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...

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The last steps of cysteine synthesis in plants involve two consecutive enzymes. The first enzyme, serine acetyltransferase, catalyses the acetylation of L-serine in the presence of acetyl-CoA to form Oacetylserine. The second enzyme, O-acetylserine (thiol) lyase, converts O-acetylserine to L-cysteine in the presence of sulfide. We have, in the present work, over-produced in Escherichia coli harboring various type of plasmids, either a plant serine acetyltransferase or this enzyme with a plant O-acetylserine (thiol) lyase. The free recombinant serine acetyltransferase (subunit mass of 34 kDa) exhibited a high propensity to form high-molecular-mass aggregates and was found to be highly unstable in solution. However, these aggregates were prevented in the presence of O-acetylserine (thiol) lyase (subunit mass of 36 kDa). Under these conditions homotetrameric serine acetyltransferase associated with two molecules of homodimeric O-acetylserine (thiol) lyase to form a bienzyme complex (molecular mass Ϸ300 kDa) called cysteine synthase containing 4 mol pyridoxal 5′-phosphate/mol complex. O-Acetylserine triggered the dissociation of the bienzyme complex, whereas sulfide counteracted the action of O-acetylserine. ProteinϪprotein interactions within the bienzyme complex strongly modified the kinetic properties of plant serine acetyltransferase : there was a transition from a typical Michaelis-Menten model to a model displaying positive kinetic co-operativity with respect to serine and acetyl-CoA. On the other hand, the formation of the bienzyme complex resulted in a very dramatic decrease in the catalytic efficiency of bound O-acetylserine (thiol) lyase. The latter enzyme behaved as if it were a structural and/or regulatory subunit of serine acetyltransferase. Our results also indicated that bound serine acetyltransferase produces a build-up of Oacetylserine along the reaction path and that the full capacity for cysteine synthesis can only be achieved in the presence of a large excess of free O-acetylserine (thiol) lyase. These findings contradict the widely held belief that such a bienzyme complex is required to channel the metabolite intermediate O-acetylserine.Keywords : cysteine synthase; serine acetyltransferase; O-acetylserine (thiol) lyase; proteinϪprotein interactions; cysteine synthesis in plants.In metabolic networks, a fine tuning of enzyme activities is verted into the final reaction product (P). Here, accumulation of I might be prevented through properly adjusted concentrations required to maintain the rates at which useful end-products are produced with regard to those at which they are needed, but also of E 2 over E 1 , so that, at steady state, the net rate of I transformation through E 2 at least equals that of synthesis through E 1 . This to avoid accumulation of toxic internal metabolites and/or to achieve efficient trapping of highly unstable reaction intermedi-situation is analogous to that encountered in enzyme assays with auxiliary enzymes [1]. ates [1Ϫ3]. Considering the simple case of two enzymes ...

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Abstract. Autophagy triggered by carbohydrate starvation was characterized at both biochemical and structural levels, with the aim to identify reliable and easily detectable marker(s) and to investigate the factors controlling this process. Incubation of suspension cells in sucrose-free culture medium triggered a marked degradation of the membrane polar lipids, including phospholipids and galactolipids. In contrast, the total amounts of sterols, which are mainly associated with plasmalemma and tonoplast membranes, remained constant. In particular, phosphatidylcholine decreased, whereas phosphodiesters including glycerylphosphorylcholine transiently increased, and phosphorylcholine (P-Cho) steadily accumulated. P-Cho exhibits a remarkable metabolic inertness and therefore can be used as a reliable biochemical marker reflecting the extent of plant cell autophagy. Indeed, whenever P-Cho accumulated, a massive regression of cytoplasm was noticed using EM. Double membrane-bounded vacuoles were formed in the peripheral cytoplasm during sucrose starvation and were eventually expelled into the central vacuole, which increased in volume and squeezed the thin layer of cytoplasm spared by autophagy.The biochemical marker P-Cho was used to investigate the factors controlling autophagy. P-Cho did not accumulate when sucrose was replaced by glycerol or by pyruvate as carbon sources. Both compounds entered the cells and sustained normal rates of respiration. No recycling back to the hexose phosphates was observed, and cells were rapidly depleted in sugars and hexose phosphates, without any sign of autophagy. On the contrary, when pyruvate (or glycerol) was removed from the culture medium, P-Cho accumulated without a lag phase, in correlation with the formation of autophagic vacuoles. These results strongly suggest that the supply of mitochondria with respiratory substrates, and not the decrease of sucrose and hexose phosphates, controls the induction of autophagy in plant cells starved in carbohydrates.

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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.

Mitochondria from pea leaves were purified by centrifugation on a self-generated Percoll gradient which contained a linear gradient of polyvinylpyrrolidone-25 (0-10010, w/v). The chlorophyll content of the purified mitochondria was less than 1 pg per mg protein. All substrates were rapidly oxidized by these mitochondria, the rate of glycine oxidation being between 200 and 300 nmol 0, min-' mg-I protein, depending on the age of the leaves used. These rates did not vary significantly over a period of 20 h, provided NAD+ was supplied exogenously, when the mitochondria were stored on ice. Respiratory control, ADP/O ratios and outer membrane integrity (always more than 95010) were also maintained during storage. The phospholipid composition of the membranes from the leaf mitochondria was virtually identical to that of mitochondria from non-photosynthetic tissues although their lipid to protein ratio was slightly lower. The polypeptide pattern of the membranes from green leaf mitochondria and those from etiolated leaves and hypocotyls were also similar, but marked differences were observed between the matrix proteins from the different tissues. In particular, intensely stained bands at 94, 51,41 and 15.5 kDa which were present in the matrix of green leaf mitochondria were missing or present in much smaller quantities in the non-photosynthetic tissues. This difference was correlated with the ability of the mitochondria to oxidize glycine, suggesting that the four polypeptides may be associated with the glycine decarboxylase complex.