Engineering of cysteine and methionine biosynthesis in potato

Summary. Methionine and cysteine, two amino acids containing reduced sulfur, are not only an important substrate of protein biosynthesis but are also precursors of various other metabolites such as glutathione, phytochelatines, S-adenosylmethionine, ethylene, polyamines, biotin, and are involved as methyl group donor in numerous cellular processes. While methionine is an essential amino acid due to an inability of monogastric animals and human beings to synthesise this metabolite, animals are still able to convert methionine consumed with their diet into cysteine. Thus, a balanced diet containing both amino acids is necessary to provide a nutritionally favourable food or feed source. Because the concentrations of methionine and cysteine are often low in edible plant sources, e.g. potato, considerable efforts in plant breeding and research have been and are still performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their synthesis, transport, and accumulation in plants. During the last decade molecular tools have enabled the isolation of most of the genes involved in cysteine and methionine biosynthesis, and the efficient plant transformation technology has allowed the creation of transgenic plants that are altered in the activity of individual genes. The physiological analysis of these transgenic plants has contributed considerably to our current understanding of how amino acids are synthesised. We focused our analysis on potato (Solanum tuberosum cv. Désirée) as this plant provides a clear separation of source and sink tissues and, for applied purposes, already constitutes a crop plant. From the data presented here and in previous work we conclude that threonine synthase and not cystathionine gamma-synthase as expected from studies of Arabidopsis constitutes the main regulatory control point of methionine synthesis in potato. This article aims to cover the current knowledge in the area of molecular genetics of sulfur-containing amino acid biosynthesis and will provide new data for methionine biosynthesis in solanaceous plants such as potato. Received December 19, 2001 Accepted January 7, 2002     

Molecular and biochemical analysis of the enzymes of cysteine biosynthesis in the plant <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Summary. Among the amino acids produced by plants cysteine plays a special role as a mediator between assimilatory sulfate reduction and provision of reduced sulfur for cell metabolism. Part of this characteristic feature is the presence of cysteine synthesis in plastids, mitochondria and cytosol. Plants are the major source of reduced sulfur for human and animal nutrition. Cysteine biosynthesis deserves special attention, since reduced sulfur is channelled from cysteine into many sulfur-containing compounds in food and feed. Recent investigations are reviewed that focus on structure and regulation of cysteine synthesis in the model plant Arabidopsis thaliana. These data indicate that cysteine synthesis is not just an intermediate reaction step but that it is part of a regulatory network that mediates between inorganic sulfur supply and the demand for reduced sulfur during plant growth and in response to environmental changes. Received December 3, 2001 Accepted December 21, 2001     

Metabolism of Sulfur Compounds in Bacillus subtilis during Sporulation

Metabolism of various sulfur compounds in Bacillus subtilis during growth and sporulation was investigated by use of tracer techniques, in an attempt to clarify the mechanism involved in the formation of cystine rich protein of the spore coat.

Methionine, homocysteine, cystathionine, cysteine and some inorganic sulfur compounds (sulfate, sulfite and thiosulfate) were utilized by this organism as sulfur sources for its growth and sporulation. Biosynthesis of methionine from sulfate during growth was more or less inhibited by the addition of cysteine, homocysteine or cystathionine to the culture.

It is suggested from these results that in Bacillus subtilis methionine is synthesized from sulfate through cysteine, cystathionine and homocysteine as is the case in Salmonella or Neurospora. The results also suggest that the metabolism of sulfur-containing amino acids in Bacillus subtilis is strongly regulated by methionine and homocysteine.     

Transgenic soybean plants overexpressing <Emphasis Type="Italic">O</Emphasis>-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and Bowman–Birk protease inhibitor in seeds

Soybeans provide an excellent source of protein in animal feed. Soybean protein quality can be enhanced by increasing the concentration of sulfur-containing amino acids. Previous attempts to increase the concentration of sulfur-containing amino acids through the expression of heterologous proteins have met with limited success. Here, we report a successful strategy to increase the cysteine content of soybean seed through the overexpression of a key sulfur assimilatory enzyme. We have generated several transgenic soybean plants that overexpress a cytosolic isoform of O-acetylserine sulfhydrylase (OASS). These transgenic soybean plants exhibit a four- to tenfold increase in OASS activity when compared with non-transformed wild-type. The OASS activity in the transgenic soybeans was significantly higher at all the stages of seed development. Unlike the non-transformed soybean plants, there was no marked decrease in the OASS activity even at later stages of seed development. Overexpression of cytosolic OASS resulted in a 58–74% increase in protein-bound cysteine levels compared with non-transformed wild-type soybean seeds. A 22–32% increase in the free cysteine levels was also observed in transgenic soybeans overexpressing OASS. Furthermore, these transgenic soybean plants showed a marked increase in the accumulation of Bowman–Birk protease inhibitor, a cysteine-rich protein. The overall increase in soybean total cysteine content (both free and protein-bound) satisfies the recommended levels required for the optimal growth of monogastric animals.     

Manipulation of thiol contents in plants

As sulfur constitutes one of the macronutrients necessary for the plant life cycle, sulfur uptake and assimilation in higher plants is one of the crucial factors determining plant growth and vigour, crop yield and even resistance to pests and stresses. Inorganic sulfate is mostly taken up as sulfate from the soil through the root system or to a lesser extent as volatile sulfur compounds from the air. In a cascade of enzymatic steps inorganic sulfur is converted to the nutritionally important sulfur-containing amino acids cysteine and methionine (Hell, 1997; Hell and Rennenberg, 1998; Saito, 1999). Sulfate uptake and allocation between plant organs or within the cell is mediated by specific transporters localised in plant membranes. Several functionally different sulfate transporters have to be postulated and have been already cloned from a number of plant species (Clarkson et al., 1993; Hawkesford and Smith, 1997; Takahashi et al., 1997; Yamaguchi, 1997). Following import into the plant and transport to the final site of reduction, the plastid, the chemically relatively inert sulfate molecule is activated through binding to ATP forming adenosine-5'-phosphosulfate (APS). This enzymatic step is controlled through the enzyme ATP-sulfurylase (ATP-S). APS can be further phosphorylated to form 3'-phosphoadenosine-5'-phosphosulfate (PAPS) which serves as sulfate donor for the formation of sulfate esters such as the biosynthesis of sulfolipids (Schmidt and J?ger, 1992). However, most of the APS is reduced to sulfide through the enzymes APS-reductase (APR) and sulfite reductase (SIR). The carbon backbone of cysteine is provided through serine, thus directly coupling photosynthetic processes and nitrogen metabolism to sulfur assimilation. L-serine is activated by serine acetyltransferase (SAT) through the transfer to an acetyl-group from acetyl coenzyme A to form O-acetyl-L-serine (OAS) which is then sulhydrylated using sulfide through the enzyme O-acetyl-L-serine thiol lyase (OAS-TL) forming cysteine. Cysteine is the central precursor of all organic molecules containing reduced sulfur ranging from the amino acid methionine to peptides as glutathione or phytochelatines, proteines, vitamines, cofactors as SAM and hormones. Cysteine and derived metabolites display essential roles within plant metabolism such as protein stabilisation through disulfide bridges, stress tolerance to active oxygen species and metals, cofactors for enzymatic reactions as e.g. SAM as major methylgroup donor and plant development and signalling through the volatile hormone ethylene. Cysteine and other metabolites carrying free sulfhydryl groups are commonly termed thioles (confer Fig. 1). The physiological control of the sulfate reduction pathway in higher plants is still not completely understood in all details. The objective of this paper is to summarise the available data on the molecular analysis and control of cysteine biosynthesis in plants, and to discuss potentials for manipulating the pathway using transgenic approaches.     

Incorporation of methylsulfonylmethane sulfur into guinea pig serum proteins

Methionine, an essential amino acid, and cysteine are the major sulfur-containing amino acids in the body and both are thought to be synthesized predominantly in plants and micro-organisms. Methylsulfonylmethane (MSM) is a natural constituent of the environment in which it is found in plants, in milk and urine of both bovines and humans, is a normal oxidation product of dimethyl sulfoxide (DMSO) also in the natural environment and may be part of the natural global sulfur cycle. To determine whether sulfur from methylsulfonylmethane (MSM) is incorporated into sulfur amino acids, I fed 35S-MSM to guinea pigs. 35S was incorporated into peptidyl methionine and cysteine of guinea pig serum proteins. The specific activity of 35S-methionine was 30% greater than for 35S-cysteine, suggesting a precursor-product relationship. Total specific activity of serum proteins was increased by only 30% with a 100% increase of administered 35S-MSM, suggesting a limiting step in synthesis. Approximately 1% of the radioactivity was recovered in serum proteins, none in the feces and most was excreted in the urine. Microorganisms of intestinal lumen may be responsible for the incorporation of the 35S of MSM into sulfur amino acids. MSM may provide a source of sulfur for essential animal methionine by mechanisms not yet elucidated in either animals or micro-organisms.     

Current understanding of the factors regulating methionine content in vegetative tissues of higher plants

Methionine is a nutritionally essential, sulfur-containing amino acid found in low levels in plants, which often limits its value as a source of dietary protein to humans and animals. Methionine is also a fundamental metabolite in plant cells since, through its first metabolite, S-adenosylmethionine (SAM), it controls the level of several key metabolites, such as ethylene, polyamines and biotin. SAM is also the primary methyl group donor that regulates different processes in plants. Despite its nutritional and regulatory significance, the factors regulating methionine content in plants are not fully known. In this review, we summarize the current knowledge and recent progress made in our understanding of the methionine metabolism. The enzymes and substrates that regulate methionine synthesis were described, as well as the influences of the catabolic pathways of methionine on its content. The current effort to tailor an improvement of methionine content in vegetative tissues with minimal interference in plant growth and productivity is described as well. The accumulated knowledge has provided new insights into the control of methionine level in plants and, in some cases, has resulted in significant improvements in the nutritional value of plants.     

Molecular aspects of methionine biosynthesis

Methionine, lysine and threonine are essential amino acids required in the diets of non-ruminant animals. Major crops, such as corn, soybean and rice, are low in one or more of these amino acids. Currently, these amino acids are supplemented to animal feed to allow optimal growth--a costly process for farmers and consumer, therefore there is a great deal of interest in increasing essential amino acids in crops. The metabolism of methionine in plants is linked to the regulation of the aspartate pathway and is important for plant growth. In recent years, several key steps of this pathway have been identified at the molecular level, enabling us to initiate transgenic approaches to engineer the methionine content of plants.     

植物硫营养代谢、调控与生物学功能

植物作为无机硫的主要还原者,在全球的硫循环中起着关键作用。植物对土壤中硫酸盐的吸收运输和同化代谢,以及一系列具有重要生物学功能的含硫代谢产物的合成,不但与植物生长发育、耐逆和抗病虫害等密切相关,而且影响农作物产量与品质。硫营养的代谢和调控非常复杂,且生物学功能众多。本文综述了近年来植物硫营养代谢及调控及其在逆境胁迫中的生物学功能等方面的新进展,同时讨论了该领域悬而未决的重要生物学问题和研究动向,进而提出硫营养在农业生产上的重要性和所面临的新问题。     

植物硫营养代谢、调控与生物学功能

植物作为无机硫的主要还原者, 在全球的硫循环中起着关键作用。植物对土壤中硫酸盐的吸收运输和同化代谢, 以及一系列具有重要生物学功能的含硫代谢产物的合成, 不但与植物生长发育、耐逆和抗病虫害等密切相关, 而且影响农作物产量与品质。硫营养的代谢和调控非常复杂, 且生物学功能众多。本文综述了近年来植物硫营养代谢及调控及其在逆境胁迫中的生物学功能等方面的新进展, 同时讨论了该领域悬而未决的重要生物学问题和研究动向, 进而提出硫营养在农业生产上的重要性和所面临的新问题。     

Systems rebalancing of metabolism in response to sulfur deprivation, as revealed by metabolome analysis of Arabidopsis plants

Sulfur is an essential macro-element in plant and animal nutrition. Plants assimilate inorganic sulfate into two sulfur-containing amino acids, cysteine and methionine. Low supply of sulfate leads to decreased sulfur pools within plant tissues. As sulfur-related metabolites represent an integral part of plant metabolism with multiple interactions, sulfur deficiency stress induces a number of adaptive responses, which must be coordinated. To reveal the coordinating network of adaptations to sulfur deficiency, metabolite profiling of Arabidopsis has been undertaken. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry techniques revealed the response patterns of 6,023 peaks of nonredundant ion traces and relative concentration levels of 134 nonredundant compounds of known chemical structure. Here, we provide a catalogue of the detected metabolic changes and reconstruct the coordinating network of their mutual influences. The observed decrease in biomass, as well as in levels of proteins, chlorophylls, and total RNA, gives evidence for a general reduction of metabolic activity under conditions of depleted sulfur supply. This is achieved by a systemic adjustment of metabolism involving the major metabolic pathways. Sulfur/carbon/nitrogen are partitioned by accumulation of metabolites along the pathway O-acetylserine to serine to glycine, and are further channeled together with the nitrogen-rich compound glutamine into allantoin. Mutual influences between sulfur assimilation, nitrogen imbalance, lipid breakdown, purine metabolism, and enhanced photorespiration associated with sulfur-deficiency stress are revealed in this study. These responses may be assembled into a global scheme of metabolic regulation induced by sulfur nutritional stress, which optimizes resources for seed production.     

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.     

Measurement of homocysteine and related metabolites in human plasma and urine by liquid chromatography electrospray tandem mass spectrometry

The sulfur amino acids, methionine and cysteine play crucial roles in cells as a substrate for protein synthesis, as a methyl donor, and for the synthesis of sulfur-containing compounds, including the key intracellular tripeptide, glutathione. Homocysteine is an intermediary metabolite formed during the metabolism of methionine to cysteine. Dysregulation of homocysteine metabolism is implicated in adverse clinical outcomes such as increased risk of cardiovascular disease, stroke, Alzheimer's disease dementia and osteoporosis. While hyperhomocysteinemia is commonly observed in those conditions, the impact on other related metabolites is condition-specific. Therefore, there exists a need to establish precise and sensitive analytical techniques that allow for the simultaneous measurement of homocysteine and related metabolites in biological samples. The current review outlines the development and use of liquid chromatography electrospray tandem mass spectrometry (LC–MS/MS) to simultaneously measure metabolites involved in sulfur amino acid metabolism. Additionally, extensions of the technique in relation to the measurement of sulfur amino acid and one-carbon kinetics in vivo are discussed. The LC–MS/MS technique has the capacity for unambiguous analyte identification and confirmation, due to its high specificity and sensitivity. It has the greatest potential of being accepted and utilized as a dedicated homocysteine and its related metabolite Standard reference method (SRM).     

Cloning and characterization of two Lactobacillus casei genes encoding a cystathionine lyase

Volatile sulfur compounds are key flavor compounds in several cheese types. To better understand the metabolism of sulfur-containing amino acids, which certainly plays a key role in the release of volatile sulfur compounds, we searched the genome database of Lactobacillus casei ATCC 334 for genes encoding putative homologs of enzymes known to degrade cysteine, cystathionine, and methionine. The search revealed that L. casei possesses two genes that putatively encode a cystathionine beta-lyase (CBL; EC 4.4.1.8). The enzyme has been implicated in the degradation of not only cystathionine but also cysteine and methionine. Recombinant CBL proteins catalyzed the degradation of L-cystathionine, O-succinyl-L-homoserine, L-cysteine, L-serine, and L-methionine to form alpha-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum.     

Plant sulfate assimilation genes: redundancy versus specialization

Sulfur is an essential nutrient present in the amino acids cysteine and methionine, co-enzymes and vitamins. Plants and many microorganisms are able to utilize inorganic sulfate and assimilate it into these compounds. Sulfate assimilation in plants has been extensively studied because of the many functions of sulfur in plant metabolism and stress defense. The pathway is highly regulated in a demand-driven manner. A characteristic feature of this pathway is that most of its components are encoded by small multigene families. This may not be surprising, as several steps of sulfate assimilation occur in multiple cellular compartments, but the composition of the gene families is more complex than simply organellar versus cytosolic forms. Recently, several of these gene families have been investigated in a systematic manner utilizing Arabidopsis reverse genetics tools. In this review, we will assess how far the individual isoforms of sulfate assimilation enzymes possess specific functions and what level of genetic redundancy is retained. We will also compare the genomic organization of sulfate assimilation in the model plant Arabidopsis thaliana with other plant species to find common and species-specific features of the pathway.     

Cysteine and Methionine Regulation of Sulfate Uptake in Potato Tuber Discs (Solanum tuberosum)

Sulfate uptake in potato tuber discs is inhibited by cysteine and methionine with an 8 h lag period. Cysteine, but not methionine, inhibition can be reversed by washing the treated discs. During the experimental period cysteine is rapidly metabolized, while methionine persists as a free amino acid. Amino acid inhibition of sulfate uptake is overcome by increasing sulfate concentration. The kinetic parameters change suggesting a loss of flexibility of the sulfate uptake system caused by sulfur amino acids.