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In vivo 31P-NMR analyses showed that the phosphate (Pi) concentration in the cytosol of sycamore (Acer pseudoplafanus) and Arabidopsis (Arabidopsis thaliana) cells was much lower than the cytoplasmic Pi concentrations usually considered (60-80 μM instead of >1 mM) and that it dropped very rapidly following the onset of Pi starvation. The Pi efflux from the vacuole was insufficient to compensate for the absence of external Pi supply, suggesting that the drop of cytosolic Pi might be the first endogenous signal triggering the Pi starvation rescue metabolism. Successive short sequences of Pi supply and deprivation showed that added Pi transiently accumulated in the cytosol, then in the stroma and matrix of organelles bounded by two membranes (plastids and mitochondria, respectively), and subsequently in the vacuole. The Pi analog methylphosphonate (MeP) was used to analyze Pi exchanges across the tonoplast. MeP incorporated into cells via the Pi carrier of the plasma membrane; it accumulated massively in the cytosol and prevented Pi efflux from the vacuole. This blocking of vacuolar Pi efflux was confirmed by in vitro assays with purified vacuoles. Subsequent incorporation of Pi into the cells triggered a massive transfer of MeP from the cytosol to the vacuole. Mechanisms for Pi exchanges across the tonoplast are discussed in the light of the low cytosolic Pi level, the cell response to Pi starvation, and the Pi/MeP interactive effects. [ABSTRACT FROM AUTHOR]

Metabolic profiling using phosphorus nuclear magnetic resonance (31P-NMR) revealed that the leaves of different herbs and trees accumulate 2- C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP), an intermediate of the methylerythritol 4-phosphate (MEP) pathway, during bright and hot days. In spinach ( Spinacia oleracea L.) leaves, its accumulation closely depended on irradiance and temperature. MEcDP was the only 31P-NMR-detected MEP pathway intermediate. It remained in chloroplasts and was a sink for phosphate. The accumulation of MEcDP suggested that its conversion rate into 4-hydroxy-3-methylbut-2-enyl diphosphate, catalysed by ( E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE), was limiting under oxidative stress. Indeed, O2 and ROS produced by photosynthesis damage this O2-hypersensitive [4Fe-4S]-protein. Nevertheless, as isoprenoid synthesis was not inhibited, damages were supposed to be continuously repaired. On the contrary, in the presence of cadmium that reinforced MEcDP accumulation, the MEP pathway was blocked. In vitro studies showed that Cd2+ does not react directly with fully assembled GcpE, but interferes with its reconstitution from recombinant GcpE apoprotein and prosthetic group. Our results suggest that MEcDP accumulation in leaves may originate from both GcpE sensitivity to oxidative environment and limitations of its repair. We propose a model wherein GcpE turnover represents a bottleneck of the MEP pathway in plant leaves simultaneously exposed to high irradiance and hot temperature. [ABSTRACT FROM AUTHOR]

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 lie 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 lie 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 lie and a potential storage form for sulfide or methyl groups, SMC. [ABSTRACT FROM AUTHOR]

Examines the biochemical and topological properties of type A monogalactosyldiacylglycerol (MGDG) synthase in both prokaryotic and eukaryotic MGDG. Description of MGDG synthase; Use of recombinant protein on type A family from Spinacia oleracea; Details on the catalyst of MGDG synthesis.

Spinach leaves were labelled with 14CO2 for subsequent isolation of radioactive chloroplasts, which were separated into envelopes and thylakoids. The analyses carried out with the lipid extracts from these membranes were concerned with the following questions: are chloroplast envelopes a functional interface between endoplasmic reticulum and thylakoids, do they also play a predominant role in galactolipid biosynthesis in vivo, and is it possible to demonstrate galactolipid export from envelopes into thylakoids? Taken together the results show that lipid export is apparently too fast in vivo to be followed by the labelling and separation technique used, since thylakoid lipids contained always far more total label than envelope counterparts, whereas the specific activity of envelope lipids was higher. Phosphatidylcholine, which has been suggested to function as acyl carrier between endoplasmic reticulum and chloroplasts, was never found labelled to any extraordinary extent in envelopes. Envelopes may be regarded as small, but rapidly turned over lipid pools. [ABSTRACT FROM AUTHOR]

We have isolated and characterized a genomic clone encoding the 41 kDa monomer T-protein. This gene called gdcT spans approximately 3 kbp and is composed of four exons interrupted by three introns (321, 691 and 114 bp). The splice sites for donor and acceptor are in agreement with the canonical GT/AG rule. Primer extension strongly suggests the presence of two major transcription start sites. The first transcription start site around 43 bases downstream of a putative TATA box was assigned the +1 position. The second (+31) is not correlated with a putative TATA box, but revealed a pyrimidine-rich region which is very similar to the initiator element. Sequence analysis of the 5′-upstream region of the gene reveals three consensus regions found in the nuclear genes encoding the chloroplastic proteins of ribulose-1,5-bisphosphate carboxylase (rbcS) and the chlorophyll a/b-binding protein (cab) such as an AT-rich sequence localized at -539 to -530, a box II core sequence GGTTAA (-123 to -118) and between -364 and -354 a tandem GATA motif. These elements are known to be involved respectively in the regulation of light-responsiveness and cell-type specificity expression of plant genes. Gel shift assays indicate that the box II core sequence could bind protein nuclear factors similar to the trans-acting factor which interact with corresponding promoter region of rbcS gene. [ABSTRACT FROM AUTHOR]

Cystathionine β-lyase, the second enzyme involved in the methionine biosynthetic pathway in plants, catalyses the synthesis of homocysteine from cystathionine. A cDNA encoding cystathionine β-lyase was cloned from an Arabidopsis thaliana expression library by complementation of an Escherichia coli mutant deficient in this enzyme. As deduced from the full-length nucleotide sequence (1.7 kb), the polypeptide contains 464 amino acids and presents a predicted M of 50372. A. thaliana cystathionine β-lyase exhibits 22% sequence identity with the E. coli corresponding enzyme and contains a 70 amino acid N-terminal additional sequence compared with the bacterial protein. Since the general features of chloroplast transit peptides could be observed in this amino-terminal extension, we propose a chloroplast localization for the cDNA-encoded enzyme. Southern blot analysis suggested that cystathionine β-lyase is encoded by a single copy gene in A. thaliana. [ABSTRACT FROM AUTHOR]

Seeds of Pisum sativum contain a biotinyl polypeptide called SBP65 that behaves as a putative sink for the free vitamin, representing more than 90% of the total protein-bound biotin in mature seeds. A cDNA encoding SBP65 was cloned and sequenced. The deduced primary structure of the protein was confirmed by protein sequencing. Peptide sequencing also indicated binding of the biotin to lysine 103. The biotinylation domain of SBP65 differs markedly from that of presently known biotin enzymes. Molecular analysis of the protein sequence reveals an extremely hydrophilic protein containing several repeated motifs. These properties, as well as the temporal and spatial patterns of expression of this protein, suggest that SBP65 belongs to the LEA (late embryogenesis-abundant) group of proteins. [ABSTRACT FROM AUTHOR]

A synthetic gene encoding the mature spinach-chloroplast O-acetylserine (thiol-)-lyase was constructed and expressed in an Escherichia coli strain carrying the T7 RNA polymerase system. The pure recombinant protein was obtained at high yield (6mg/l cell culture) using a new purification procedure that includes affinity chromatography on Green A agarose. Its specific activity was of the order of 1000 U/mg, and its physical properties were similar to those previously reported for the natural enzyme isolated from spinach chloroplasts. In particular the recombinant enzyme, as for the natural enzyme, behaved as a homodimer composed of two identical subunits each of MΓ 35 000. From steady-state kinetic studies using sulfide or 5-thio(2-nitrobenzoate) (Nbs) as alternative nucleophilic co-substrates, the enzyme exhibited positive kinetic co-operativity with respect to O-acetylserine [Ser(Ac)] in the presence of sulfide and a negative kinetic co-operativity in the presence of Nbs. Binding of Ser(Ac) to the enzyme was also investigated by absorbance and fluorescence measurements to obtain insight into the role of pyridoxal 5'-phosphate and of the single tryptophan residue (Trp176) present in the enzyme molecule. Addition of Ser(Ac) to the enzyme provoked the disappearance of the 409-nm absorbance bands, the one located between 320 nm and 360 nm and the other centered at 470 nm. Also, the fluorescence emission of the pyridoxal 5'-phosphate Schiff base was quenched upon addition of Ser(Ac) to the enzyme. These changes were most presumably due to the formation of a Schiff base intermediate between α-aminoacrylate and the pyridoxal 5'-phosphate cofactor. The fluorescence emission of Trp176 was also quenched upon Ser(Ac) binding to the enzyme. Quantitative analysis of the absorbance and fluorescence equilibrium data disclosed a cooperative behaviour in Ser(Ac) binding, in agreement with the steady-state kinetic results. Fluorescence quenching experiments with acrylamide and iodide revealed that the indole ring of Trp176 was largely exposed and located within the pyridoxal 5'-phosphate active site. These results are consistent with the finding that the native enzyme is composed of two identical subunits. Yet, presumably due to subunit —subunit interactions, the enzyme exhibits two non-equivalent pyridoxal 5'-phosphate-containning active sites. [ABSTRACT FROM AUTHOR]

A synthetic gene encoding the entire mature H protein of the glycine decarboxylase complex from pea (Pisum sativum L.) was constructed and expressed in Escherichia coli. The recombinant H protein, which after the induction period constituted more than half of the E. coli protein, was found in a soluble form. Activity measurements and mass-spectrometry analysis of the purified protein showed that, in the absence or presence of 5[3-(1,2)-dithiolanyl]pentanoic acid (lipoic acid) in the culture medium, recombinant H protein could be produced as the unlipoylated apoform or as the lipoylated form, respectively. Addition of chloramphenicol to the culture medium after induction increased the proportion of lipoylated H protein. High rates of lipoylation of the H apoprotein were measured in vivo and in vitro, revealing that the recombinant pea H protein was an excellent substrate for the E. coli lipoyl-ligase. The three-dimensional structure of the recombinant H apoprotein was determined at a 0.25-nm resolution. It was almost identical to the structure of the native pea leaf enzyme, which indicates that the recombinant protein folds properly in E. coli and that the lipoyl-ligase recognizes a three-dimensional structure in order to add lipoic acid to its specific lysine residue. It is postulated that the high level of expression and lipoylation of recombinant H protein may be due to the protein retaining the structure of the original enzyme. [ABSTRACT FROM AUTHOR]

Steady-state kinetics of the 220-kDa form of acetyl-CoA carboxylase (ACC220), as purified from mature pea seeds, have been investigated with respect to the substrate specificity and inhibition by quizalofop, a herbicide of the aryloxyphenoxypropionate type. The enzyme showed a dual specificity, being able to carboxylate propionyl-CoA at a maximal rate approximately 20% that measured in the presence of the acetyl-CoA substrate. These two reactions occur at separate sites on the enzyme. One site binds either acetyl-CoA (Km = 226 μM) or propionyl-CoA (Km = 38 μM) and is strongly inhibited by quizalofop (Ki = 25 μM and 9.3 μM for the acetyl-CoA and propionyl-CoA substrates, respecgtively). The other is specific for acetyl-CoA (Km = 11 μM) and is much less inhibited by quizalofop (Ki = 256 μM). Owing to the existence of these two catalytically different sites, the enzyme obeyed Michaelis-Menten kinetics with propionyl-CoA, but exhibited kinetic co-operativity in the presence of acetyl-CoA. Also, kinetics of propionyl-CoA carboxylase activity of ACC220 exhibited hyperbolic inhibition in the presence of quizalofop, but co-operative inhibition when following the ACC activity of the enzyme. The results suggest that the higher the substrate specificity, the lower the quizalofop sensitivity of the active site. Similar kinetic behaviour was observed with ACC220 purified from pea leaves. Also, the apparent correlation between the substrate specificity and the sensitivity of ACC towards quizalofop was confirmed by kinetic analyses of the low-molecular-mass form of ACC present in chloroplats of young pea leaves. This enzyme was insensitive to quizalofop inhibition and was not able to carboxylate propionyl-CoA. No other propionyl-CoA carboxylase activity, different from that catalysed by ACC220, could be detected from either reproductive or vegetative organs of pea plants at any stage of development. [ABSTRACT FROM AUTHOR]

We have followed the dynamic evolution of intracellular pH and of the intracellular concentration of nucleotides (NDP, NTP), Pi and lactate in maize root tips during the course of normoxia and anoxia transition. The intracellular pH, determined from the 31P-NMR chemical shift of the cytoplasmic Pi peak, dropped from 7.5 to 6.9 during the first few minutes after anaerobiosis. It increased again, then settled to a steady-state value of 7.1 - 7.2, 25 min after the beginning of the anoxic treatment. Following oxygenation, the chemical shift of the cyloplasmic P1 peak drilled gradually to its initial value. The cytoplasmic pH followed an oscillatory time course which was almost identical to the time course of NTP. Intracellular lactate accumulated steadily during the first 30 min after anaerobiosis, then its intracellular concentration remained almost constant. Following oxygenation, the intracellular concentration of lactate decreased slowly. The cytoplasmic pH followed a time course which was not identical to the time course of lactate. Following hypoxia, the pH dropped to low values long before the intracellular lactate concentration reached a steady-state equilibrium. Conversely, subsequent to oxygenation, the pH returned to normal values long before lactate. These results do not agree with the statement that cytoplasmic acidification in hypoxic maize root tips is necessarily associated with lactic acid synthesis. [ABSTRACT FROM AUTHOR]

Focuses on the advances in plasmid envelope membranes. Characteristics of plant cell; Major plastid glycolipids; Composition of plasmid envelope membranes.

The mitochondrial sn-glycerol-3-phosphate and 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from potato tubers and pea leaves were investigated with respect to their intraorganelle localization, their positional and substrate specificities, and their fatty acid selectivities. In mitochondria from potato tubers both enzymes were found to be located in the outer membrane. The 1-acyl-sn-glycerol-3-phosphate O-acyltransferase of pea mitochondria showed the same intraorganelle localization whereas the sn-glycerol-3-phosphate O-acyltransferase behaved like a soluble protein of the intermembrane space. The sn-glycerol-3-phosphate O-acyltransferase of both potato and pea mitochondria used sn-glycerol-3-phosphate but not dihydroxyacetone phosphate as acyl acceptor and exclusively catalyzed the formation of 1-acyl-sn-glycerol-3-phosphate which subsequently served as substrate for the second acylation reaction at its C-2 position. Both acyltransferases of potato as well as pea mitochondria showed higher activities with acyl-CoA than with the corresponding acyl-(acyl carrier protein) thioesters. When different acyl-CoA thioesters were offered separately, the sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed no fatty acid specificity whereas the enzyme of pea mitochondria revealed one for saturated acyl groups. On the other hand, the mitochondrial 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from both potato tubers and pea leaves were more active on unsaturated than on saturated acyl-CoA thioesters. Furthermore, these enzymes preferentially used oleoyl- and linoleoyl-CoA when they were offered in a mixture with saturated ones, although the fatty acid selectivity of the pea enzyme was less pronounced than that of the potato enzyme. The sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed a slight preference for saturated acyl groups. [ABSTRACT FROM AUTHOR]

The primary structure of acetohydroxy acid isomeroreductase from Arabidopsis thaliana was deduced from two overlapping cDNA. The full-length cDNA sequence predicts an amino acid sequence for the protein precursor of 591 residues including a putative transit peptide of 67 amino acids. Comparison of the A. thaliana and spinach acetohydroxy acid isomeroreductases reveals that the sequences are conserved in the mature protein regions, but divergent in the transit peptides and around their putative processing site. [ABSTRACT FROM AUTHOR]

Presents information on a study which investigated the mechanisms involved in homeostatic regulation of amino acids in plants with specific features of methionine biosynthesis. Details on the de novo synthesis of methionine; Information on S-adensoyl-methionine (AdoMet) recycling during transmethylation reactions; Conclusions drawn from the results of the study.

Acetohydroxy acid isomeroreductase catalyzes the conversion of acetohydroxy acids into dihydroxy valerates. This reaction is the second in the synthetic pathway of the essential branched side chain amino acids valine and isoleucine. Because this pathway is absent from animals, the enzymes involved in it are good targets for a systematic search for herbicides. The crystal structure of acetohydroxy acid isomeroreductase complexed with cofactor NADPH, Mg2+ ions and a competitive inhibitor with herbicidal activity, N-hydroxy-N-isopropyloxamate, was solved to 1.65 Å resolution and refined to an R factor of 18.7% and an R free of 22.9%. The asymmetric unit shows two functional dimers related by non-crystallographic symmetry. The active site, nested at the interface between the NADPH-binding domain and the all-helical C-terminus domain, shows a situation analogous to the transition state. It contains two Mg2+ ions interacting with the inhibitor molecule and bridged by the carboxylate moiety of an aspartate residue. The inhibitor-binding site is well adjusted to it, with a hydrophobic pocket and a polar region. Only 24 amino acids are conserved among known acetohydroxy acid isomeroreductase sequences and all of these are located around the active site. Finally, a 140 amino acid region, present in plants but absent from other species, was found to make up most of the dimerization domain. [ABSTRACT FROM AUTHOR]

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 bi-functional 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. [ABSTRACT FROM AUTHOR]

Lipid mixtures from chloroplast envelope and thylakoid membranes were isolated after different labelling times in vivo and in vitro and separated into major components. The isolated compounds were subjected to analyses such as separation of molecular species, determination of radioactivity in fatty acids and water-soluble hydrolysis products and radio gas-liquid chromatography of fatty acid mixtures. In the case of monogalactosyl and digalactosyl diacylglycerol these procedures were also applied to several individual molecular species. To investigate the extent of de novo synthesis these species were also used for methylation studies and their fatty acids subjected to α-oxidation. In envelope membranes diacylglycerols and monogalactosyl diacylglycerols may each be separated into several distinct and non-mixing pools. Molecules made de novo with oligoene fatty acids are very efficient substrates for galactosylation in vivo. The time-dependent changes in patterns of galactolipid molecular species may indicate a desaturation of acyl chains operating in close contact to intact lipids. After isolation, envelopes incorporated UDP-[14C]galactose into completely different patterns of galactolipids and molecular species pointing to changed properties of this membrane system or to a loss of regulatory factors. [ABSTRACT FROM AUTHOR]