JXB Advance Access originally published online on July 2, 2004
Journal of Experimental Botany 2004 55(404):1799-1808; doi:10.1093/jxb/erh139
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RESEARCH PAPER |
Current understanding of the regulation of methionine biosynthesis in plants
Max-Planck-Institute of Molecular Plant Physiology, Department of Molecular Physiology, Am Muehlenberg 1, D-14476 Golm, Germany
* To whom correspondence should be addressed. Fax: +49 331 56789 8247. E-mail: hesse{at}mpimp-golm.mpg.de
Received 12 January 2004; Accepted 23 February 2004
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Abstract |
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Plants can provide most of the nutrients for the human diet. However, the major crops are often deficient in some of the nutrients. Thus, malnutrition, with respect to micronutrients such as vitamin A, iron, and zinc, but also macronutrients such as the essential amino acids lysine and methionine, affects more than 40% of the world's population. Recent advances in molecular biology, but also the grasp of biochemical pathways, metabolic fluxes, and networks can now be exploited to produce crops enhanced in key nutrients to increase the nutritional value of plant-derived foods and feeds. Some of the predictions appear to be accurate, while others not, reflecting the fact that plant metabolism is more complex than presently understood. A good example for a complex regulation is the methionine biosynthetic pathway in plants. The nutritional importance of Met and cysteine has motivated extensive studies of their roles in plant molecular physiology, especially regarding to their transport, synthesis, and accumulation in plants. Recent studies have demonstrated that Met metabolism is regulated differently in various plant species.
Key words:
Cystathionine 
-synthase, cystathionine ß-lyase, methionine biosynthesis, methionine synthase
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Introduction |
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Methionine (Met) is used at multiple levels in cellular metabolism: as a protein constituent, in the initiation of mRNA translation, and as a regulatory molecule in the form of S-adenosylmethionine (SAM). Thus, it can be assumed that Met synthesis, accumulation, and consumption are under stringent regulatory control (Matthews, 1999
; Hesse and Hoefgen, 2003). SAM itself has key functions as a primary methyl-group donor and as a precursor for metabolites such as ethylene, polyamines, vitamin B1, 3-dimethylsulphoniopropionate (an osmoprotectant), and as a source of atmospheric sulphur: dimethylsulphide (Amir et al., 2002). A derivative of Met, S-methylmethionine (SMM), is used as a major transport molecule for reduced sulphur in some plant, connecting sink and source organs (Bourgis et al., 1999). Furthermore, Met is an essential amino acid required in the diet of non-ruminant animals. Human and monogastric mammals can synthesize only half of the 20 proteinogenic amino acids and, therefore, must obtain the others from their diets. Major crops, such as cereals (e.g. corn, rice, etc.) and legumes, are low in Met (Tabe and Higgins, 1998; Hesse et al., 2001). Improved nutritional quality may help to solve problems encountered in cases where plant foods are the major or sole source of protein, such as in many developing countries, as well as plant feeds for livestock which are subsequently used as human food. To meet the essential amino acid requirements in animal feeds, supplements may be added or various plant sources are combined. The manipulation of essential amino acid levels in crops is, therefore, of high interest to breeders, feed producers, and, eventually, the consumer (Galili and Höfgen, 2002). For such manipulation to be effective, an understanding of the underlying metabolic regulation of these amino acids is essential. The use of mutants or transgenic plants altered with respect to the activity of a single enzyme allows its function to be analysed in vivo. Furthermore, ectopic expression of foreign enzymes enables the introduction of new pathways, resulting in the manipulation of metabolite concentrations and/or end products. |
Biosynthesis of methionine |
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Met, a sulphur-containing amino acid, and the amino acids lysine, threonine, and isoleucine constitute the aspartate family (Fig. 1). In plants, the branch point intermediate of threonine and Met synthesis is O-phosphohomoserine (OPH), which represents the common substrate for both threonine synthase (TS) and cystathionine gamma-synthase (CgS). OPH is either directly converted to threonine by TS or, in a three-step mechanism, to Met through condensation of cysteine and OPH to cystathionine, which is subsequently converted to homocysteine and then to Met by the enzymes CgS cystathionine ß-lyase (CbL), and methionine synthase (MS), respectively (Matthews, 1999
; Hesse and Höfgen, 2003). Eventually, about 20% of the Met is incorporated into proteins while 80% is converted to SAM, which comprises de facto the end-product of the Met biosynthetic pathway (Giovanelli et al., 1985). The use of mutants or transgenic plants altered with respect to the activity of single enzymes led to improved knowledge of the synthesis of sulphur-containing amino acids (Saito, 2000, and references therein). Recent studies suggest that Met synthesis in plants is controlled at the level of competition between CgS and TS for their common substrate OPH (Bartlem et al., 2000; Gakière et al., 2000a; Zeh et al., 2001).
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Get together: cystathionine formation |
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The first committed step of de novo Met synthesis in plants is the formation of the thioether cystathionine catalysed by cystathionine
-synthase (CgS) from the substrates cysteine and O-phosphohomoserine. The reaction involves a trans-sulphuration process via a -replacement reaction. This step separates Met synthesis from the other amino acids belonging to the aspartate family because of its connection with the sulphur assimilation pathway. Furthermore, the carbon precursor of Met synthesis is distinct from that in yeast and bacteria. In yeast, Met is synthesized by direct sulphydration of O-acetylhomoserine; in bacteria, it is through a different pathway with succinyl-homoserine as substrate. In micro-organisms, homoserine is the branch point intermediate leading to the synthesis of Met and threonine, whereas, in plants, O-phosphohomoserine is the last common intermediate to synthesize threonine and Met (Datko et al., 1974). In humans, Met synthesis is omitted and only the yeast-like conversion of feed methionine to cysteine occurs. Therefore, CgS is the branch point enzyme leading to Met synthesis competing with threonine synthase for the pathway intermediate, phosphohomoserine. Furthermore, for plant CgS enzymes a minor activity has been described in which sulphide is used as a substrate instead of cysteine to form homocysteine directly (Thompson et al., 1982b; Kreft et al., 1994; Ravanel et al., 1995b, 1998b). However, this alternative pathway seems to have only a minor physiological significance in plant cell metabolism regarding the entry of reduced sulphur into Met biosynthesis (MacNicol et al., 1981; Thompson et al., 1982a, b).
CgS has been purified from various plants (Aarnes, 1980; Kreft et al., 1994; Ravanel et al., 1995b, 1998b) and displays monomer sizes between 34.5 kDa (wheat) and 53 kDa (spinach) with native molecular masses between 155 kDa and 215 kDa, respectively, for the CgS homo-tetramer. Enzyme activity reaches a pH optimum at pH 7.5. The enzyme requires pyridoxalphosphate as a coenzyme for activity and operates by a hybrid ping-pong mechanism. cDNAs encoding CgS have been isolated from several plant species (Table 1; Ravanel et al., 1995a; Kim and Leustek, 1996; Hesse et al., 1999; Hughes et al., 1999; Nam et al., 1999; Riedel et al., 1999). The predicted proteins show high homology to each other and even to the corresponding bacterial genes. Southern blots suggest that soybean and potato CgSs are encoded by single or low copy number genes, respectively (Hughes et al., 1999; Hesse et al., 1999; Riedel et al., 1999).
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As demonstrated by feeding studies, CgS is not an allosteric enzyme since enzyme activity is not inhibited by Met (Thompson et al., 1982a
). Mutants and transgenic plants have been used to display the central role of CgS in the synthesis of Met. The characterization of mto1 Arabidopsis mutants, in which CgS modifications result in transcripts resistant to Met-dependent degradation, suggests that CgS is auto-regulated at the post-transcriptional level, presumably via a mechanism involving the N-terminal region of the AtCgS protein (Inaba et al., 1994; Chiba et al., 1999; Ominato et al., 2002). Mto1 mutants accumulate high levels of soluble Met (up to 40-fold) in young rosette leaves. Notably, these levels decrease during flowering, at the same time that levels rise in flowers, thus indicating a spatial and developmental regulation of the size of the soluble Met pool in Arabidopsis. Moreover, studies of Arabidopsis plants over-expressing CgS revealed that accumulation of Met (and SMM) inversely correlates with CgS levels only in tissues of flowering plants and not in young plants, suggesting that additional and perhaps more important factors contribute to the regulation of the size of the Met pool during plant development (Gakière et al., 2000b; Kim and Leustek, 2000). Taken together, these findings demonstrate that CgS is transcriptionally and post-transcriptionally regulated by Met or one of its metabolites and that the unregulated expression of CgS leads to an increase in Met content. This could not be supported by investigations of potato in which CgS over-expression was unable to increase the flow of carbon towards Met synthesis and in which an antisense approach resulted neither in phenotypic changes nor in disturbance of the free and bound amino acid composition (Kreft et al., 2003). However, in a corresponding experiment, endogenous CgS mRNA and protein levels were reduced by expression of a CgS antisense RNA in Arabidopsis. Transgenic plants with up to 9-fold less CgS activity revealed Met auxotrophy and developmental abnormalities resulting in severe growth stunting and an inability to flower. Furthermore, while increased free Met levels are able to regulate CgS in Arabidopsis trancriptionally and post-transcriptionally, potato CgS transcript stability is not influenced by Met (Kreft et al., 2003). This finding has been supported recently for Arabidopsis. Goto et al. (2002) identified an S-adenosylmethionine synthetase mutant (mto3-2) with elevated Met levels. Despite this fact, the endogenous CgS mRNA level did not respond to the mto3-2 phenotype by transcript reduction. |
Separating again: homocysteine formation through trans-sulphurylation |
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 TopAbstractIntroductionBiosynthesis of methionineGet together: cystathionine... Separating again: homocysteine... Coming to an end:...Regulation of methionine...Looking into the future:...References |
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Cystathionine ß-lyase (CbL) catalysis the ß-cleavage of cystathionine to homocysteine. CbL has been purified from Spinacia oleracea, Arabidopsis thaliana, and Echinochloa colonum (Droux et al., 1995
; Ravanel et al., 1996, 1998a; Turner et al., 1998). The native pyridoxalphosphate-dependent CbL protein with a molecular mass of 170 kDa consists of four identical subunits with an optimal pH range between pH 8.3 and 9.0. CbL was cloned from Arabidopsis and potato by complementation for the E. coli Met auxotroph GUC41, which lacks CbL activity (Ravanel et al., 1995a; Maimann et al., 2000) or by homologous screening (A. thaliana: Bork and Hell, 1997). Both predicted proteins contain an N-terminal extension showing features of a plastidial targeting sequence. More sequences are available under www.ncbi.nlm.nih.gov (Table 2).
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There is evidence of only one gene encoding CbL in Arabidopsis and of a low copy gene family in potato. Its essential role was demonstrated through the isolation of a Met–mutant from Nicotiana plumbaginifolia protoplast cultures by Negrutiu et al. (1985)
. The mutant shows a severe phenotype, stunted in growth and development exhibiting CbL activity 50% below the wild type. Higher inhibition rates proved to be lethal. Supplying homocysteine and Met in spraying experiments were able to restore the growth to the wild type. Further indications of the essential role of CbL were demonstrated in transgenic potato plants expressing antisense RNA (Maimann et al., 2000) showing the same severe phenotype as the tobacco mutant. Intriguingly, depending on the remaining CbL activity, metabolites of the aspartate pathway and sulphur assimilation are affected by this modification. Met decreases in content whereas cysteine, homoserine, and cystathionine accumulate, demonstrating the reduced flow of Met precursors towards Met synthesis. Unexpectedly homocysteine increases in content. Explanations for this phenomenon remain speculative. Although it has been suggested that CgS may catalyse homocysteine formation (Thompson et al., 1982b; Kreft et al., 1994; Ravanel et al., 1995b, 1998b), this assumption is unlikely for two reasons. First, homocysteine formation only takes place in the absence of cysteine, which actually accumulates in the transgenic plants. Second, this direct sulphydration pathway participates in only 3% of total homocysteine synthesis and has seemingly no physiological significance (Giovanelli et al., 1978; MacNicol et al., 1981). The accumulating homocysteine should be methylated to Met by Met synthase (MS). However, a decrease in Met was observed, while homocysteine increased. Analysis of MS using RNA and protein blots revealed that neither expression level nor protein amount of MS was altered compared with the wild type. As an alternative explanation, one might assume specific compartmentalization of the homocysteine pool, derived from a spontaneous degradation of cystathionine under the acidic conditions of the vacuole, occluding it from the cytosolically localized MS. On the other hand, the expression of CbL did not lead to an increase in flux to Met. Although transgenic Arabidopsis (Gakière et al., 2000b) and potato (Maimann et al., 2001) plants accumulated both CbL transcript and protein, and contained subsequently increased enzymatic activity (e.g. for potato up to 2.5-fold), no significant changes in content of amino acids and pathway intermediates could be shown when transgenic and wild-type plants were compared. This assumption is supported by increasing leaf Met levels when cystathionine is supplied via petioles to detached leaves of potato in both wild-type and transgenic plants over expressing CbL. Thus, CbL seems to have a low control coefficient for the carbon flow towards Met synthesis and is further hampered by an insufficient supply of substrate. This supports the assumption that the availability of OPH might act as the flux-controlling metabolite in the Met synthesis pathway.
These results are further supported by the recent findings of Frankard et al. (2002) for the above-mentioned Nicotiana plumbaginifolia mutant complemented by targeting the E. coli CbL homologue, metC, to chloroplasts resulting in full complementation of the mutant phenotype and restoration to the wild-type-like plants. Met levels were restored to wild-type levels, but no accumulation of Met or its derivatives was achieved.
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Coming to an end: a methyl group transfer finishes the synthesis of methionine |
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The last step of Met synthesis is localized in the cytosol (Wallsgrove et al., 1983
) and catalysed by methionine synthase (MS), which methylates homocysteine to form Met using N5-methyltetrahydrofolate as a methylgroup-donor. The function of this enzyme is on the one hand the de novo synthesis of Met and on the other the regeneration of the methyl group of S-adenosylmethionine. So far, the molecular and biochemical characterization of methionine synthase from plants is still limited. One reason for this is the low amount of protein present in plants and another is the substrate specificity of the enzyme. While bacteria are able to use monoglutameric methyltetrahydrofolate, Catharanthus roseus (Eichel et al., 1995) accepts only the triglutameric isoform that is not freely available. Neither SAM nor cobalamin is required for activity of the cobalamin-independent methionine synthase as demonstrated for MS from C. roseus.
Full-length cDNAs have been described from, for example, Coleus blumei (Petersen et al., 1995), Mesembryanthemum crystallinum (U84889
[GenBank]
), Chlamydomonas reinhardtii (Kurvari et al., 1995), and Solanum tuberosum (Zeh et al., 2001). In addition, sequences of MS from other plants are available under www.ncni.nlm.nih.gov (Table 3). Full-length cDNAs code for proteins with a molecular mass of about 85 kDa with no recognizable signal peptide.
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Intensive studies on MS expression revealed that MS is a low copy gene differentially expressed, at least in potato organs, with elevated levels in flowers, basal levels in sink and source leaves, roots, and stolons, and low levels in stems and tubers. This is in good agreement with protein data except that the protein content in leaves was less than expected from the RNA data. Western blot analysis of subcellular fractions revealed that the protein is located in the cytosol. However, the changing pattern of gene expression during the day/night period implied a light-dependent control of MS-transcription normally seen for enzymes localized in plastids. The expression of MS was shown to be light-inducible with its highest expression at midday, while, during the night, expression dropped to a low, basal mRNA level. These RNA data were not confirmed at the protein level since the protein content remained constant over the whole day. Feeding experiments using detached leaves revealed that sucrose or sucrose-derived products are responsible for the induction of StMS1 gene expression, but with no effect on the protein level in C. roseus and S. tuberosum (Eckermann et al., 2000
; Zeh et al., 2001). The localization of the final step can be debated (Ravanel et al., 1998a). Observations based on enzyme measurements have been made in pea chloroplasts and mitochondria suggesting that the de novo plastidial synthesis of Met is favoured (Shah and Cossins, 1970; Clandinin and Cossins, 1974). By contrast, the photosynthetic protozoan E. gracilis Z. expresses three isoforms of cobalamin-dependent enzymes located in chloroplasts, mitochondria, and cytosol in addition to a cytosolic cobalamin-independent Met synthase (Isegawa et al., 1994). None of the identified plant cDNAs code for a signal peptide, nor could a plastidial localization be shown via western blot analysis (Zeh et al., 2002).
To investigate the role of MS in the synthesis of Met further, homologous expression of MS sense and antisense constructs has been performed in potato (Nikiforova et al., 2002). Among transgenic plants exhibiting no phenotypic changes, only a few plants with reduced expression of MS were observed, and even these plants exhibited no change in protein content. MS over-expressing plants exhibited a moderate increase of MS mRNA signal as indicated using northern blot analysis, but again these changes were not reflected by changes in protein content. Enzymatic measurements of MS activity were not performed due to the unavailability of the substrate. Analyses of pathway-relevant amino acids, such as Met, threonine, isoleucine, lysine, and aspartate resulted in insignificant minor changes (e.g. Thr by factor 1.8; Nikiforova et al., 2002). This indicates that genetic modulation of MS resulted in only minor changes. It can be speculated that over-expression of MS leads to a slight increase of Met, probably camouflaged by conversion of Met to SAM. Elevated SAM levels might be responsible for an increase in threonine through a SAM-induced increase of TS activity. A determination of metabolites downstream of Met, such as SAM or S-methylmethionine (Bourgis et al., 1999) has not been performed. The fact that only moderate alterations of MS expression and protein levels could be achieved through over-expression and antisense inhibition indicates the essential role of MS for Met biosynthesis, but probably even more the importance of the plant one carbon methylation cycle. MS activity is essential for Met synthesis and recycling of SAM and seems to be tightly controlled within narrow borders. Hence, one can speculate that greater alterations are not tolerated without affecting plant growth, thus prohibiting the regeneration of genetically altered plants with grossly changed MS activity. This result is supported by Gallardo et al. (2002) who investigated the proteosome of germinating Arabidopsis seeds. Met synthase and S-adenosylmethionine synthetase were identified as proteins, probably having fundamental controlling function in plants, indicating a superposition of SAM and Met in plant metabolism.
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Regulation of methionine synthesis |
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Several lines of recent evidence show that the branch point between CgS and TS plays a major regulatory role in the flux of carbon into Met (Fig. 2), and that CgS competes fairly weakly with TS for their common substrate O-phosphohomoserine. The enzymatic activity of plant TS is strongly stimulated by SAM, the end-product of the competing pathway (Curien et al., 1996
). Because Km values of fully activated TS for OPH have been shown to be 250–500-fold lower than those of CgS (Curien et al., 1998; Ravanel et al., 1998a), carbon flux is directed into the Thr branch when Met and, hence, SAM levels are high. Characterization of transgenic and mutant plants altered in aspartate kinase activity or in the CgS to TS ratio has provided important evidence for the essential function of this competition for the flow of carbon into either Met or Thr synthesis. Plants overproducing a bacterial feedback-insensitive aspartate kinase exhibited higher levels of threonine, but Met levels increased only slightly (Galili and Höfgen, 2002). Arabidopsis plants expressing CgS antisense mRNA revealed a 4–7-fold increase in Thr levels accompanied by severe morphological aberrations due to reduced Met synthesis capacity (Gakière et al., 2000b; Kim and Leustek, 2000).
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More interesting findings were obtained when TS activity was reduced: an Arabidopsis mutant (mto2), deficient in TS enzymatic activity, exhibited an accumulation of free Met in young rosette leaves (20-fold), accompanied by comparably reduced soluble Thr contents (down to 6%), but not in mature plants (Bartlem et al., 2000
). These results suggest that in young Arabidopsis plants, the regulation of Met synthesis is mainly dependent on the dynamic interplay between changing biochemical properties of CgS and TS (Thompson et al., 1982a; Bartlem et al., 2000). Moreover, when the CgS to TS ratio is altered in favour of CgS, autoregulation of CgS alone is not sufficient to maintain the net rate of Met synthesis. This suggests that Met regulates its own synthesis through SAM (Fig. 2). When a high level of Met, and hence SAM, is produced, TS activity is increased, causing reduced O-phosphohomoserine availability for Met synthesis (Curien et al., 1996). Similarly, a reduction in TS levels by an antisense approach in potato plants caused a reduction of 45% in threonine levels, whereas Met levels increased by up to 239 times compared with non-transformed plants (Zeh et al., 2001). Notably, a reduction of TS activity in both Arabidopsis and potato caused a far stronger molar increase in Met levels than the molar decrease in threonine levels. These results suggest that a reduction in TS levels causes an increased flux of the carbon skeleton from aspartate to Met, although possible effects of decreased TS level on a reduction of Met catabolism cannot be ruled out. The complex regulation of the TS-branch-point can be further illustrated by a study in which a bacterial TS was produced in transgenic tobacco plants (Muhitch, 1997). This expression resulted in a 5-fold increase in threonine, but no decrease in Met, even though CgS activity increased by 3.5 times in these transgenic plants, apparently to compensate for the increased competition of TS for the common substrate, O-phosphohomoserine (Muhitch, 1997).
Recent results from transgenic Arabidopsis plants manipulated in CgS enzymatic activity levels gave rise to the hypothesis that CgS exerts major flux control for Met metabolism in Arabidopsis (Gakière et al., 2000a, 2002; Kim and Leustek, 2000; Kim et al., 2002). This hypothesis is supported by studies indicating that Arabidopsis CgS is feedback-regulated by Met itself or derivatives at the post-transcriptional level (Chiba et al., 1999, 2003; Bartlem et al., 2000). Up to now, molecular investigations of CgS regulation have been focused on a stretch of 39 amino acids, encoded by exon 1 of AtCgS and designated as the MTO1 region, which is believed to act in cis to destabilize its own transcript in a process that involves Met or related metabolites such as SAM, at least in Arabidopsis (Chiba et al., 1999, 2003; Suzuki et al., 2001; Lambein et al., 2003). Accordingly, AtCgS mRNA levels and enzymatic activities are reduced in the presence of excess Met in Arabidopsis (Inaba et al., 1994; Chiba et al., 1999; Bartlem et al., 2000). By contrast, it could be shown that increasing the soluble Met pool in potato leaves was not accompanied by changes in levels of CgS transcript or activity (Kreft et al., 2003). Even though this paradoxical observation is difficult to explain, it cannot exclusively be attributed to the polypeptide encoded by CgS exon 1. The amino acid sequence in the MTO1 region is almost perfectly conserved among plant species including both potato CgS isoforms, thus indicating a general motif with a functional role (Chiba et al., 1999; Ominato et al., 2002). Transfection experiments with wild-type and mutant forms of CgS in Arabidopsis suggest a novel post-transcriptional control of CgS gene expression in which amino acids encoded by the MTO1 mRNA region act in cis to destabilize the CgS mRNA in response to high levels of Met or one of its metabolites (Chiba et al., 1999, 2003; Lambein et al., 2003). The regulatory mechanism is not known, but computer analysis predicts that mRNA sequences near the MTO1 region can form stable stem-loop structures (Amir et al., 2002), supporting a model of post-transcriptional control by this region (Chiba et al., 1999; Kim et al., 2002; Lambein et al., 2003). In this model it is proposed that the regulation occurs during translation when the nascent polypeptide of CgS and its mRNA are in close proximity. This model predicts that inhibition of translation abolishes the regulation.
The results obtained for the mto1 mutant also support previous reports from feeding experiments in Lemna (Thompson et al., 1982a), which indicated that Met might regulate its own synthesis by repressing CgS gene expression. Notably, the post-transcriptional regulation machinery predicted for Arabidopsis CgS might not exist in potato. Transgenic potato plants expressing a TS antisense construct significantly overproduced free Met, similar to the mto2 mutant of Arabidopsis, which also has reduced TS activity (Bartlem et al., 2000; Zeh et al., 2001).
Alignment of the amino acid sequences from different organisms revealed that the plant enzyme has an extended N-terminal region that is not found in the bacterial enzyme. To test whether this N-terminal region plays a regulatory role in Met metabolism, in addition to post-transcriptional regulation by the MTO1 region, transgenic Arabidopsis and tobacco plants overproducing a truncated CgS that lacks this region and the natural form of CgS were analysed (Hacham et al., 2002). These plants exhibited increases in free Met levels (up to 40-fold) and SMM contents (up to 25-fold) as a result of expressing either full-length or N-terminally truncated AtCgS proteins, respectively (Gakière et al., 2002; Hacham et al., 2002; Kim et al., 2002). Furthermore, these plants emitted 20 times more of the Met catabolic products dimethyl sulphide and carbon disulphide, and also contained 40 times more of the Met-derived hormone ethylene. A similar change in plant phenotype, the emission of Met catabolic products, was also detected in transgenic plants expressing a SAM-synthase antisense construct (Boerjan et al., 1994; Shen et al., 2002). Thus, despite the unambiguous role of the MTO1 region, ectopic over-expression of wild type or mutated CgS might still outrun the RNA destabilization, eventually resulting in the observed accumulation of downstream metabolites. Yet, over-expression of CgS in potato did not lead to accumulation of downstream metabolites (Kreft et al., 2003). As a simple explanation, one might assume that substrates required for cystathionine synthesis are not available in sufficient amounts to cope with increased CgS activity.
When taking into account changes taking place during different developmental stages, such as decreased Met biosynthesis in rosette leaves upon the onset of flowering (Chiba et al., 1999), further superimposed regulatory mechanisms acting on Met synthesis have to be assumed.
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Looking into the future: biotechnological aspects |
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A major target of academician and industry has been the improvement of the amino acid composition of seed proteins. Analysis of seed proteins in crops revealed that, in most cases, the amino acid composition with respect to nutritional parameters is unbalanced, especially for the amino acids Met, lysine, and threonine, and, in some cases, tryptophan (Mertz et al., 1964
), which subsequently reduces nutritional quality or increases waste nitrogen release. For example, although corn meals used in animal feeds are usually supplemented with legume meals to increase the level of lysine, this corn–legume mixture remains deficient in Met. Thus, sales of synthetic Met to animal producers for supplementation are direct expenses for farmers and are therefore passed on to consumers. Furthermore, it is likely that the Met level of the human cereal–legume diet is inadequate in many developing countries (Tabe and Higgins, 1998). Therefore, methods of genetically increasing Met levels in soybean and maize varieties are needed. Sulphur-rich proteins are thought to be good targets for crop improvement since important cereals and legume seeds often lack the desired levels of sulphur-containing amino acids.
One approach for manipulating amino acid composition in plants is the introduction of a heterologous gene into the target host that encodes a protein with a balanced amino acid profile or that is enriched in the amino acid that is under-represented. For this purpose, different plant species have been transformed with genes encoding sulphur-rich proteins. Examples are canola (Brassica napus, Altenbach et al., 1992), narbon bean (Vicia narbonensis, Saalbach et al., 1995), narrow leaf lupin (Lupinus angustifolius, Molvig et al., 1997), maize (Anthony et al., 1997; Lai and Messing, 2002), and rice (Oryza sativa; Hagan et al., 2003). These transformants accumulate SSA up to approximately 7% of total protein reaching 80% of the FAO standard for Met in mature grains or tubers (seed albumin from Amaranthus hypochondriacus; Chakraborty et al., 2000). In some cases, it could be shown that accumulation of sulphur-rich proteins results in the diversion of limited sulphur reserves away from the synthesis of endogenous proteins, resulting in no or only minor net changes in the total balance. The most likely mediator of such a response is the mechanism that modulates seed storage protein composition in response to sulphur and nitrogen availability (Tabe et al., 2002).
A major problem with foreign proteins is their allergenicity. In the case of the transformation of soybean with the high-Met Brazil nut protein, commercialization did not occur because of the protein's allergenic properties (Nordlee et al., 1996). As an alternative approach to using a heterologous protein, the expression of an endogenous protein can be altered. Using a seedling screen on lysine-plus-threonine supplemented media, a natural variant of maize was found with elevated protein-bound Met in the form of a 10 kDa 
-zein seed storage protein Dzs10 in its seeds (Phillips and McClure, 1985; Phillips et al., 1981).
Although expression of genes for methionine-rich proteins seems to be a promising approach to increasing overall methionine availability in foods and feeds, it is still not enough to increase methionine content to 100% of the FAO recommendation. Thus, this ‘pull’ approach might well be complemented synergistically through ‘push’ approaches aimed at increasing the biosynthetic rate of the desired amino acids (Fig. 3). This can be accomplished through over-expression of homologous or heterologous pathway genes, or pathway genes mutated in properties such as feedback inhibition, the introduction of entire new pathways from other organisms, or the down-regulation of competing pathways through, for example, antisense (Galili and Höfgen, 2002). With respect to sulphur-containing amino acids, push approaches have been successful with respect to overproducing serine-acetyl transferase (SAT) or APS reductase with bacterial genes up to 4-fold in free cysteine levels (Harms et al., 2000; Blaszczyk et al., 1999, 2002; Tsakraklides et al., 2002). Partial inhibition of threonine synthase (Zeh et al., 2001) resulted in Met over-accumulation (up to 240-fold), as well as the previously discussed examples on CgS over-expression (40-fold) and SAM synthetase inhibition (up to 450-fold).
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-TS, antisense threonine synthase; CgS, cystathionine The next logical step is the application of this approach to improve seed storage protein composition. In maize, push approaches resulted in increased accumulation of endogenous sulphur-containing proteins indicating a previous limitation of Met and Cys supply, while in other cases sulphur-rich seed proteins (Tabe et al., 2002
; Chakraborty et al., 2000; Galili and Höfgen, 2002) need to be expressed in the respective push background. Notably, the limited investigations on maize, potato, and Arabidopsis to date have revealed that differences in the various crops, or perhaps even cultivars, have to be taken into account when manipulating a metabolic process as highly regulated as amino acid biosynthesis. A general formula is still not at hand, despite the progress achieved in the field. Strategies are in place, however, and research continues to move ever closer to the development of more nutritious crops.
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Acknowledgements |
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The authors' work has been supported by the European Union (Bio4CT 97-2182, QLRT-2000-00103), the Deutsche Forschungsgemeinschaft, DFG, and the Max-Planck Society.
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References |
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Amir R, Hacham Y, Galili G. 2002. Cystathionine
-synthase and threonine synthase operate in concert to regulate carbon flow towards methionine in plants. Trends in Plant Science 7, 153–156.[CrossRef][ISI][Medline]Aarnes H. 1980. Biosynthesis of the thioether cystathionine in barley seedlings. Plant Science Letters 19, 81–89.[CrossRef]
Altenbach SB, Chiung-Chi K, Staraci LC, Pearson KW, Wainwright C, Georgescu A, Townsend J. 1992. Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine. Plant Molecular Biology 18, 235–245.[CrossRef][ISI][Medline]
Anthony A, Brown W, Buhr D, Ronhovde G, Genovesi D, Lane T, Yingling R, Aves K, Rosato M, Anderson P. 1997. Transgenic maize with elevated 10 kDa zein and methionine. In: Cram WJ, De Kok LJ, Stulen I, Brunold C, Rennenberg H, eds. Sulphur metabolism in higher plants. Leiden: Backhuys Publishers, 295–297.
Bartlem D, Lambein I, Okamoto T, Itaya A, Uda Y, Kijima F, Tamaki Y, Nambara E, Naito S. 2000. Mutation in the threonine synthase gene results in an over-accumulation of soluble Met in Arabidopsis. Plant Physiology 123, 101–110.
Blaszczyk A, Brodzik R, Sirko A. 1999. Increased resistance to oxidative stress in transgenic tobacco plants overexpressing bacterial serine acetyltransferase. The Plant Journal 20, 237–243.[CrossRef][ISI][Medline]
Blaszczyk A, Sirko L, Hawkesford MJ, Sirko A. 2002. Biochemical analysis of transgenic tobacco lines producing bacterial serine acetyltransferase. Plant Science 162, 589–597.[CrossRef]
Boerjan W, Bauw G, Van Montagu M, Inzé D. 1994. Distinct phenotypes generate by over-expression and suppression of S-adenosyl-L-methionine synthetase reveal developmental patterns of gene silencing in tobacco. The Plant Cell 6, 1401–1414.[Abstract]
Bork C, Hell R. 1997. Cloning and expression of CBL1 gene (Accession No. AJ001148) encoding cystathionine ß-lyase from Arabidopsis thaliana. Plant Physiology 115, 864.
Bourgis F, Roje S, Nuccio ML, et al. 1999. S-methylmethionine plays a major role in phloem sulphur transport and is synthesized by a novel type of methyltransferase. The Plant Cell 11, 1485–1497.
Chakraborty S, Chakraborty N, Datta A. 2000. Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus. Proceedings of the National Academy of Sciences, USA 97, 3724–3729.
Chiba Y, Ishikawa M, Kijima F, Tyson RH, Kim J, Yamamoto A, Nambara E, Leustek T, Wallsgrove RM, Naito S. 1999. Evidence for autoregulation of cystathionine -synthase mRNA stability in Arabidopsis. Science 286, 1371–1374.
Chiba Y, Sakurai R, Yoshino M, Ominato K, Ishikawa M, Onouchi H, Naito S. 2003. S-adenosyl-L-methionine is an effector in the post-transcriptional autoregulation of the cystathionine -synthase gene in Arabidopsis. Proceedings of the National Academy of Sciences, USA 100, 10225–10230.
Clandinin MT, Cossins EA. 1974. Methionine biosynthesis in isolated Pisum sativum mitochondria. Phytochemistry 13, 585–591.[CrossRef]
Curien G, Dumas R, Ravanel S, Douce R. 1996. Characterization of an Arabidopsis thaliana cDNA encoding an S-adenosylmethionine-sensitive threonine synthase—threonine synthase from higher plants. FEBS Letters 390, 85–90.[CrossRef][ISI][Medline]
Curien G, Job D, Douce R, Dumas R. 1998. Allosteric activation of Arabidopsis threonine synthase by S-adenosylmethionine. Biochemistry 37, 13212–13221.[CrossRef][Medline]
Datko AH, Giovanelli J, Mudd SH. 1974. Homocysteine biosynthesis in green plants. O-phosphohomoserine is the physiological substrate for cystathionine -synthase. Journal of Biological Chemistry 249, 1139–1155.
Droux M, Ravanel S, Douce R. 1995. Methionine biosynthesis in higher plants. II. Purification and characterisation of cystathionine ß-lyase from spinach chloroplasts. Archives of Biochemistry and Biophysics 316, 585–595.[CrossRef][ISI][Medline]
Eichel J, González JC, Hotze M, Matthews RG, Schröder J. 1995. Vitamin-B12-independent methionine synthase from higher plant (Catharanthus roseus). Molecular characterisation, regulation, heterologous expression, and enzyme properties. European Journal of Biochemistry 230, 1053–1058.[ISI][Medline]
Eckermann C, Eichel J, Schröder J. 2000. Plant methionine synthase: new insights into properties and expression. Journal of Biological Chemistry 381, 695–703.
Frankard V, Ispas G, Hesse H, Jacobs M, Höfgen R. 2002. A defect in cystathionine beta-lyase activity causes the severe phenotype of a Nicotiana plumbaginifolia methionine auxotroph. Plant Science 162, 607–614.[CrossRef]
Gakière B, Denis L, Droux M, Job D. 2002. Over-expression of cystathionine -synthase in Arabidopsis thaliana leads to increased levels of methionine and S-methylmethionine. Plant Physiology and Biochemistry 40, 119–126.
Gakière B, Denis L, Droux M, Ravanel S, Job D. 2000b. Methionine synthesis in higher plants: sense strategy applied to cystathionine -synthase and cystathionine ß-lyase in Arabidopsis thaliana. In: Brunold C, ed. Sulphur nutrition and sulphur assimilation in higher plants. Bern, Switzerland: Paul Haupt, 313–315.
Gakière B, Ravanel S, Droux M, Douce R, Job D. 2000a. Mechanisms to account for maintenance of the soluble methionine pool in transgenic Arabidopsis plants expressing antisense cystathionine -synthase cDNA. CR Academie de Science III 323, 841–851.
Gallardo K, Job C, Groot SPC, Puype M, Demol H, Vandekerckhove J, Job D. 2002. Importance of methionine biosynthesis for Arabidopsis seed germination and seedling growth. Physiologia Plantarum 116, 238–247.[CrossRef][Medline]
Galili G, Höfgen R. 2002. Metabolic engineering of amino acids and storage proteins in plants. Metabolic Engineering 4, 3–11.[CrossRef][ISI][Medline]
Goto DB, Ogi M, Kijima F, Kumagai T, van Werven F, Onouchi H, Naito S. 2002. A single-nucleotide mutation in a gene encoding S-adenosylmethionine synthetase is associated with methionine over-accumulation phenotype in Arabidopsis thaliana. Genes and Genetic Systems 77, 89–95.
Giovanelli J, Mudd SH, Datko AH. 1978. Homocysteine biosynthesis in green plants. Physiological importance of the transsulphuration pathway in Chlorella sorokiniana growing under steady-state conditions with limiting sulfate. Journal of Biological Chemistry 253, 665–677.
Giovanelli J, Mudd SH, Datko AH. 1985. Quantitative analysis of pathways of methionine metabolism and their regulation in Lemna. Plant Physiology 78, 555–560.
Hacham Y, Avraham T, Amir R. 2002. The N-terminal region of Arabidopsis cystathionine -synthase plays an important role in methionine metabolism. Plant Physiology 128, 454–462.
Hagan ND, Upadhyaya N, Tabe LM, Higgins TJV. 2003. The redistribution of protein sulphur in transgenic rice expressing a gene for a foreign, sulphur-rich protein. The Plant Journal 34, 1–11.[CrossRef][ISI][Medline]
Harms K, von Ballmoos P, Brunold C, Höfgen R, Hesse H. 2000. Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione. The Plant Journal 22, 335–343.[CrossRef][ISI][Medline]
Hesse H, Basner A, Willmitzer L, Höfgen R. 1999. Cloning and characterisation of a cDNA (Accession No. AF082892) encoding a second cystathionine gamma-synthase in potato (Solanum tuberosum L.). Plant Physiology 121, 1057.
Hesse H, Kreft O, Maimann S, Zeh M, Willmitzer L, Höfgen R. 2001. Approaches towards understanding methionine biosynthesis in higher plants. Amino Acids 20, 281–289.[CrossRef][ISI][Medline]
Hesse H, Höfgen R. 2003. Molecular aspects of methionine biosynthesis in Arabidopsis and potato. Trends in Plant Science 8, 259–262.[CrossRef][ISI][Medline]
Hughes CA, Gebhardt JS, Reuss A, Matthews BF. 1999. Identification of a cDNA encoding cystathionine -synthase in soybean. Plant Science 146, 69–79.[CrossRef]
Inaba K, Fujiwara T, Hayashi H, Chino M, Komeda Y, Naito S. 1994. Isolation of an Arabidopsis thaliana mutant, mto1, that overaccumulates soluble methionine. Plant Physiology 104, 881–887.[Abstract]
Isegawa Y, Watanabe F, Kitaoka S, Nakano Y. 1994. Subcellular distribution of cobalamin-dependent methionine synthase in Euglena gracilis Z. Phytochemistry 35, 59–61.[CrossRef]
Kim J, Lee M, Chalam R, Martin MN, Leustek T, Boerjan W. 2002. Constitutive overexpression of cystathionine -synthase in Arabidopsis leads to accumulation of soluble methionine and S-methylmethionine. Plant Physiology 128, 95–107.
Kim J, Leustek T. 1996. Cloning and analysis of the gene for cystathionine -synthase from Arabidopsis thaliana. Plant Molecular Biology 32, 1117–1124.[CrossRef][ISI][Medline]
Kim J, Leustek T. 2000. Repression of cystathionine -synthase in Arabidopsis thaliana produces partial methionine auxotrophy and developmental abnormalities. Plant Science 151, 9–18.[CrossRef]
Kreft O, Höfgen R, Hesse H. 2003. Functional analysis of cystathionine -synthase in genetically engineered potato plants. Plant Physiology 131, 1843–1854.
Kreft BD, Townsend A, Pohlenz HD, Laber B. 1994. Purification and properties of cystathionine -synthase from wheat (Triticum aestivum L.). Plant Physiology 104, 1215–1220.[Abstract]
Kurvari V, Qian F, Snell WJ. 1995. Increased transcript levels of a methionine synthase during adhesion-induced activation of Chlamydomonas reinhardtii gametes. Plant Molecular Biology 29, 1235–1252.[CrossRef][ISI][Medline]
Lai J, Messing J. 2002. Increasing maize seed methionine by mRNA stability. The Plant Journal 30, 395–402.[CrossRef][ISI][Medline]
Lambein I, Chiba Y, Onouchi H, Naito S. 2003. Decay kinetics of autogenously regulated CgS1 mRNA that codes for cystathionine -synthase in Arabidopsis thaliana. Plant and Cell Physiology 44, 893–900.
MacNicol PK, Datko AH, Giovanelli J, Mudd SH. 1981. Homocysteine biosynthesis in green plants: physiological importance of the transsulphuration pathway in Lemna paucicostata. Plant Physiology 68, 619–625.
Maimann S, Höfgen R, Hesse H. 2001. Enhanced cystathionine ß-lyase activity in transgenic potato plants does not force metabolite flow towards methionine. Planta 214, 163–170.[ISI][Medline]
Maimann S, Wagner C, Kreft O, Zeh M, Willmitzer L, Höfgen R, Hesse H. 2000. Transgenic potato plants reveal the indispensable role of cystathionine ß-lyase in plant growth and development. The Plant Journal 23, 747–758.[CrossRef][ISI][Medline]
Matthews BF. 1999. Lysine, threonine and methionine biosynthesis. In: Singh BK, ed. Plant amino acids: biochemistry and biotechnology. New York: Marcel Dekker, 205–225.
Mertz ET, Bates LS, Nelson OE. 1964. Mutant gene that changes protein composition and increases lysine content of