Metabolic Engineering of Amino Acids and Storage Proteins in Plants

Galili, Gad; Höfgen, Rainer

doi:10.1006/mben.2001.0203

Cited by 167 publications

(96 citation statements)

References 76 publications

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“…As shown in Fig. 1A, equal amounts of CH 3 SH, NH 3 , and ␣KB were detected, demonstrating that the protein has an MGL activity and may therefore be termed AtMGL. The recombinant At-MGL obeyed Michaelis-Menten kinetics with a K m value for Met of 10.6 Ϯ 0.9 mM.…”

Section: Resultsmentioning

confidence: 82%

“…In these organisms, Met is metabolized through the reverse transsulfuration pathway where the ␥-cleavage of the cystathionine intermediate, a reaction that does not exist in plants, allows synthesis of cysteine (Cys) (2). Therefore, Met must be supplied from the diet and, because it is the most limiting essential amino acid in legume seeds, metabolic engineering strategies have been developed to increase the carbon flux into free Met as well as the incorporation of this amino acid into proteins (3).…”

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confidence: 99%

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Methionine catabolism in Arabidopsis cells is initiated by a γ-cleavage process and leads to S -methylcysteine and isoleucine syntheses

Rébeillé

Jabrin

Bligny

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

134

112

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methionine ␥-lyase ͉ NMR profiling ͉ plant T he sulfur-containing amino acid methionine (Met) is an essential metabolite in all living organisms (1). Besides its role as protein constituent, Met is the precursor of S-adenosylmethionine (AdoMet), which is the major methyl-group donor in transmethylation reactions and an intermediate in the biosynthesis of biotin, polyamines, and the phytohormone ethylene. Met is synthesized de novo by plants, fungi, and microbes. It is the only sulfur-containing amino acid that is essential for human and monogastric livestock. In these organisms, Met is metabolized through the reverse transsulfuration pathway where the ␥-cleavage of the cystathionine intermediate, a reaction that does not exist in plants, allows synthesis of cysteine (Cys) (2). Therefore, Met must be supplied from the diet and, because it is the most limiting essential amino acid in legume seeds, metabolic engineering strategies have been developed to increase the carbon flux into free Met as well as the incorporation of this amino acid into proteins (3).In higher plants, the neo-synthesized Met molecule originates from three convergent pathways with the sulfur atom deriving from Cys, the nitrogen͞carbon backbone from aspartate, and the methyl moiety from the ␤-carbon of serine via the pool of folates (1). De novo Met synthesis consists of three consecutive reactions localized in plastids (4) and catalyzed by cystathionine ␥-synthase, cystathionine ␤-lyase, and methionine synthase. Several recent studies have indicated that Met synthesis and accumulation are subject to complex regulatory controls in which cystathionine ␥-synthase plays a crucial role (5-7).Studies of metabolic fates of Met using the aquatic plant Lemna paucicostata indicated that the synthesis and turnover of AdoMet accounts for Ϸ80% of Met metabolism, whereas the synthesis of proteins drives Ϸ20% of Met (for a review, see ref. 8). More than 90% of AdoMet then is used for transmethylation, leading to nucleic acid, protein, lipid, and other metabolites modifications. The resulting homocysteinyl moiety is recycled back to Met and AdoMet by a set of cytosolic reactions designated as the activated methyl cycle. The use of AdoMet for the synthesis of polyamines and, in some plant tissues, ethylene also is accompanied by recycling of the methylthio moiety and regeneration of Met. The last known metabolic fate of Met is unique to plants and leads to the production of S-methylmethionine (SMM), a compound resulting from the AdoMet-dependent methylation of Met. SMM then can donate a methyl group to homocysteine (Hcy) through the reaction catalyzed by Hcy S-methyltransferase, yielding two molecules of Met. The SMM cycle might serve to achieve short-term control of AdoMet levels in plant cells, thus controlling the commitment of one-carbon units into methyl-group synthesis (9-11).Despite recent progress in elucidating the synthesis of Met and its complex regulation, little is known about the catabolism of this amino acid in plant cells. Several authors have ...

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Section: Resultsmentioning

confidence: 82%

mentioning

confidence: 99%

Methionine catabolism in Arabidopsis cells is initiated by a γ-cleavage process and leads to S -methylcysteine and isoleucine syntheses

Rébeillé

Jabrin

Bligny

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

134

112

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“…In acid-hydrolyzed samples, mol % Asx was increased (Table I), consistent with the results in developing siliques (Table II). Asx provides the substrate for the Asp amino acid biosynthetic pathway (products including essential amino acids such as Lys, Met, Thr, and Ile; Galili and Hofgen, 2002). An alternation in the relative percentage of free Asx may have strong implications concerning the amino acid metabolism in the sink tissues.…”

Section: Discussionmentioning

confidence: 99%

“…To apply this result (increase of Asx) to seed quality improvement, further manipulation is needed. In particular, deregulation of end production feedback inhibition and other transcriptional and posttranscriptional control mechanisms in the biosynthesis of the Asp family amino acids should be achieved (Galili and Hofgen, 2002). In addition, the presence of effective sinks must be engineered (e.g.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of the ASN1 Gene Enhances Nitrogen Status in Seeds of Arabidopsis

et al. 2003

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In wild-type Arabidopsis, levels of ASN1 mRNA and asparagine (Asn) are tightly regulated by environmental factors and metabolites. Because Asn serves as an important nitrogen storage and transport compound used to allocate nitrogen resources between source and sink organs, we tested whether overexpression of the major expressed gene for Asn synthetase, ASN1, would lead to changes in nitrogen status in the ultimate storage organ for metabolites-seeds. Transgenic Arabidopsis constitutively overexpressing ASN1 under the cauliflower mosaic virus 35S promoter were constructed (35S-ASN1). In seeds of the 35S-ASN1 lines, three observations support the notion that the nitrogen status was enhanced: (a) elevations of soluble seed protein contents, (b) elevations of total protein contents from acid-hydrolyzed seeds, and (c) higher tolerance of young seedlings when grown on nitrogen-limiting media. Besides quantitative differences, changes in the relative composition of the seed amino acid were also observed. The change in seed nitrogen status was accompanied by an increase of total free amino acids (mainly Asn) allocated to flowers and developing siliques. In 35S-ASN1 lines, sink tissues such as flowers and developing siliques exhibit a higher level of free Asn than source tissues such as leaves and stems, despite significantly higher levels of ASN1 mRNA observed in the source tissues. This was at least partially due to an enhanced transport of Asn from source to sink via the phloem, as demonstrated by the increased levels of Asn in phloem exudates of the 35S-ASN1 plants.In higher plants, nitrogen is acquired from the environment via nitrate reduction (Crawford and Arst, 1993), ammonia uptake, or nitrogen fixation (Burris and Roberts, 1993). All inorganic nitrogen must first be reduced to ammonia by the action of nitrate and nitrite reductase before being assimilated into amino acids Miflin and Lea, 1980;Lea et al., 1990). In plants, the majority of ammonia is assimilated into organic form via the combined action of Gln synthetase and Gln 2-oxoglutarate aminotransferase. The products of Gln synthetase-Gln 2-oxoglutarate aminotransferase cycle, Gln, and Glu are highly reactive and are used in various anabolic pathways. Through the action of Asn synthetase (AS; EC 6.3.5.4), Gln will react with Asp to form Asn and Glu. Because all the substrates and products of the AS-catalyzed reaction are major nitrogen carriers in plant metabolism for transporting nitrogen in the phloem, the AS enzyme is believed to play an important role in regulating the flow of nitrogen into the organic nitrogen pool (Lam et al., 1994). Asn is an especially important nitrogen transport amino acid because it has a high nitrogen to carbon ratio (relative to the other amide amino acid Gln) and is relatively inert compared with the other nitrogen-transporting amino acids (Glu, Asp, and Gln). Therefore, Asn can be used for long-range nitrogen transport and storage, which is vital to physiological processes such as germination and nitrogen assimilation Siecie...

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“…The import of cytosolic SAM into plastids is crucial for plastid metabolism ( Figure 1). SAM is an allosteric activator of plastid Thr synthase (Madison and Thompson, 1976;Aarnes, 1978;Giovanelli et al, 1984;Curien et al, 1998) and, thus, potentially controls the flux of O-phosphohomoserine into the biosynthesis of Thr and Met (Giovanelli et al, 1989;Galili and Hofgen, 2002) (Figure 1). Imported SAM is also used by several plastid methyltransferases, the substrates of which range from small molecules such as prenyllipids (e.g., chlorophylls, plastoquinone, tocopherol, and phylloquinone) (Bouvier et al, 2005;DellaPenna, 2005) and the diterpene antioxidant carnosic acid (Munne-Bosch and Alegre, 2001) to macromolecules such as plastid DNA (Nishiyama et al, 2002) and Rubisco (Black et al, 1987;Grimm et al, 1997;Ying et al, 1999;Trievel et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

ArabidopsisSAMT1 Defines a Plastid Transporter Regulating Plastid Biogenesis and Plant Development

et al. 2006

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S-Adenosylmethionine (SAM) is formed exclusively in the cytosol but plays a major role in plastids; SAM can either act as a methyl donor for the biogenesis of small molecules such as prenyllipids and macromolecules or as a regulator of the synthesis of aspartate-derived amino acids. Because the biosynthesis of SAM is restricted to the cytosol, plastids require a SAM importer. However, this transporter has not yet been identified. Here, we report the molecular and functional characterization of an Arabidopsis thaliana gene designated SAM TRANSPORTER1 (SAMT1), which encodes a plastid metabolite transporter required for the import of SAM from the cytosol. Recombinant SAMT1 produced in yeast cells, when reconstituted into liposomes, mediated the counter-exchange of SAM with SAM and with S-adenosylhomocysteine, the by-product and inhibitor of transmethylation reactions using SAM. Insertional mutation in SAMT1 and virus-induced gene silencing of SAMT1 in Nicotiana benthamiana caused severe growth retardation in mutant plants. Impaired function of SAMT1 led to decreased accumulation of prenyllipids and mainly affected the chlorophyll pathway. Biochemical analysis suggests that the latter effect represents one prominent example of the multiple events triggered by undermethylation, when there is decreased SAM flux into plastids.

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Metabolic Engineering of Amino Acids and Storage Proteins in Plants

Cited by 167 publications

References 76 publications

Methionine catabolism in Arabidopsis cells is initiated by a γ-cleavage process and leads to S -methylcysteine and isoleucine syntheses

Methionine catabolism in Arabidopsis cells is initiated by a γ-cleavage process and leads to S -methylcysteine and isoleucine syntheses

Overexpression of the ASN1 Gene Enhances Nitrogen Status in Seeds of Arabidopsis

ArabidopsisSAMT1 Defines a Plastid Transporter Regulating Plastid Biogenesis and Plant Development

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