Plants, unlike other higher eukaryotes, possess all the necessary enzymatic equipment for de novo synthesis of methionine, an amino acid that supports additional roles than simply serving as a building block for protein synthesis. This is because methionine is the immediate precursor of S -adenosylmethionine (AdoMet), which plays numerous roles of being the major methyl-group donor in transmethylation reactions and an intermediate in the biosynthesis of polyamines and of the phytohormone ethylene. In addition, AdoMet has regulatory function in plants behaving as an allosteric activator of threonine synthase. Among the AdoMet-dependent reactions occurring in plants, methylation of cytosine residues in DNA has raised recent interest because impediment of this function alters plant morphology and induces homeotic alterations in flower organs. Also, AdoMet metabolism seems somehow implicated in plant growth via an as yet fully understood link with plant-growth hormones such as cytokinins and auxin and in plant pathogen interactions. Because of this central role in cellular metabolism, a precise knowledge of the biosynthetic pathways that are responsible for homeostatic regulation of methionine and AdoMet in plants has practical implications, particularly in herbicide design.

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 B(12)-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.

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.

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 K _m of the enzyme for Met. Second, [ ¹³ C]-NMR profiling of Arabidopsis cells fed with [ ¹³ C]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.

Plastids are semiautonomous organelles derived from cyanobacterial ancestors. Following endosymbiosis, plastids have evolved to optimize their functions, thereby limiting metabolic redundancy with other cell compartments. Contemporary plastids have also recruited proteins produced by the nuclear genome of the host cell. In addition, many genes acquired from the cyanobacterial ancestor evolved to code for proteins that are targeted to cell compartments other than the plastid. Consequently, metabolic pathways are now a patchwork of enzymes of diverse origins, located in various cell compartments. Because of this, a wide range of metabolites and ions traffic between the plastids and other cell compartments. In this review, we provide a comprehensive analysis of the well-known, and of the as yet uncharacterized, chloroplast/cytosol exchange processes, which can be deduced from what is currently known about compartmentation of plant-cell metabolism.

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.