Lysine enhances methionine content by modulating the expression of S-adenosylmethionine synthase

Lysine and methionine are two essential amino acids whose levels affect the nutritional quality of cereals and legume plants. Both amino acids are synthesized through the aspartate family biosynthesis pathway. Within this family, lysine and methionine are produced by two different branches, the lysine branch and the threonine-methionine branch, which compete for the same carbon/amino substrate. To elucidate the relationship between these biosynthetic branches, we crossed two lines of transgenic tobacco plants: one that overexpresses the feedback-insensitive bacterial enzyme dihydrodipicolinate synthase (DHPS) and contains a significantly higher level of lysine, and a second that overexpresses Arabidopsis cystathionine gamma-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Significantly higher levels of methionine and its metabolite, S-methylmethionine (SMM), accumulated in the newly produced plants compared with plants overexpressing AtCGS alone, while the level of lysine remained the same as in those overexpressing DHPS alone. The increased levels of methionine and SMM were correlated with increases in the mRNA and protein levels of AtCGS and a reduced mRNA level for the genes encoding S-adnosylmethionine (SAM) synthase, which converts methionine to SAM. Reduction in SAMS expression level leads most probably to the reduction of SAM found in plants that feed with lysine. As SAM is a negative regulator of CGS, this reduction leads to higher expression of CGS and consequently to an increased level of methionine. Elucidating the relationship between lysine and methionine synthesis may lead to new ways of producing transgenic crop plants containing increased methionine and lysine levels, thus improving their nutritional quality.     

Mechanisms to account for maintenance of the soluble methionine pool in transgenic Arabidopsis plants expressing antisense cystathionine gamma-synthase cDNA

To investigate the role of cystathionine gamma-synthase (CGS) in the regulation of methionine synthesis Arabidopsis plants were transformed with a full-length antisense CGS cDNA and transformants analysed. Plants that were heterozygous for the transgene showed a 20-fold reduction of CGS activity that was accompanied by severe growth retardation and morphological abnormalities, from germination to flowering. Application of exogenous methionine to the transgenic lines restored normal growth. Surprisingly, transformed Arabidopsis plants exhibited a modest decrease in methionine content (35% reduction of the wild-type level) but a seven-fold decrease in the soluble pool of S-methylmethionine (SMM), a compound that plays a major role in storage and transport of reduced sulphur and labile methyl moieties. Several mechanisms can account for the maintenance of the soluble pool of methionine. First, the observed 20-fold increase in O-phosphohomoserine, a substrate of CGS, could compensate for the depressed level of CGS polypeptide by increasing the net rate of catalysis supported by the remaining enzyme. Second, the transgenic plants exhibited a two-fold increased level of cystathionine beta-lyase, the second enzyme in the methionine biosynthetic pathway. This indicates that enzymes other than CGS are subjected to a regulatory control by methionine or one of its metabolites. In addition to these mechanisms affecting de novo methionine synthesis, the recruitment of SMM to produce methionine may account for the small change of methionine levels in transgenic lines.     

Soluble methionine enhances accumulation of a 15 kDa zein, a methionine-rich storage protein, in transgenic alfalfa but not in transgenic tobacco plants

With the general aim of elevating the content of the essential amino acid methionine in vegetative tissues of plants, alfalfa (Medicago sativa L.) and tobacco plants, as well as BY2 tobacco suspension cells, were transformed with a beta-zein::3HA gene under the 35S promoter of cauliflower mosaic virus encoding a rumen-stable methionine-rich storage protein of 15 kDa zein. To examine whether soluble methionine content limited the accumulation of the 15 kDa zein::3HA, methionine was first added to the growth medium of the different transgenic plants and the level of the alien protein was determined. Results demonstrated that the added methionine enhanced the accumulation of the 15 kDa zein::3HA in transgenic alfalfa and tobacco BY2 cells, but not in whole transgenic tobacco plants. Next, the endogenous levels of methionine were elevated in the transgenic tobacco and alfalfa plants by crossing them with plants expressing the Arabidopsis cystathionine gamma-synthase (AtCGS) having significantly higher levels of soluble methionine in their leaves. Compared with plants expressing only the 15 kDa zein::3HA, transgenic alfalfa co-expressing both alien genes showed significantly enhanced levels of this protein concurrently with a reduction in the soluble methionine content, thus implying that soluble methionine was incorporated into the 15 kDa zein::3HA. Similar phenomena also occurred in tobacco, but were considerably less pronounced. The results demonstrate that the accumulation of the 15 kDa zein::3HA is regulated in a species-specific manner and that soluble methionine plays a major role in the accumulation of the 15 kDa zein in some plant species but less so in others.     

Functional characterization of a methionine gamma-lyase in Arabidopsis and its implication in an alternative to the reverse trans-sulfuration pathway

Methionine gamma-lyase (MGL) catalyzes the degradation of L-methionine to alpha-ketobutyrate, methanethiol and ammonia. The Arabidopsis (Arabidopsis thaliana) genome includes a single gene (At1g64660) encoding a protein (AtMGL) with approximately 35% identity to bacterial and protozoan MGLs. When overexpressed in Escherichia coli, AtMGL allowed growth on L-methionine as sole nitrogen source and conferred a high rate of methanethiol emission. The purified recombinant protein exhibited a spectrum typical of pyridoxal 5'-phosphate enzymes, and had high activity toward l-methionine, L-ethionine, L-homocysteine and seleno-L-methionine, but not L-cysteine. Quantitation of mRNA showed that the AtMGL gene is expressed in aerial organs and roots, and that its expression in leaves was increased 2.5-fold by growth on low sulfate medium. Emission of methanethiol from Arabidopsis plants supplied with 10 mM L-methionine was undetectable (<0.5 nmol min(-1) g(-1) FW), suggesting that AtMGL is not an important source of volatile methanethiol. Knocking out the AtMGL gene significantly increased leaf methionine content (9.2-fold) and leaf and root S-methylmethionine content (4.7- and 7-fold, respectively) under conditions of sulfate starvation, indicating that AtMGL carries a significant flux in vivo. In Arabidopsis plantlets fed L-[(35)S]methionine on a low sulfate medium, label was incorporated into protein-bound cysteine as well as methionine, but incorporation into cysteine was significantly (30%) less in the knockout mutant. These data indicate that plants possess an alternative to the reverse trans-sulfuration pathway (methionine-->homocysteine-->cystathionine-->cysteine) in which methanethiol is an intermediate.     

Restricting glutathione biosynthesis to the cytosol is sufficient for normal plant development

Glutathione (GSH) homeostasis in plants is essential for cellular redox control and efficient responses to abiotic and biotic stress. Compartmentation of the GSH biosynthetic pathway is a unique feature of plants. The first enzyme, γ-glutamate cysteine ligase (GSH1), responsible for synthesis of γ-glutamylcysteine (γ-EC), is, in Arabidopsis, exclusively located in the plastids, whereas the second enzyme, glutathione synthetase (GSH2), is located in both plastids and cytosol. In Arabidopsis, gsh2 insertion mutants have a seedling lethal phenotype in contrast to the embryo lethal phenotype of gsh1 null mutants. This difference in phenotype may be due to partial replacement of GSH functions by γ-EC, which in gsh2 mutants hyperaccumulates to levels 5000-fold that in the wild type and 200-fold wild-type levels of GSH. In situ labelling of thiols with bimane and confocal imaging in combination with HPLC analysis showed high concentrations of γ-EC in the cytosol. Feedback inhibition of Brassica juncea plastidic GSH1 by γ-EC in vitro strongly suggests export of γ-EC as functional explanation for hyperaccumulation. Complementation of gsh2 mutants with the cytosol-specific GSH2 gave rise to phenotypically wild-type transgenic plants. These results support the conclusion that cytosolic synthesis of GSH is sufficient for plant growth. The transgenic lines further show that, consistent with the exclusive plastidic localization of GSH1, γ-EC is exported from the plastids to supply the cytosol with the immediate precursor for GSH biosynthesis, and that there can be efficient re-import of GSH into the plastids to allow effective control of GSH biosynthesis through feedback inhibition of GSH1.     

Regulation of glutathione synthesis in leaves of transgenic poplar (Populus tremula X P. alba) overexpressing glutathione synthetase

The poplar hybrid Populus tremula X P. alba was transformed with the Escherichia coli gene for glutathione synthetase ( gsh II ) targetted to the cytosol. Leaves of five lines of transgenic plants exhibited glutathione synthetase activities 15- to 60-fold higher than those of wild-type plants. Total glutathione levels and GSH/GSSG ratios were similar in transgenic and wild-type plants. Precursor feeding experiments with cysteine and γ-glutamylcysteine suggest that glutathione synthesis in the cytoplasm is controlled by a multistep procedure that includes (i) the availability of cysteine, (ii) the availability of γ-glutamylcysteine, and (iii) regulation of the activities of both γ-glutamylcysteine synthetase and glutathione synthetase. However step (ii) may set an upper limit for the cellular glutathione content.     

Protein accumulation and rumen stability of wheat γ‐gliadin fusion proteins in tobacco and alfalfa

The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine‐rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine‐rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ‐gliadin‐δ‐zein and γ‐δ‐zein, as well as δ‐zein co‐expressed with β‐zein, all formed protein bodies. However, the γ‐gliadin‐δ‐zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ‐gliadin‐δ‐zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ‐gliadin‐δ‐zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ‐gliadin‐GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ‐gliadin‐δ‐zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ‐gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants.     

TILLING to detect induced mutations in soybean

Background

Potato is a staple food in the diet of the world's population and also being used as animal feed. Compared to other crops, however, potato tubers are relatively poor in the essential amino acid, methionine. Our aim was to increase the methionine content of tubers by co-expressing a gene involved in methionine synthesis with a gene encoding a methionine-rich storage protein in potato plants.

Results

In higher plants, cystathionine γ-synthase (CgS) is the first enzyme specific to methionine biosynthesis. We attempted to increase the methionine content of tubers by expressing the deleted form of theArabidopsis CgS (CgS _Δ90), which is not regulated by methionine, in potato plants. To increase the incorporation of free methionine into a storage protein theCgS _Δ90was co-transformed with the methionine-rich15-kD β-zein. Results demonstrated a 2- to 6-fold increase in the free methionine content and in the methionine content of the zein-containing protein fraction of the transgenic tubers. In addition, in line with higher methionine content, the amounts of soluble isoleucine and serine were also increased. However, all of the lines with high level of CgS_Δ90 expression were phenotypically abnormal showing severe growth retardation, changes in leaf architecture and 40- to 60% reduction in tuber yield. Furthermore, the colour of the transgenic tubers was altered due to the reduced amounts of anthocyanin pigments. The mRNA levels of phenylalanine ammonia-lyase (PAL), the enzyme catalysing the first step of anthocyanin synthesis, were decreased.

Conclusion

Ectopic expression of CgS_Δ90 increases the methionine content of tubers, however, results in phenotypic aberrations in potato. Co-expression of the 15-kD β-zein with CgS_Δ90 results in elevation of protein-bound methionine content of tubers, but can not overcome the phenotypical changes caused by CgS_Δ90 and can not significantly improve the nutritional value of tubers. The level ofPAL mRNA and consequently the amount of anthocyanin pigments are reduced in the CgS_Δ90 transgenic tubers suggesting that methionine synthesis and production of anthocyanins is linked.     

Salinity-induced glutathione synthesis in Brassica napus

The role of S-assimilation and the biosynthesis of cysteine and glutathione were studied during the response to salt stress of wild-type and salt-tolerant transgenic Brassica napus L. (canola) plants overexpressing a vacuolar Na+/H+ antiporter. A 3-fold increase in cysteine and glutathione content was observed in wild-type plants exposed to salt stress, but not in the transgenic plants. The induction of cysteine and glutathione synthesis during salt stress in the wild-type plants suggests a possible protective mechanism against salt-induced oxidative damage. On the other hand, the salt-tolerant transgenic plants did not show significant changes in either cysteine or glutathione content, confirming the role of vacuolar Na+ accumulation and ion homeostasis in salt tolerance.     

The cytosolic O-acetylserine(thiol)lyase gene is regulated by heavy metals and can function in cadmium tolerance

Regulation of the expression of the cytosolic O-acetylserine(thiol)lyase gene (Atcys-3A) from Arabidopsis thaliana under heavy metal stress conditions has been investigated. Northern blot analysis of Atcys-3A expression shows a 7-fold induction after 18 h of cadmium treatment. Addition of 50 microm CdCl(2) to the irrigation medium of mature Arabidopsis plants induces a rapid accumulation of the mRNA throughout the leaf lamina, the root and stem cortex, and stem vascular tissues when compared with untreated plants, as observed by in situ hybridization. High pressure liquid chromatography analysis of GSH content shows a transient increase after 18 h of metal treatment. Our results are compatible with a high cysteine biosynthesis rate under heavy metal stress required for the synthesis of GSH and phytochelatins, which are involved in the plant detoxification mechanism. Arabidopsis-transformed plants overexpressing the Atcys-3A gene by up to 9-fold show increased tolerance to cadmium when grown in medium containing 250 microm CdCl(2), suggesting that increased cysteine availability is responsible for cadmium tolerance. In agreement with these results, exogenous addition of cystine can, to some extent, also favor the growth of wild-type plants in cadmium-containing medium. Cadmium accumulates to higher levels in leaves of tolerant transformed lines than in wild-type plants.     

Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress

Phytochelatin (PC) plays an important role in heavy metal detoxification in plants and other living organisms. Therefore, we overexpressed an Arabidopsis PC synthase (AtPCS1) in transgenic Arabidopsis with the goal of increasing PC synthesis, metal accumulation, and metal tolerance in these plants. Transgenic Arabidopsis plants were selected, designated pcs lines, and analyzed for tolerance to cadmium (Cd). Transgenic pcs lines showed 12- to 25-fold higher accumulation of AtPCS1 mRNA, and production of PCs increased by 1.3- to 2.1-fold under 85 microM CdCl(2) stress for 3 d when compared with wild-type plants. Cd tolerance was assessed by measuring root length of plants grown on agar medium containing 50 or 85 microM CdCl(2). Pcs lines paradoxically showed hypersensitivity to Cd stress. This hypersensitivity was also observed for zinc (Zn) but not for copper (Cu). The overexpressed AtPCS1 protein itself was not responsible for Cd hypersensitivity as transgenic cad1-3 mutants overexpressing AtPCS1 to similar levels as those of pcs lines were not hypersensitive to Cd. Pcs lines were more sensitive to Cd than a PC-deficient Arabidopsis mutant, cad1-3, grown under low glutathione (GSH) levels. Cd hypersensitivity of pcs lines disappeared under increased GSH levels supplemented in the medium. Therefore, Cd hypersensitivity in pcs lines seems due to the toxicity of PCs as they existed at supraoptimal levels when compared with GSH levels.     

Expression of Arabidopsis plastidial phosphoglucomutase in tobacco stimulates photosynthetic carbon flow into starch synthesis

Phosphoglucomutase (PGM, EC 2.7.5.1) is one of the enzymes constituting the carbohydrate synthesis pathway in higher plants. It catalyzes the reversible conversion of glucose 6-phosphate (Glc6P) to glucose 1-phosphate (Glc1P). Previously, metabolic turnover analysis using (13)CO(2) in tobacco leaves demonstrated that conversion of Glc6P to Glc1P may limit carbon flow into carbohydrate synthesis. In order to assess the effects of PGM, Arabidopsis thaliana cytosolic or plastidial PGM was expressed under the control of cauliflower mosaic virus 35S promoter in tobacco plants (Nicotiana tabacum cv. Xanthi) and phenotypic analysis was performed. The transgenic plants expressing Arabidopsis plastidial PGM showed 3.5-8.2-fold higher PGM activity than that of wild-type, and leaf starch and sucrose contents increased 2.3-3.2-fold and 1.3-1.4-fold, respectively over wild-type levels. In vivo(13)C-labeling experiments indicated that photosynthetically fixed carbon in the transgenic plants could be converted faster to Glc1P and adenosine 5'-diphosphate glucose than in wild-type, suggesting that elevation of plastidial PGM activity should accelerate conversion of Glc6P to Glc1P in chloroplasts and increase carbon flow into starch. On the other hand, transgenic plants expressing Arabidopsis cytosolic PGM showed a 2.1-3.4-fold increase in PGM activity over wild-type and a decrease of leaf starch content, but no change in sucrose content. These results suggest that plastidial PGM limits photosynthetic carbon flow into starch.     

Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione

The coding sequence of the wild-type, cys-sensitive, cysE gene from Escherichia coli, which encodes an enzyme of the cysteine biosynthetic pathway, namely serine acetyltransferase (SAT, EC 2.3.1. 30), was introduced into the genome of potato plants under the control of the cauliflower mosaic virus 35S promoter. In order to target the protein into the chloroplast, cysE was translationally fused to the 5'-signal sequence of rbcS from Arabidopsis thaliana. Transgenic plants showed a high accumulation of the cysE mRNA. The chloroplastic localisation of the E. coli SAT protein was demonstrated by determination of enzymatic activities in enriched organelle fractions. Crude leaf extracts of these plants exhibited up to 20-fold higher SAT activity than those prepared from wild-type plants. The transgenic potato plants expressing the E. coli gene showed not only increased levels of enzyme activity but also exhibited elevated levels of cysteine and glutathione in leaves. Both were up to twofold higher than in control plants. However, the thiol content in tubers of transgenic lines was unaffected. The alterations observed in leaf tissue had no effect on the expression of O-acetylserine(thiol)-lyase, the enzyme which converts O-acetylserine, the product of SAT, to cysteine. Only a minor effect on its enzymatic activity was observed. In conclusion, the results presented here demonstrate the importance of SAT in plant cysteine biosynthesis and show that production of cysteine and related sulfur-containing compounds can be enhanced by metabolic engineering.     

Evidence of a significant role of glutathione reductase in the sulfur assimilation pathway

With the objective of studying the role of glutathione reductase (GR) in the accumulation of cysteine and methionine, we generated transgenic tobacco and Arabidopsis lines overexpressing the cytosolic AtGR1 and the plastidic AtGR2 genes. The transgenic plants had higher contents of cysteine and glutathione. To understand why cysteine levels increased in these plants, we also used gr1 and gr2 mutants. The results showed that the transgenic plants have higher levels of sulfite, cysteine, glutathione and methionine, which are downstream to adenosine 5′ phosphosulfate reductase (APR) activity. However, the mutants had lower levels of these metabolites, while the sulfate content increased. A feeding experiment using ³⁴SO₄^2– also showed that the levels of APR downstream metabolites increased in the transgenic lines and decreased in gr1 compared with their controls. These findings, and the results obtained from the expression levels of several genes related to the sulfur pathway, suggest that GR plays an essential role in the sulfur assimilation pathway by supporting the activity of APR, the key enzyme in this pathway. GR recycles the oxidized form of glutathione (GSSG) back to reduce glutathione (GSH), which serves as an electron donor for APR activity. The phenotypes of the transgenic plants and the mutants are not significantly altered under non‐stress and oxidative stress conditions. However, when germinating on sulfur‐deficient medium, the transgenic plants grew better, while the mutants were more sensitive than the control plants. The results give substantial evidence of the yet unreported function of GR in the sulfur assimilation pathway.