Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

<p>Abstract</p> <p>Background</p> <p>Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in <it>Medicago truncatula</it>.</p> <p>Results</p> <p>This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2) in <it>M. truncatula</it>. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene.</p> <p>Conclusions</p> <p>This study shows that <it>Medicago truncatula </it>contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate that this gene duplication represents a functional innovation of plastid located GS related to storage protein accumulation exclusive to legume seed metabolism.</p

Influence of mitochondrial genome rearrangement on cucumber leaf carbon and nitrogen metabolism

The MSC16 cucumber (Cucumis sativus L.) mitochondrial mutant was used to study the effect of mitochondrial dysfunction and disturbed subcellular redox state on leaf day/night carbon and nitrogen metabolism. We have shown that the mitochondrial dysfunction in MSC16 plants had no effect on photosynthetic CO2 assimilation, but the concentration of soluble carbohydrates and starch was higher in leaves of MSC16 plants. Impaired mitochondrial respiratory chain activity was associated with the perturbation of mitochondrial TCA cycle manifested, e.g., by lowered decarboxylation rate. Mitochondrial dysfunction in MSC16 plants had different influence on leaf cell metabolism under dark or light conditions. In the dark, when the main mitochondrial function is the energy production, the altered activity of TCA cycle in mutated plants was connected with the accumulation of pyruvate and TCA cycle intermediates (citrate and 2-OG). In the light, when TCA activity is needed for synthesis of carbon skeletons required as the acceptors for NH4+ assimilation, the concentration of pyruvate and TCA intermediates was tightly coupled with nitrate metabolism. Enhanced incorporation of ammonium group into amino acids structures in mutated plants has resulted in decreased concentration of organic acids and accumulation of Glu

Biochemical Characterisation of an Aldoxime-Forming Flavoprotein involved in 2-Phenylethylglucosinolate Biosynthesis in Brassica Species

L-Homophenylalanine (L-HPhe) is the precursor of 2-phenylethylglucosinolate, a secondary metabolite present in some Brassica and related species. A key step in its biosynthesis is the oxidative decarboxylation of L-HPhe to its aldoxime. The enzyme catalysing this reaction has been shown to be a NADPH- and O-2-dependent microsomal flavoprotein (L-HPhe FP; EC unclassified). Inhibition studies using Phe homologs and HPhe analogs (alpha-amino-, alpha-carboxyl- and ring-substituted), and specific amino acid modifications, were carried out to determine the possible active site structure and catalytic mechanism of L-HPhe FP. Activity with L-HPhe was inhibited by the two higher homologs, but not by L-Phe. Methylation of the substrate alpha-amino group, or replacement of the alpha-carboxyl group with a phosphonic acid group, significantly reduced the inhibition. Ring substitutions had varying effects: single methyl substitutions had only minor effects on binding to the active site, whereas di- or tri-methyl, methoxy or halide substitutions significantly reduced inhibition. Simple amines had no significant effect on L-HPhe FP activity. Binding to the active site of the enzyme appears to require a minimum chain length, plus an aromatic ring at one end of the molecule and unmodified alpha-amino acid moiety at the other. Chemical modification of amino acids on the protein implied there was no requirement for thiol groups (-SH), Ser/Thr hydroxyl groups, or L-Arg in the active site of L-HPhe FP. However, there was evidence for the presence of essential His and Tyr residues, and the involvement of Glu or Asp residues at or near the active site. (C) Elsevier, Paris.</p

High Performance Liquid-Chromatography Separation of Natural and Synthetic Desulfoglucosinolates and Their Chemical Validation by Spectroscopic, NMR and CI-MS Methods

Methods are described for the optimised extraction, desulphation and HPLC separation of desulphoglucosinolates, These methods provide rapid separation, identification and quantitative measurements of glucosinolates extracted from Brassica napus L and related crops, of unusual glucosinolates found in crucifer weed species, and also of synthetic alkylglucosinolates, The desulphoglucosinolates used in these studies were either chemically synthesised (at least one example from each major structural class), or purified from various plant sources. Validation of the identities of the desulphoglucosinolates was by comparison of retention times with standards, and by UV,H-1- and C-13-NMR and chemical ionisation MS analysis. A list of useful species, and the specific tissues, from which high concentrations of standards can be extracted is included. Copyright (C) 2001 John Wiley &amp; Sons, Ltd.</p

Methionine Biosynthesis in Higher Plants .II. Purification and Characterization of Cystathionine β-Lyase from Spinach Chloroplasts

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