Plant Folate Research Articles

Evolution of folate biosynthesis and metabolism across algae and land plant lineages

Tetrahydrofolate and its derivatives, commonly known as folates, are essential for almost all living organisms. Besides acting as one-carbon donors and acceptors in reactions producing various important biomolecules such as nucleic and amino acids, as well as pantothenate, they also supply one-carbon units for methylation reactions. Plants along with bacteria, yeast and fungi synthesize folates de novo and therefore constitute a very important dietary source of folates for animals. All the major steps of folate biosynthesis and metabolism have been identified but only few have been genetically characterized in a handful of model plant species. The possible differences in the folate pathway between various plant and algal species have never been explored. In this study we present a comprehensive comparative study of folate biosynthesis and metabolism of all major land plant lineages as well as green and red algae. The study identifies new features of plant folate metabolism that might open new directions to folate research in plants.

Bioengineering of crop plants for improved tetrahydrofolate production

ABSTRACTDe novo synthesis of folates in plants is tightly regulated through feedback-regulation of certain pathway catalysts. Recently, we investigated the prospects of incessant production of folates in an evolutionary conjunction, through the overexpression of feedback targeted and evolutionarily conserved heterologous E.coli dihydroneopterin aldolase (EcDHNA) in tobacco.1 The enhanced production of folates in the transgenic lines was associated with differential allosteric regulatory cavities accessible at EcDHNA surface having critical amino-acid differences as Ile 64 (His_63), Val 70 (Phe_69), His 75 (Arg_78) and Arg 79 (Glu_72). These structural characteristics are indicative of evolutionary signatures of the catalytic feedback-regulation of folate manufacturing. We exploited the biotechnological potential of such allosterically diverged trans-DHNA for improved folate production in plants. Nonetheless, genetic manipulation of single enzymes modulating complex pathways such as folate biosynthesis is often inadequate to achieve desired phenotypes; therefore, multi-gene integration with explicit genic-combination for folate enrichment in plants has also been projected for future folate agri-biofortification schemes.

Folates in Plants: Research Advances and Progress in Crop Biofortification.

Folates, also known as B9 vitamins, serve as donors and acceptors in one-carbon (C1) transfer reactions. The latter are involved in synthesis of many important biomolecules, such as amino acids, nucleic acids and vitamin B5. Folates also play a central role in the methyl cycle that provides one-carbon groups for methylation reactions. The important functions fulfilled by folates make them essential in all living organisms. Plants, being able to synthesize folates de novo, serve as an excellent dietary source of folates for animals that lack the respective biosynthetic pathway. Unfortunately, the most important staple crops such as rice, potato and maize are rather poor sources of folates. Insufficient folate consumption is known to cause severe developmental disorders in humans. Two approaches are employed to fight folate deficiency: pharmacological supplementation in the form of folate pills and biofortification of staple crops. As the former approach is considered rather costly for the major part of the world population, biofortification of staple crops is viewed as a decent alternative in the struggle against folate deficiency. Therefore, strategies, challenges and recent progress of folate enhancement in plants will be addressed in this review. Apart from the ever-growing need for the enhancement of nutritional quality of crops, the world population faces climate change catastrophes or environmental stresses, such as elevated temperatures, drought, salinity that severely affect growth and productivity of crops. Due to immense diversity of their biochemical functions, folates take part in virtually every aspect of plant physiology. Any disturbance to the plant folate metabolism leads to severe growth inhibition and, as a consequence, to a lower productivity. Whereas today's knowledge of folate biochemistry can be considered very profound, evidence on the physiological roles of folates in plants only starts to emerge. In the current review we will discuss the implication of folates in various aspects of plant physiology and development.

Sulfamethazine Suppresses Epigenetic Silencing in Arabidopsis by Impairing Folate Synthesis

DNA methylation is a critical, dynamically regulated epigenetic mark. Small chemicals can be valuable tools in probing cellular processes, but the set of chemicals with broad effects on epigenetic regulation is very limited. Using the Arabidopsis thaliana repressor of silencing1 mutant, in which transgenes are transcriptionally silenced, we performed chemical genetic screens and found sulfamethazine (SMZ) as a chemical suppressor of epigenetic silencing. SMZ treatment released the silencing of transgenes as well as endogenous transposons and other repetitive elements. Plants treated with SMZ exhibit substantially reduced levels of DNA methylation and histone H3 Lys-9 dimethylation, but heterochromatic siRNA levels were not affected. SMZ is a structural analog and competitive antagonist to p-aminobenzoic acid (PABA), which is a precursor of folates. SMZ decreased the plant folate pool size and caused methyl deficiency, as demonstrated by reductions in S-adenosylmethionine levels and in global DNA methylation. Exogenous application of PABA or compounds downstream in the folate biosynthesis pathway restored transcriptional silencing in SMZ-treated plants. Together, our results revealed a novel type of chemical suppressor of epigenetic silencing, which may serve as a valuable tool for studying the roles and mechanisms of epigenetic regulation and underscores an important linkage between primary metabolism and epigenetic gene regulation.

Folate Biosynthesis, Turnover, and Transport in Plants

Folates are essential cofactors for one-carbon transfer reactions and are needed in the diets of humans and animals. Because plants are major sources of dietary folate, plant folate biochemistry has long been of interest but progressed slowly until the genome era. Since then, genome-enabled approaches have brought rapid advances: We now know (a) all the plant folate synthesis genes and some genes of folate turnover and transport, (b) certain mechanisms governing folate synthesis, and (c) the subcellular locations of folate synthesis enzymes and of folates themselves. Some of this knowledge has been applied, simply and successfully, to engineer folate-enriched food crops (i.e., biofortification). Much remains to be discovered about folates, however, particularly in relation to homeostasis, catabolism, membrane transport, and vacuolar storage. Understanding these processes, which will require both biochemical and -omics research, should lead to improved biofortification strategies based on transgenic or conventional approaches.

A central role for gamma-glutamyl hydrolases in plant folate homeostasis

Most cellular folates carry a short poly-γ-glutamate tail, and this tail is believed to affect their efficacy and stability. The tail can be removed by γ-glutamyl hydrolase (GGH; EC 3.4.19.9), a vacuolar enzyme whose role in folate homeostasis remains unclear. In order to probe the function of GGH, we modulated its level of expression and subcellular location in Arabidopsis plants and tomato fruit. Three-fold overexpression of GGH in vacuoles caused extensive deglutamylation of folate polyglutamates and lowered the total folate content by approximately 40% in Arabidopsis and tomato. No such effects were seen when GGH was overexpressed to a similar extent in the cytosol. Ablation of either of the major Arabidopsis GGH genes (AtGGH1 and AtGGH2) alone did not significantly affect folate status. However, a combination of ablation of one gene plus RNA interference (RNAi)-mediated suppression of the other (which lowered total GGH activity by 99%) increased total folate content by 34%. The excess folate accumulated as polyglutamate derivatives in the vacuole. Taken together, these results suggest a model in which: (i) folates continuously enter the vacuole as polyglutamates, accumulate there, are hydrolyzed by GGH, and exit as monoglutamates; and (ii) GGH consequently has an important influence on polyglutamyl tail length and hence on folate stability and cellular folate content.

Folate biofortification of tomato fruit

Folate deficiency leads to neural tube defects and other human diseases, and is a global health problem. Because plants are major folate sources for humans, we have sought to enhance plant folate levels (biofortification). Folates are synthesized from pteridine, p-aminobenzoate (PABA), and glutamate precursors. Previously, we increased pteridine production in tomato fruit up to 140-fold by overexpressing GTP cyclohydrolase I, the first enzyme of pteridine synthesis. This strategy increased folate levels 2-fold, but engineered fruit were PABA-depleted. We report here the engineering of fruit-specific overexpression of aminodeoxychorismate synthase, which catalyzes the first step of PABA synthesis. The resulting fruit contained an average of 19-fold more PABA than controls. When transgenic PABA- and pteridine-overproduction traits were combined by crossing, vine-ripened fruit accumulated up to 25-fold more folate than controls. Folate accumulation was almost as high (up to 15-fold) in fruit harvested green and ripened by ethylene-gassing, as occurs in commerce. The accumulated folates showed normal proportions of one-carbon forms, with 5-methyltetrahydrofolate the most abundant, but were less extensively polyglutamylated than controls. Folate concentrations in developing fruit did not change in controls, but increased continuously throughout ripening in transgenic fruit. Pteridine and PABA levels in transgenic fruit were >20-fold higher than in controls, but the pathway intermediates dihydropteroate and dihydrofolate did not accumulate, pointing to a flux constraint at the dihydropteroate synthesis step. The folate levels we achieved provide the complete adult daily requirement in less than one standard serving.

Comparative genomics of bacterial and plant folate synthesis and salvage: predictions and validations

BackgroundFolate synthesis and salvage pathways are relatively well known from classical biochemistry and genetics but they have not been subjected to comparative genomic analysis. The availability of genome sequences from hundreds of diverse bacteria, and from Arabidopsis thaliana, enabled such an analysis using the SEED database and its tools. This study reports the results of the analysis and integrates them with new and existing experimental data.ResultsBased on sequence similarity and the clustering, fusion, and phylogenetic distribution of genes, several functional predictions emerged from this analysis. For bacteria, these included the existence of novel GTP cyclohydrolase I and folylpolyglutamate synthase gene families, and of a trifunctional p-aminobenzoate synthesis gene. For plants and bacteria, the predictions comprised the identities of a 'missing' folate synthesis gene (folQ) and of a folate transporter, and the absence from plants of a folate salvage enzyme. Genetic and biochemical tests bore out these predictions.ConclusionFor bacteria, these results demonstrate that much can be learnt from comparative genomics, even for well-explored primary metabolic pathways. For plants, the findings particularly illustrate the potential for rapid functional assignment of unknown genes that have prokaryotic homologs, by analyzing which genes are associated with the latter. More generally, our data indicate how combined genomic analysis of both plants and prokaryotes can be more powerful than isolated examination of either group alone.

Folates in plants: biosynthesis, distribution, and enhancement

Folates are crucial intermediates for a set of reactions that involve the transfer of single‐carbon units (C1 metabolism). They are directly involved in the synthesis of nucleic acids, methionine, pantothenate, glycine and serine, and indirectly, through S‐adenosyl methionine, in all methylation reactions. Humans cannot synthesize folates de novo. In these organisms, folate deficiency has severe effects on health and affects large population groups around the world. Because plants are the main source of dietary folates, there are great concerns to select plant food having high concentrations of folates or to engineer their folate metabolism to increase the initial amount. All these attempts rely on what we know about the metabolism of folates. During these last 10 years, the complex pathway leading to the synthesis of folates has been deciphered. Our knowledge about folate synthesis and distribution during plant growth and development also increased substantially. However, important aspects of folate metabolism remain unclear, such as catabolism, transport and regulation of the homeostasis. The aim of this review was to summarize our recent findings, to describe the few attempts reported in the literature to engineer folate level in plants, and to discuss potential strategies that could be used for enhancement.

Free and total para‐aminobenzoic acid analysis in plants with high‐performance liquid chromatography/tandem mass spectrometry

para-Aminobenzoic acid (PABA), a precursor in the synthesis of folates in plants, is determined by liquid chromatography/tandem mass spectrometry (LC/MS/MS). In plants PABA can be converted into its beta-D-glucopyranosyl ester (PABA-Glc) and can also exist in its free form. In this work, we developed and validated a quantitative method to study free and total PABA in plants. The total PABA (free PABA plus PABA-Glc) can be evaluated after acid hydrolysis at 80 degrees C for 2 hours. The plant material is homogenized and the PABA content is quantified using the standard addition procedure. The validated method is selective, sensitive, simple, accurate, has a recovery between 99.6 to 102.5%, is reproducible (RSD between 1.4 and 4.4%), and is linear between 2.5 and 1538 ng/mL. Free and total PABA determinations in five vegetables showed that different plant species had different amounts of free and total PABA, and that the ratios of total versus free PABA were also variable. This new method could be valuable for studies of folate synthesis in plants.

Folate Synthesis and Metabolism in Plants and Prospects For Biofortification

Folates are essential cofactors for one‐carbon transfer reactions in most living organisms and are required for the biosynthesis of nucleic acids, amino acids, and pantothenate. Unlike plants and microorganisms, humans cannot synthesize folates de novo and must acquire them from the diet, primarily from plant foods. However, lack of folates is the most common vitamin deficiency in the world and has serious health consequences, including increased risk of neural tube defects in infants, cancers, and vascular diseases. Consequently, there is much interest in engineering plants with enhanced folate content (biofortification). In this review, we outline progress in defining the plant folate synthesis pathway and its unique compartmentation and point out sectors of folate metabolism that have yet to be elucidated, including transport and catabolism. We also consider possible strategies to enhance plant folate synthesis and accumulation by metabolic engineering.

Enhancement of folates in plants through metabolic engineering.

Humans depend on plants as a major source of dietary folates. Inadequate dietary levels of the vitamin folate can lead to megaloblastic anemia, birth defects, impaired cognitive development, and increased risk of cardiovascular disease and cancer. The biofortification of folate levels in food crops is a target for metabolic engineering. Folates are synthesized de novo from pterins and para-amino benzoic acid, which are subsequently combined to form dihydropteroate, the direct precursor to dihydrofolate. We postulated that GTP cyclohydrolase-1, which catalyzes the first committed step in pterin biosynthesis, was a rate-limiting step in pterin synthesis in plants and, therefore, in folate synthesis. On this basis, we proposed that the expression of an unregulated bacterial GTP cyclohydrolase-1 in plants would increase pterin biosynthesis with a concomitant enhancement of folate levels. The folE gene encoding GTP cyclohydrolase-1 was cloned from Escherichia coli and introduced into Arabidopsis thaliana through plant transformation. The expression of bacterial GTP cyclohydrolase-1 in transgenic Arabidopsis resulted in a 1,250-fold and 2- to 4-fold enhancement of pterins and folates, respectively. These results helped to identify other potential factors regulating folate synthesis, suggesting ways to further enhance folate levels in food crops.

Folic acid and folates: the feasibility for nutritional enhancement in plant foods

In mammalian and plant cells the folates play a key role in the methylation cycle and in the DNA biosynthesis cycle (in the de novo biosynthesis of purines and pyrimidines). Deficiency of folate in the diet is likely to result in a reduction in the capacity to synthesise DNA and maintain the usual rate of cell division. This most evidently results in the production of anaemia from folate deficiency due to a reduction in the biosynthesis of cells in the bone marrow. In addition it has been shown that high levels of plasma homocysteine occur which is an important risk factor in cardiovascular disease. Furthermore it has been shown that reduced maternal folate status is associated with the progressive increase in neural tube defects in infants. There is good evidence for folate deficiency in a considerable number of the population in developed countries, even where folate supplementation of foods is practised, and for a level of intake in excess of the recommended dietary allowance. This paper reviews the principal routes of folate biosynthesis in plants and the potential for increasing the levels of natural folates through conventional plant breeding and biotechnological means. The effect of the major food preservation processes on the levels of folates in plants is also reviewed. The current information about the bioavailability of folates from plant food sources, and how this might be improved, is also summarised. The important health benefits that would arise from increasing folate intake in the diet provide a strong incentive for considering how this might be achieved. © 2000 Society of Chemical Industry