Fabrice Rébeillé

The glycine decarboxylase system: a fascinating complex

The mitochondrial glycine decarboxylase multienzyme system, connected to serine hydroxymethyltransferase through a soluble pool of tetrahydrofolate, consists of four different component enzymes, the P-, H-, T- and L-proteins. In a multi-step reaction, it catalyses the rapid destruction of glycine molecules flooding out of the peroxisomes during the course of photorespiration. In green leaves, this multienzyme system is present at tremendously high concentrations within the mitochondrial matrix. The structure, mechanism and biogenesis of glycine decarboxylase are discussed. In the catalytic cycle of glycine decarboxylase, emphasis is given to the lipoate-dependent H-protein that plays a pivotal role, acting as a mobile substrate that commutes successively between the other three proteins. Plant mitochondria possess all the necessary enzymatic equipment for de novo synthesis of tetrahydrofolate and lipoic acid, serving as cofactors for glycine decarboxylase and serine hydroxymethyltransferase functioning.

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

Methionine metabolism in plants: chloroplasts are autonomous for de novo methionine synthesis and can import S-adenosylmethionine from the cytosol.

The subcellular distribution of Met and S-adenosylmethionine (AdoMet) metabolism in plant cells discloses a complex partition between the cytosol and the organelles. In the present work we show that Arabidopsis contains three functional isoforms of vitamin B12-independent methionine synthase (MS), the enzyme that catalyzes the methylation of homocysteine to Met with 5-methyltetrahydrofolate as methyl group donor. One MS isoform is present in chloroplasts and is most likely required to methylate homocysteine that is synthesized de novo in this compartment. Thus, chloroplasts are autonomous and are the unique site for de novo Met synthesis in plant cells. The additional MS isoforms are present in the cytosol and are most probably involved in the regeneration of Met from homocysteine produced in the course of the activated methyl cycle. Although Met synthesis can occur in chloroplasts, there is no evidence that AdoMet is synthesized anywhere but the cytosol. In accordance with this proposal, we show that AdoMet is transported into chloroplasts by a carrier-mediated facilitated diffusion process. This carrier is able to catalyze the uniport uptake of AdoMet into chloroplasts as well as the exchange between cytosolic AdoMet and chloroplastic AdoMet or S-adenosylhomocysteine. The obvious function for the carrier is to sustain methylation reactions and other AdoMet-dependent functions in chloroplasts and probably to remove S-adenosylhomocysteine generated in the stroma by methyltransferase activities. Therefore, the chloroplastic AdoMet carrier serves as a link between cytosolic and chloroplastic one-carbon metabolism.

ATP control on state transitions in vivo in Chlamydomonas reinhardtii

Abstract We have attempted to depress the ATP content in dark adapted cells of the unicellular green algae, C. reinhardtii, by inhibiting ATP synthesis coupled to mitochondrial electron flow. Whether we used uncouplers, ATP synthase inhibitors, inhibitors of mitochondrial electron transport or a mutant altered in mitochondrial cytochrome b, we observed a fluorescence quenching at room temperature associated with an increased reduction of the intersystem electron carriers of the photosynthetic apparatus. This fluorescence quenching reflected a genuine transition to state II, since: (i) it was associated with an increase in light-harvesting complex phosphorylation and (ii) it did not occur in mutants lacking the b 6 f complex which are blocked in State I. The complete reversion to state I occurred only into experimental conditions which allowed both ATP synthesis and reoxidation of intersystem electron carriers. We conclude that the intracellular demand for ATP controls state transitions in vivo. The interplay between intracellular ATP concentrations and the redox state of the kinase activator at the thylakoid membrane level is discussed.

Folate biosynthesis in higher plants: purification and molecular cloning of a bifunctional 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase/7,8‐dihydropteroate synthase localized in mitochondria

In pea leaves, the synthesis of 7,8‐dihydropteroate, a primary step in folate synthesis, was only detected in mitochondria. This reaction is catalyzed by a bifunctional 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase/7,8‐dihydropteroate synthase enzyme, which represented 0.04–0.06% of the matrix proteins. The enzyme had a native mol. wt of 280–300 kDa and was made up of identical subunits of 53 kDa. The reaction catalyzed by the 7,8‐dihydropteroate synthase domain of the protein was Mg2+‐dependent and behaved like a random bireactant system. The related cDNA contained an open reading frame of 1545 bp and the deduced amino acid sequence corresponded to a polypeptide of 515 residues with a calculated Mr of 56 454 Da. Comparison of the deduced amino acid sequence with the N‐terminal sequence of the purified protein indicated that the plant enzyme is synthesized with a putative mitochondrial transit peptide of 28 amino acids. The calculated Mr of the mature protein was 53 450 Da. Southern blot experiments suggested that a single‐copy gene codes for the enzyme. This result, together with the facts that the protein is synthesized with a mitochondrial transit peptide and that the activity was only detected in mitochondria, strongly supports the view that mitochondria is the major (unique?) site of 7,8‐dihydropteroate synthesis in higher plant cells.

Methionine catabolism in Arabidopsis cells is initiated by a γ-cleavage process and leads to S-methylcysteine and isoleucine syntheses

Despite recent progress in elucidating the regulation of methionine (Met) synthesis, little is known about the catabolism of this amino acid in plants. In this article, we present several lines of evidence indicating that the cleavage of Met catalyzed by Met γ-lyase is the first step in this process. First, we cloned an Arabidopsis cDNA coding a functional Met γ-lyase (AtMGL), a cytosolic enzyme catalyzing the conversion of Met into methanethiol, α-ketobutyrate, and ammonia. AtMGL is present in all of the Arabidopsis organs and tissues analyzed, except in quiescent dry mature seeds, thus suggesting that AtMGL is involved in the regulation of Met homeostasis in various situations. Also, we demonstrated that the expression of AtMGL was induced in Arabidopsis cells in response to high Met levels, probably to bypass the elevated Km of the enzyme for Met. Second, [13C]-NMR profiling of Arabidopsis cells fed with [13C]Met allowed us to identify labeled S-adenosylmethionine, S-methylmethionine, S-methylcysteine (SMC), and isoleucine (Ile). The unexpected production of SMC and Ile was directly associated to the function of Met γ-lyase. Indeed, we showed that part of the methanethiol produced during Met cleavage could react with an activated form of serine to produce SMC. The second product of Met cleavage, α-ketobutyrate, entered the pathway of Ile synthesis in plastids. Together, these data indicate that Met catabolism in Arabidopsis cells is initiated by a γ-cleavage process and can result in the formation of the essential amino acid Ile and a potential storage form for sulfide or methyl groups, SMC.

Tetrahydrofolate biosynthesis in plants: Molecular and functional characterization of dihydrofolate synthetase and three isoforms of folylpolyglutamate synthetase in Arabidopsis thaliana

Tetrahydrofolate coenzymes involved in one-carbon (C1) metabolism are polyglutamylated. In organisms that synthesize tetrahydrofolate de novo, dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) catalyze the attachment of glutamate residues to the folate molecule. In this study we isolated cDNAs coding a DHFS and three isoforms of FPGS from Arabidopsis thaliana. The function of each enzyme was demonstrated by complementation of yeast mutants deficient in DHFS or FPGS activity, and by measuring in vitro glutamate incorporation into dihydrofolate or tetrahydrofolate. DHFS is present exclusively in the mitochondria, making this compartment the sole site of synthesis of dihydrofolate in the plant cell. In contrast, FPGS is present as distinct isoforms in the mitochondria, the cytosol, and the chloroplast. Each isoform is encoded by a separate gene, a situation that is unique among eukaryotes. The compartmentation of FPGS isoforms is in agreement with the predominance of γ-glutamyl-conjugated tetrahydrofolate derivatives and the presence of serine hydroxymethyltransferase and C1-tetrahydrofolate interconverting enzymes in the cytosol, the mitochondria, and the plastids. Thus, the combination of FPGS with these folate-mediated reactions can supply each compartment with the polyglutamylated folate coenzymes required for the reactions of C1 metabolism. Also, the multicompartmentation of FPGS in the plant cell suggests that the transported forms of folate are unconjugated.

Glycerolipids in photosynthesis: Composition, synthesis and trafficking☆

Glycerolipids constituting the matrix of photosynthetic membranes, from cyanobacteria to chloroplasts of eukaryotic cells, comprise monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol and phosphatidylglycerol. This review covers our current knowledge on the structural and functional features of these lipids in various cellular models, from prokaryotes to eukaryotes. Their relative proportions in thylakoid membranes result from highly regulated and compartmentalized metabolic pathways, with a cooperation, in the case of eukaryotes, of non-plastidic compartments. This review also focuses on the role of each of these thylakoid glycerolipids in stabilizing protein complexes of the photosynthetic machinery, which might be one of the reasons for their fascinating conservation in the course of evolution. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.

Relationship between the cytoplasm and the vacuole phosphate pool in Acer pseudoplatanus cells

The Pi concentration of Acer pseudoplatanus cells in the two major intracellular compartments, the cytoplasm and the vacuole, has been studied using 31P NMR. For sycamore cells containing approximately 2 mM of total Pi, the cytoplasmic Pi and the vacuolar Pi concentrations were approximately 6 and 1.5 mM, respectively. When the cells were transferred to a phosphate-deficient medium, the vacuolar Pi decreased rapidly while the cytoplasmic Pi decreased slowly during the first 48 h, indicating that Pi in the cytoplasm was maintained at the expense of the vacuolar Pi. When the Pi-starved cells (i.e., those containing less than 0.5 mumol of total Pi/g wet wt) were transferred to a medium containing 300 microM Pi, Pi entered the cells rapidly and accumulated in the cytoplasm. Once the cytoplasmic Pi pool was filled, Pi was taken up in the vacuole until the vacuole Pi pool was filled. On the contrary when the non-Pi-starved cells were transferred to a phosphate-rich medium (i.e., containing 45 mM Pi), Pi entered the cells slowly by diffusion and accumulated in the vacuole but not in the cytoplasm. These results demonstrate that the Pi content of the cytoplasm is maintained at the expense of the vacuolar Pi pool when sycamore cells are transferred to either a phosphate-deficient or a phosphate-rich medium.

One-Carbon Metabolism in Plants. Regulation of Tetrahydrofolate Synthesis during Germination and Seedling Development

Tetrahydrofolate (THF) is a central cofactor for one-carbon transfer reactions in all living organisms. In this study, we analyzed the expression of dihydropterin pyrophosphokinase-dihydropteroate synthase (HPPK-DHPS) in pea (Pisum sativum) organs during development, and so the capacity to synthesize dihydropteroate, an intermediate in the de novo THF biosynthetic pathway. During seedling development, all of the examined organs/tissues contain THF coenzymes, collectively termed folate, and express the HPPK-DHPS enzyme. This suggests that each organ/tissue is autonomous for the synthesis of THF. During germination, folate accumulates in cotyledons and embryos, but high amounts of HPPK-DHPS are only observed in embryos. During organ differentiation, folate is synthesized preferentially in highly dividing tissues and in photosynthetic leaves. This is associated with high levels of the HPPK-DHPS mRNA and protein, and a pool of folate 3- to 5-fold higher than in the rest of the plant. In germinating embryos and in meristematic tissues, the high capacity to synthesize and accumulate folate correlates with the general resumption of cell metabolism and the high requirement for nucleotide synthesis, major cellular processes involving folate coenzymes. The particular status of folate synthesis in leaves is related to light. Thus, when illuminated, etiolated leaves gradually accumulate the HPPK-DHPS enzyme and folate. This suggests that folate synthesis plays an important role in the transition from heterotrophic to photoautotrophic growth. Analysis of the intracellular distribution of folate in green and etiolated leaves indicates that the coenzymes accumulate mainly in the cytosol, where they can supply the high demand for methyl groups.