Changes in the levels of major sulfur metabolites and free amino acids in pea cotyledons recovering from sulfur deficiency

Changes in levels of sulfur metabolites and free amino acids were followed in cotyledons of sulfur-deficient, developing pea seeds (Pisum sativum L.) for 24 hours after resupply of sulfate, during which time the legumin mRNA levels returned almost to normal. Two recovery situations were studied: cultured seeds, with sulfate added to the medium, and seeds attached to the intact plant, with sulfate added to the roots. In both situations the levels of cysteine, glutathione, and methionine rose rapidly, glutathione exhibiting an initial lag. In attached but not cultured seeds methionine markedly overshot the level normally found in sulfur-sufficient seeds. In the cultured seed S-adenosylmethionine (AdoMet), but not S-methylmethionine, showed a sustained rise; in the attached seed the changes were slight. The composition of the free amino acid pool did not change substantially in either recovery situation. In the cultured seed the large rise in AdoMet level occurred equally in nonrecovering seeds. It was accompanied by 6-fold and 10-fold increases in γ-aminobutyrate and alanine, respectively. These effects are attributed to wounding resulting from excision of the seed. ³⁵S-labeling experiments showed that there was no significant accumulation of label in unidentified sulfur-containing amino compounds in either recovery situation. It was concluded from these results and those of other workers that, at the present level of knowledge, the most probable candidate for a `signal' compound, eliciting recovery of legumin mRNA level in response to sulfur-feeding, is cysteine.     

Increased sulfur amino acids in soybean plants overexpressing the maize 15 kDa zein protein

Summary Four transgenic soybean [Glycine max (L.) Merrill] lines were generated containing the maize 15 kDa zein protein gene using somatic embryogenic protocols. The zein gene was inserted behind the β-phaseolin promoter for seed-specific expression. All four lines represent separate transformation events as they were generated in different experiments at different locations. Two of the transformation events produced multiple plants, and these produced identical Southern hybridization patterns (UKY/Z1, UKY/Z2 and UKY/Z3 from the first; and OSU/Z4, OSU/Z8 and OSU/Z10 from the second). Molecular characterization revealed that multiple copies of the zein gene were present in all of the transgenic lines. Immunoblot analysis confirmed the accumulation of the zein protein product in the seeds of the UKY/Z1, UKY/Z2, UKY/Z3, OSU/Z4, OSU/Z8 and OSU/Z10 transgenic lines. However, there was no accumulation of zein protein in the UGA/Z1 line and Northern analysis confirmed that the zein transgene was silenced in this line. It was not possible to analyze the zein expression in the seeds of the UKY/Z4 line, as it was sterile. Amino acid analysis of the UKY and OSU lines confirmed that there was a 12–20% increase in methionine, and 15–35% increase in cysteine content in these lines compared to the control. There were no consistent changes in the content of the other amino acids in the transgenic lines. These results suggest that while the increase in methionine content in these lines is modest, it is possible to increase the methionine content without adversely affecting the protein composition of soybean.     

Synthesis of the sulfur amino acids: cysteine and methionine

This review will assess new features reported for the molecular and biochemical aspects of cysteine and methionine biosynthesis in Arabidopsis thaliana with regards to early published data from other taxa including crop plants and bacteria (Escherichia coli as a model). By contrast to bacteria and fungi, plant cells present a complex organization, in which the sulfur network takes place in multiple sites. Particularly, the impact of sulfur amino-acid biosynthesis compartmentalization will be addressed in respect to localization of sulfur reduction. To this end, the review will focus on regulation of sulfate reduction by synthesis of cysteine through the cysteine synthase complex and the synthesis of methionine and its derivatives. Finally, regulatory aspects of sulfur amino-acid biosynthesis will be explored with regards to interlacing processes such as photosynthesis, carbon and nitrogen assimilation.     

含硫氨基酸的抗氧化作用

抗氧化剂是维持动物体内自由基平衡和稳定的重要物质。甲硫氨酸(Met)和半胱氨酸(Cys) 是组成蛋白质的2种含硫氨基酸,基于其独特的结构,在动物体内起着与动物营养与免疫相关的重要生理功能,而其抗氧化能力越来越受关注。综述了蛋白质Met残基的氧化还原作用和含硫氨基酸的代谢产物谷胱甘肽(GSH)的抗氧化作用。     

Neoaplectana glaseri: essential amino acids

Required for a nematode's reproduction in a chemically defined medium are the nine mammalian essential amino acids (sensu strictu). Needed in addition to lysine, tryptophane, histidine, phenylalanine, leucine, isoleucine, threonine, methionine, and valine is arginine which is marginally essential for mammals. Tyrosine, nonessential for mammals, is not absolutely required by the nematode but is beneficial in terms of onset time and quantity of reproduction. Aspartic acid, nonessential for the nematode and mammals, is toxic for adult nematodes at and above 4.8 mg/ml medium. The nematode, Neoaplectana glaseri, is parasitic in insect grubs but the strain used has been cultivated in species isolation on kidney slices continuously since (1944).     

The aspartate-family pathway of plants: Linking production of essential amino acids with energy and stress regulation

The Asp family pathway of plants is highly important from a nutritional standpoint because it leads to the synthesis of the four essential amino acids Lys, Thr, Met and Ile. These amino acids are not synthesized by human and its monogastric livestock and should be supplemented in their diets. Among the Asp-family amino acids, Lys is considered as the nutritionally most important essential amino acid because its level is most limiting in cereal grains, representing the largest source of plant foods and feeds worldwide. Metabolic engineering approaches led to significant increase in Lys level in seeds by enhancing its synthesis and reducing its catabolism. However, results from the model plant Arabidopsis showed that this approach may retard seed germination due to a major negative effect on the levels of a number of TCA cycle metabolites that associate with cellular energy. In the present review, we discuss the regulatory metabolic link of the Asp-family pathway with the TCA cycle and its biological significance upon exposure to stress conditions that cause energy deprivation. In addition, we also discuss how deep understanding of the regulatory metabolic link of the Asp-family pathway with energy and stress regulation can be used to improve Lys level in seeds of important crop species, minimizing the interference with the cellular energy status and plant-stress interaction. This review thus provides an example showing how deep understanding the inter-regulation of metabolism with plant stress physiology can lead to successful nutritional improvements with minimal negative effect on plant growth and response to stressful environments.Key words: Lysine, metabolic engineering, essential amino acids, plants energy, TCA cycle     

Effect of sulfur availability on the integrity of amino acid biosynthesis in plants

Summary. Amino acid levels in plants are regulated by a complex interplay of regulatory circuits at the level of enzyme activities and gene expression. Despite the diversity of precursors involved in amino acid biosynthesis as providing the carbon backbones, the amino groups and, for the amino acids methionine and cysteine, the sulfhydryl group and despite the involvement of amino acids as substrates in various downstream metabolic processes, the plant usually manages to provide relatively constant levels of all amino acids. Here we collate data on how amino acid homeostasis is shifted upon depletion of one of the major biosynthetic constituents, i.e., sulfur. Arabidopsis thaliana seedlings exposed to sulfate starvation respond with a set of adaptation processes to achieve a new balance of amino acid metabolism. First, metabolites containing reduced sulfur (cysteine, glutathione, S-adenosylmethionine) are reduced leading to a number of downstream effects. Second, the relative excess accumulation of N over S triggers processes to dump nitrogen in asparagine, glutamine and further N-rich compounds like ureides. Third, the depletion of glutathione affects the redox and stress response system of the glutathione-ascorbate cycle. Thus, biosynthesis of aromatic compounds is triggered to compensate for this loss, leading to an increased flux and accumulation of aromatic amino acids, especially tryptophan. Despite sulfate starvation, the homeostasis is kept, though shifted to a new state. This adaptation process keeps the plant viable even under an adverse nutritional status.     

Identification of essential amino acids in the bacterial alpha -mannosyltransferase aceA

The alpha-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues included Glu-287 and Glu-295, which comprise an EX(7)E motif and have been proposed to be involved in catalysis. Alanine replacements of each conserved residue were constructed by site-directed mutagenesis. The mannosyltransferase activity of each mutant was examined by both an in vitro transferase assay using recombinant mutant AceA expressed in Escherichia coli and by an in vivo rescue assay by expressing the mutant AceA in a Xanthomonas campestris gumH(-) strain. We found that only mutants K211A and E287A lost all detectable activity both in vitro and in vivo, whereas E295A retained residual activity in the more sensitive in vivo assay. H127A and S162A each retained reduced but significant activities both in vitro and in vivo. Secondary structure predictions of AceA and subsequent comparison with the crystal structures of the T4 beta-glucosyltransferase and MurG suggest that AceA Lys-211 and Glu-295 are involved in nucleotide sugar donor binding, leaving Glu-287 of the EX(7)E as a potential catalytic residue.     

Ion-exchange chromatography of sulfur amino acids on a single-column amino acid analyzer.

Examination of the extent of production of the ninhydrin-colored derivative, Ruhemann's purple, under automated conditions of a single-column amino acid analyzer by several classes of sulfur-containing amino acids revealed a wide variation in the color factors relative to leucine. These ranged from 0.02 for the methyl ester of cysteine to 2.19 for D-homocystine. Color yields obtained by the manual ninhydrin reaction are generally lower than the corresponding values obtained on the amino acid analyzer. The elution positions ranged from 5.12 min for cysteic acid to 84.9 min for l-cystine dimethyl ester. The observed behavior of these compounds in the ninhydrin reaction is rationalized in terms of structural and electronic factors which they exhibit in reacting with ninhydrin to form the visible dye. Such an analysis should make it possible to predict ninhydrin color factors, and possibly also elution times, of structurally related compounds.     

Identification of essential amino acids in the Streptococcus mutans glucosyltransferases.

A comparison of the amino acid sequences of the glucosyltransferases (GTFs) of mutans streptococci with those from the alpha-amylase family of enzymes revealed a number of conserved amino acid positions which have been implicated as essential in catalysis. Utilizing a site-directed mutagenesis approach with the GTF-I enzyme of Streptococcus mutans GS-5, we identified three of these conserved amino acid positions, Asp413, Trp491, and His561, as being important in enzymatic activity. Mutagenesis of Asp413 to Thr resulted in a GTF which expressed only about 12% of the wild-type activity. In contrast, mutagenesis of Asp411 did not inhibit enzyme activity. In addition, the D413T mutant was less stable than was the parental enzyme when expressed in Escherichia coli. Moreover, conversion of Trp491 or His561 to either Gly or Ala resulted in enzymes devoid of GTF activity, indicating the essential nature of these two amino acids for activity. Furthermore, mutagenesis of the four Tyr residues present at positions 169 to 172 which are part of a subdomain with homology to the direct repeating sequences present in the glucan-binding domain of the GTFs had little overall effect on enzymatic activity, although the glucan products appeared to be less adhesive. These results are discussed relative to the mechanisms of catalysis proposed for the GTFs and related enzymes.     

Construction and expression of synthetic DNA fragments coding for polypeptides with elevated levels of essential amino acids

Summary Polypeptides, with elevated levels of essential amino acids, could be useful as partial protein supplements to food and feeds. To obtain DNA fragments coding for these polymers, oligonucleotides were constructed by random synthesis of a mixture of appropriate codon pairs and inserted into a bacterial plasmid in E. coli. Two of the isolated fragments were subjected to DNA sequence analysis and theoretically code for polypeptides containing up to 23% lysine, 12% tryptophan, 12% methionine, 6% isoleucine, and 6% threonine. These five amino acids make up 60% of the total amino acid content of the peptide, compared with 25% for the same amino acids in lactalbumin, a milk protein considered to be high in essential amino acids. These fragments, when fused to an active bacterial promoter, which directs the synthesis of chloramphenicol acetyl transferase (CAT), cause bacteria, harboring these modified genes, to take up more lysine as compared to control cells and produce commensurately larger CAT polypeptides. This method of gene synthesis may permit production of polypeptides with a specified amino acid composition to supplement specific diets low in the essential amino acids.     

Effect of natural amino alcohols on the yield of essential amino acids and the amino acid pattern in stressed barley

Summary By application with 2-aminoethanol (2-AE) and choline chloride (CC) in pot experiments the effect of drought stress on barley plants was diminished. In treated plants an increase of the grain yield by 14% (2-AE) and 40% (CC) and a decrease of the stress metabolites glycine betaine and trigonelline was observed. Additionally, treated barley plants showed higher yields of essential amino acids as well. The contents of proline (stress indicator) and arginine (precursor of the stress metabolite putrescine) of treated plants were by 12% and 22% respectively, lower than in untreated plants.     

Voltammetric investigation of the interactions between superoxide ion and some sulfur amino acids

In this paper, we analyze a group of amino acids containing sulfur, either as thiols or as disulfides and sulfides (l-cysteine, l-cystine, S-carboxymethyl-l-cysteine, N-acetyl-l-cysteine, reduced and oxidized l-glutathione) for their interaction with superoxide ion in protic and aprotic media. Superoxide ion was electrochemically generated by reduction of oxygen molecules dissolved in the solutions. concentration changes due to the presence of interacting species and was easily monitored under mild and controlled experimental conditions, in the presence or in the absence of the selected substrate, in protic and aprotic media. The reactions were followed by cyclic (CV) or linear (LSV) voltammetry, depending on the medium and the electrochemical techniques widely used to study the antioxidant capacity of biological and chemical systems. According to the experimental results, it was concluded that substrates having a free thiol group react with ion giving disulfides and can be considered as antioxidants for the superoxide ion in the following decreasing order: N-acetyl-l-cysteine > l-cysteine > reduced glutathione. Compounds having protected sulfur (l-cystine, S-carboxymethyl-l-cysteine, oxidized glutathione) do not react with the superoxide ion and do not form compounds with a superior S oxidation number, such as sulfoxide and sulfone.