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) Methionine is the only sulfur-containing amino acid that is essential for mammals and must therefore be derived entirely from the diet. In contrast, methionine is synthesized de novo by plants and most microorganisms after the initial steps of inorganic sulfate assimilation and cysteine or homocysteine (Hcy) syntheses (1-4). Because of its central importance in cellular metabolism, the metabolic sequence ensuring the conversion of cysteine into methionine has been extensively studied in enteric bacteria. The pioneer work of John Giovanelli (5) established that the enzymatic reactions leading to methionine were similar in the plant kingdom (1). In plants, as in bacteria, methionine belongs to the aspartate family of amino acids, which also comprises lysine, threonine, and isoleucine (1-4). These biosynthetic pathways deserve considerable attention for several reasons. First, the study of their biochemical and molecular control would provide new insights into the mechanisms involved in the homeostatic regulation of amino acids in plants. Second, these metabolic pathways give rise to essential amino acids that limit the nutritional quality of crop plants as diet for human beings and monogastric animals because seeds of cereals and legumes are deficient in lysine and methionine, respectively (6). Third, the inhibition of acetolactate synthase, an enzyme involved in the biosynthesis of isoleucine, valine, and leucine, by sulfonylureas or imidazolinones is lethal for plants (7). Thus, it is anticipated that key regulatory enzymes of the aspartate-derived amino acid branches also would be suitable targets for efficient herbicides.,The synthesis of aspartate-derived amino acids as well as the assimilation of sulfate and the synthesis of sulfur amino acids in plants have been covered recently in several reviews (7-10). The present article is intended to serve three purposes. The first is to provide a general background on the physiology of methionine synthesis in higher plants. The second is to highlight some recent findings linked to the metabolism of Sadenosylmethionine (AdoMet) in plants due to its regulatory influence on the aspartate pathway and its implication in plant growth and plant-pathogen interactions. The third is to present and discuss an integrative view of our present understanding of the functioning of the methionine and AdoMet biosynthetic͞recycling pathways, notably in relation with the unique compartmentation of metabolism in higher plants. The trends that emerge here open new research directions for the study of these essential metabolites in plants.De Novo Synthesis of Methionine. The methionine molecule originates from three convergent pathways: the carbon backbone deriving from...

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 ␥-glutamylconjugated 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.

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.The synthesis of numerous biological compounds and the regulation of many metabolic processes require the addition or removal of one-carbon units (C1 metabolism). Tetrahydrofolate (THF) coenzymes mediate these C1 transfer reactions that are involved in several major cellular processes, including the synthesis of purines and thymidylate, amino acid metabolism, pantothenate synthesis, mitochondrial and chloroplastic protein biogenesis, and Met synthesis (Fig. 1). Met is the direct precursor of S-adenosyl-Met (Ado-Met), which in turn is the source of methyl units for the synthesis of a myriad of molecules such as choline, chlorophyll, or lignin (for reviews, see Cossins, 2000;Scott et al., 2000;Hanson and Roje, 2001). In plants, THF is also involved in the photorespiratory cycle, a specific pathway that occurs at very high rates in green leaves from C3 plants. Photorespiration relies on two THF-dependent enzymes present in the matrix space of leaf mitochondria, the Gly decarboxylase complex (GDC) and Ser hydroxymethyltransferase (SHMT; for reviews, see Oliver, 1994;Douce et al., 2001).THF is composed of three distinct parts, namely a pterin ring, a p-aminobenzoic acid, and a poly-Glu chain of variable length (1-8 residues). Its function is to bind, transport, and donate C1 units that differ i...

It is not known how plants synthesize the p-aminobenzoate (PABA) moiety of folates. In Escherichia coli, PABA is made from chorismate in two steps. First, the PabA and PabB proteins interact to catalyze transfer of the amide nitrogen of glutamine to chorismate, forming 4-amino-4-deoxychorismate (ADC). The PabC protein then mediates elimination of pyruvate and aromatization to give PABA. Fungi, actinomycetes, and Plasmodium spp. also synthesize PABA but have proteins comprising fused domains homologous to PabA and PabB. These bipartite proteins are commonly called ''PABA synthases,'' although it is unclear whether they produce PABA or ADC. Genomic approaches identified Arabidopsis and tomato cDNAs encoding bipartite proteins containing fused PabA and PabB domains, plus a putative chloroplast targeting peptide. These cDNAs encode functional enzymes, as demonstrated by complementation of an E. coli pabA pabB double mutant and a yeast PABA-synthase deletant. The partially purified recombinant Arabidopsis protein did not produce PABA unless the E. coli PabC enzyme was added, indicating that it forms ADC, not PABA. The enzyme behaved as a monomer in size-exclusion chromatography and was not inhibited by physiological concentrations of PABA, its glucose ester, or folates. When the putative targeting peptide was fused to GFP and expressed in protoplasts, the fusion protein appeared only in chloroplasts, indicating that PABA synthesis is plastidial. In the pericarp of tomato fruit, the PabA-PabB mRNA level fell drastically as ripening advanced, but there was no fall in total PABA content, which stayed between 0.7 and 2.3 nmol⅐g ؊1 fresh weight.

Cystathionine gamma-synthase catalyses the first reaction specific for methionine biosynthesis in plants, the gamma-replacement of the phosphoryl substituent of O-phosphohomoserine by cysteine. A cDNA encoding cystathionine gamma-synthase from Arabidopsis thaliana has been cloned and used to overexpress the enzyme in Escherichia coli. The native recombinant enzyme is a homotetramer composed of 53 kDa subunits, each being tightly associated with one molecule of pyridoxal 5'-phosphate that binds at lysine-379 of the protein precursor. The replacement reaction follows a Ping Pong mechanism with a Vmax of 33.6 units/mg and Km values of 2.5 mM and 460 microM for O-phosphohomoserine and cysteine respectively. The protective effect of O-phosphohomoserine against enzyme inactivation by propargylglycine indicated that the Kd for the substrate is approx. 1/2500 of its Km value. Thus most of these biochemical properties are similar to those previously reported for plant and bacterial cystathionine gamma-synthases. However, the plant enzyme differs markedly from its enterobacterial counterparts because it catalyses a very faint gamma-elimination of O-phosphohomoserine in the absence of cysteine, this process being about 1/2700 as fast as the gamma-replacement reaction and approx. 1/1500 as fast as the gamma-elimination catalysed by the E. coli enzyme. This huge difference could be attributed to the inability of the A. thaliana cystathionine gamma-synthase to accumulate a long-wavelength-absorbing species that is characteristic for the efficient gamma-elimination reaction catalysed by the enterobacterial enzyme.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b 6 f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b 6 f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b 6 f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LH-CII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.State transition has been originally described as a process whereby organisms performing oxygenic photosynthesis respond to changes in the spectral quality of light by changing the relative sizes of their photosystem I (PSI) 1 and photosystem II (PSII) antennae (for review, see Ref. 1). This leads to a redistribution of excitation energy between these two photosystems, which optimizes the photosynthetic yield. The reorganization of the antennae involves the displacement of the mobile fraction of a peripheral antenna complex, the light-harvesting antenna of photosystem II (LHCII), within the thylakoid membrane. During transition from state I to state II induced by PSII light (650 nm), the mobile fraction of LHCII is displaced from the PSII-enriched grana region to the PSI-enriched stromal lamellae. In the presence of PSI light (700 nm), the reverse migration of LHCII from PSI to PSII occurs. The redistribution of excitation energy between the photosystems results in changes in room temperature fluorescence with high and low fluorescence levels in state I and II, respectively, and in drastic alterations of the fluorescence emission spectrum at low temperature. As a consequence of this redistribution of energy between the two photosystems, state transition regulates the...

methionine ␥-lyase ͉ NMR profiling ͉ plant T he sulfur-containing amino acid methionine (Met) is an essential metabolite in all living organisms (1). Besides its role as protein constituent, Met is the precursor of S-adenosylmethionine (AdoMet), which is the major methyl-group donor in transmethylation reactions and an intermediate in the biosynthesis of biotin, polyamines, and the phytohormone ethylene. Met is synthesized de novo by plants, fungi, and microbes. It is the only sulfur-containing amino acid that is essential for human and monogastric livestock. In these organisms, Met is metabolized through the reverse transsulfuration pathway where the ␥-cleavage of the cystathionine intermediate, a reaction that does not exist in plants, allows synthesis of cysteine (Cys) (2). Therefore, Met must be supplied from the diet and, because it is the most limiting essential amino acid in legume seeds, metabolic engineering strategies have been developed to increase the carbon flux into free Met as well as the incorporation of this amino acid into proteins (3).In higher plants, the neo-synthesized Met molecule originates from three convergent pathways with the sulfur atom deriving from Cys, the nitrogen͞carbon backbone from aspartate, and the methyl moiety from the ␤-carbon of serine via the pool of folates (1). De novo Met synthesis consists of three consecutive reactions localized in plastids (4) and catalyzed by cystathionine ␥-synthase, cystathionine ␤-lyase, and methionine synthase. Several recent studies have indicated that Met synthesis and accumulation are subject to complex regulatory controls in which cystathionine ␥-synthase plays a crucial role (5-7).Studies of metabolic fates of Met using the aquatic plant Lemna paucicostata indicated that the synthesis and turnover of AdoMet accounts for Ϸ80% of Met metabolism, whereas the synthesis of proteins drives Ϸ20% of Met (for a review, see ref. 8). More than 90% of AdoMet then is used for transmethylation, leading to nucleic acid, protein, lipid, and other metabolites modifications. The resulting homocysteinyl moiety is recycled back to Met and AdoMet by a set of cytosolic reactions designated as the activated methyl cycle. The use of AdoMet for the synthesis of polyamines and, in some plant tissues, ethylene also is accompanied by recycling of the methylthio moiety and regeneration of Met. The last known metabolic fate of Met is unique to plants and leads to the production of S-methylmethionine (SMM), a compound resulting from the AdoMet-dependent methylation of Met. SMM then can donate a methyl group to homocysteine (Hcy) through the reaction catalyzed by Hcy S-methyltransferase, yielding two molecules of Met. The SMM cycle might serve to achieve short-term control of AdoMet levels in plant cells, thus controlling the commitment of one-carbon units into methyl-group synthesis (9-11).Despite recent progress in elucidating the synthesis of Met and its complex regulation, little is known about the catabolism of this amino acid in plant cells. Several authors have ...

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