Regulation of One-Carbon Metabolism in Arabidopsis: The N-Terminal Regulatory Domain of Cystathionine -Synthase Is Cleaved in Response to Folate Starvation

Laboratoire de Physiologie Cellulaire Végétale, Institut National de la Recherche Agronomique, Université Joseph Fourier Grenoble I, Grenoble, France.
Plant physiology (Impact Factor: 6.84). 11/2007; 145(2):491-503. DOI: 10.1104/pp.107.105379
Source: PubMed


In all organisms, control of folate homeostasis is of vital importance to sustain the demand for one-carbon (C1) units that are essential in major metabolic pathways. In this study we induced folate deficiency in Arabidopsis (Arabidopsis thaliana) cells by using two antifolate inhibitors. This treatment triggered a rapid and important decrease in the pool of folates with significant modification in the distribution of C1-substituted folate coenzymes, suggesting an adaptive response to favor a preferential shuttling of the flux of C1 units to the synthesis of nucleotides over the synthesis of methionine (Met). Metabolic profiling of folate-deficient cells indicated important perturbation of the activated methyl cycle because of the impairment of Met synthases that are deprived of their substrate 5-methyl-tetrahydrofolate. Intriguingly, S-adenosyl-Met and Met pools declined during the initial period of folate starvation but were further restored to typical levels. Reestablishment of Met and S-adenosyl-Met homeostasis was concomitant with a previously unknown posttranslational modification that consists in the removal of 92 amino acids at the N terminus of cystathionine gamma-synthase (CGS), the first specific enzyme for Met synthesis. Rescue experiments and analysis of different stresses indicated that CGS processing is specifically associated with perturbation of the folates pool. Also, CGS processing involves chloroplastic serine-type proteases that are expressed in various plant species subjected to folate starvation. We suggest that a metabolic effector, to date unidentified, can modulate CGS activity in vivo through an interaction with the N-terminal domain of the enzyme and that removal of this domain can suppress this regulation.

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    • "Inhibitors of dihydrofolate reductase (DHFR; reaction 10 in Fig. 6) mimic pteridine or pyrimidine structures and are used as therapeutic agents for cancer (methotrexate), as antibiotics (trimethoprim), and, synergistically with sulphonamides , in treatments against parasites of the Apicomplexa phylum (pyrimethamine, cycloguanil). Note that methotrexate is also used to manipulate the folate and AdoMet pools in plants (Loizeau et al., 2007, 2008; Prabhu et al., 1998; van Wilder et al., 2009). "
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    ABSTRACT: Tetrahydrofolate and its derivatives, collectively termed folates or vitamin B9, are essential cofactors for one-carbon metabolism. They transport and donate C1-units for the synthesis of pantothenate, purines, thymidylate, serine, glycine, methionine and formylmethionyl-tRNA. Also, recent studies indicate that folates can act as electron donors in major cellular processes. Plants and many microorganisms synthesize folates de novo through a complex metabolic route that is now fully elucidated. In contrast, humans and other vertebrates lack a complete biosynthetic pathway and thus need dietary folates, of which plants are major sources. Folate deficiency is widespread in rich and developing countries and is associated with severe health problems. Supplementation of foods with synthetic folic acid and biofortification is an alternative strategy to fight folate deficiency. Encouraging pilot metabolic engineering studies in plants enabled significant enhancement of folate contents. In the next future, increasing our knowledge about the mechanisms controlling folates homeostasis in plants will provide the keys towards efficient biofortification of plant foods.
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    • "CH 3 - THF pool was selected for analysis in this experiment , as previous studies have indi - cated that , under folate deficiency , most folate - dependent transcriptional and metabolic alterations are correlated with the cellular levels of this folate species ( Loizeau et al . , 2007 , 2008"
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    ABSTRACT: Cellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis.
    The Plant Journal 10/2010; 64(2):267-79. DOI:10.1111/j.1365-313X.2010.04336.x · 5.97 Impact Factor
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    • "Taken as a whole, these results are indicative of important modifications within the methyl cycle activity, Hcy and AdoHcy concentrations being the most affected by the MTX treatment. Similar observations were previously reported for Arabidopsis cells having a folate concentration lowered by 25-fold (Loizeau et al., 2007). Our present data indicate that these events also occur with a moderate decrease of the folate content, suggesting a tight coupling between methyl cycle and folate synthesis. "
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    ABSTRACT: * Tetrahydrofolate derivatives are central cofactors of C1 metabolism. Using methotrexate as a specific inhibitor of folate biosynthesis, we altered the folate status in 10-d-old etiolated pea (Pisum sativum) leaves and followed the rate of chlorophyll synthesis upon illumination. * In our conditions, the folate concentration decreased only from 5.7 to 4.2 nmol g(-1) FW, but the amount of chlorophyll after 24 h of illumination was reduced 2.5 times. Folate status and rate of chlorophyll synthesis were apparently correlated through the methyl cycle. * Indeed, we observed that methyl-tetrahydrofolate was the folate derivative most affected by the treatment; the decrease of methyl-tetrahydrofolate was associated with a sharp rise in homocysteine and S-adenosylhomocysteine concentrations, which are normally maintained at very low values, shifting the methylation index (S-adenosylmethionine/S-adenosylhomocysteine ratio) from 7 to 1; the decrease of the methylation index reduced by a factor of 3 the activity of the Mg-protoporphyrin IX methyltransferase (CHLM), an essential enzyme for chlorophyll synthesis. CHLM gene expression and protein concentration remained unchanged, suggesting that this inhibition relied essentially on metabolic regulation. * These results point out that an even moderate change in the folate status may affect plant development and adaptation.
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