylpropanoids, and flavonoids. Phenylalanine hydroxylase (PheH, EC 1.14.16.1) is not detected by sequence similarity in angiosperms, but it is present in gymnosperms and mosses where it is known to hydroxylate phenylalanine to produce tyrosine in vitro. However, its physiological role in gymnosperms is unclear. This study aimed to clarify the role of the gene encoding PheH in Pinus taeda (PtPheH), an economically and ecologically important tree native to the southern USA. The PtPheH gene is present as a single copy and consists of five exons and four introns. Comparison of two PtPheH alleles in P. taeda revealed a mobile element specific to the genus Pinus that was upstream of the transcriptional start site. PtPheH transcript abundance increased after introduction of exogenous phenylalanine and during water limitation. Similar highly and statistically significant shifts in transcript abundance were found for the genes involved in tyrosine synthesis and in the homogentisate pathway, but not for genes of phenylpropanoid metabolism. The coordinated accumulation of transcripts implies that under conditions of limited water availability, PtPheH may reroute phenylalanine away from lignin synthesis and into the degradative homogentisate pathway. Because PtPheH has homologs in pine, moss, and animals, it is presumably an ancient gene that has been lost in the angiosperm lineage of plants.