Pathway Analysis of Sulfur Containing Amino Acids

Methionine Biosynthesis

Methionine is synthesised from cysteine and phosphohomoserine via the enzymes Cystathionine gamma-synthase, Cystathionine beta-lyase (CbL), and Methionine synthase. We have cloned and characterised these three genes in potato. All three have been over-expressed or antisensed in transgenic potato in order to improve our understanding of the enzymes and metabolites at play in methionine metabolism. For example, depending on the level of inhibition, CbL antisense plants exhibit lethal or severely retarded growth phenotypes due to methionine starvation. Furthermore, CbL antisense inhibition results in reduced steady state CbL mRNA, protein, and enzyme activity levels. Increases in the levels of many pathway intermediates (such as huge increases in the precursors cystathionine, cysteine, and homoserine) occur in these plants, whereas methionine is depleted. Even though CbL over-expression is not in itself able to enhance metabolic flux towards methionine biosynthesis, it is clear that CbL is essential for plant growth and development. Our objective is now to deepen our understanding of methionine biosynthetic regulation and to exploit what we learn in order to improve the nutritional quality of crop plants.

The characterisation of transgenic and mutant plants with altered expression of genes in methionine biosynthesis led to the conclusion that the branch between both enzymes is the major regulatory site in carbon flux to methionine. The enzymes further downstream, CbL and MS, seem not to influence the flow of metabolites even when over-expressed. The enzymatic activity of plant threonine synthase (TS) is strongly stimulated by SAM causing Km values of fully activated TS for OPHS to be 250 to 500-fold lower than those of CgS, directing the carbon flux into the Thr branch when methionine and, hence, SAM levels are high. Arabidopsis plants expressing CgS antisense mRNA revealed a four- to seven-fold increase in Thr levels accompanied by severe morphological aberrations observed in correlation with reduced methionine synthesis capacity. A similar change in plant phenotype and the emission of methionine catabolic products was also detected in transgenic plants expressing a SAM-synthase antisense construct or containing a mutated SAMS.

Additional interesting findings were obtained when TS activity was reduced. An Arabidopsis mutant (mto2) deficient in TS enzymatic activity exhibited an accumulation of free methionine in young rosette leaves (20-fold), accompanied by comparably reduced soluble Thr (down to 6 %) contents. Similarly, a reduction in TS levels by an antisense approach in potato plants caused a threonine reduction of 45% in threonine levels, whereas methionine levels increased by up to 239 times compared with non-transformed plants. Notably, a reduction of TS activity in both Arabidopsis and potato caused a far stronger increase in methionine levels than the decrease in threonine levels indicating an increased flux of the carbon skeleton from aspartate to methionine. Even when a bacterial TS was expressed in transgenic tobacco plants the methionine level remained unaffected although a fivefold increase in threonine level was observed.

Results from transgenic plants manipulated in CgS enzymatic activity levels indicated that Arabidopsis CgS is feedback-regulated by methionine or a derivative thereof at the post-transcriptional level. Molecular investigations of Arabidopsis CgS regulation were focused on a stretch of 39 amino acids, encoded by exon 1 of CgS and designated as the MTO1 region, which is believed to act in cis to destabilize its own transcript in a process that involves methionine or related metabolites. Accordingly, CgS mRNA levels and enzymatic activities are reduced in the presence of excess methionine in Arabidopsis. In contrast, it could be shown that increasing the soluble methionine pool in potato leaves was not accompanied by changes in levels of CgS transcript or activity. Accordingly, the post-transcriptional regulation machinery predicted for Arabidopsis CgS seems not to exist in potato. This difference, though, cannot 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. The mechanism of this regulation is not known but a computer analysis predicts that mRNA sequences near the MTO1 region can form stable stem-loop structures, supporting post-transcriptional control by this region additional to the MTO1 motif.

Future studies will be directed at elucidating the mechanism of this post-transcriptional control of CgS gene expression as well as CgS's post-translational interactions with other factors regulating methionine metabolism. In addition, it will be important to discover whether the mechanisms regulating CgS expression and function in Arabidopsis as depicted in figure 2 are ubiquitous among plant species. In contrast to the situation seen in Arabidopsis, the down-regulation of CgS activity when methionine accumulated was not observed in potato. Besides the understanding of the regulatory node of this branch the cross talk between different compartments with respect to exchange of metabolites such as pathway intermediates and SAM will be analysed. Finally, although there is irrefutable evidence in plants for the cytosolic localisation of methionine synthase, the localisation of other isoforms needs further exploration.

Further reading

H Hesse and R Hoefgen (2003) Molecular aspects of methionine biosynthesis. TIPS, 8, 259-262

Kreft, R Hoefgen, and H Hesse (2003) Functional Analysis of Cystathionine gamma-Synthase in Genetically Engineered Potato Plants. Plant Physiology 131(1843-1854

M Zeh, AP Casazza, O Kreft, U Roessner, K Bieberich, L Willmitzer, R Hoefgen, and H Hesse (2001) Antisense Inhibition of Threonine Synthase Leads to High Methionine Content in Transgenic Potato Plants. Plant Physiol. 127(792-802

V Frankard, G Ispas, H Hesse, M Jacobs, R Höfgen (2002) Defect in cystathionine b-lyase activity causes the severe phenotype of a Nicotiana plumbaginifolia methionine auxotroph. Plant Sci., 162, 607-614

M Zeh, G Leggewie, R Höfgen, H Hesse (2002) Cloning and characterization of a cDNA encoding a cobalamin-independent methionine synthase from potato (Solanum tuberosum L.). Plant Mol. Biol. 48, 255-265

H Hesse, O Kreft, S Maimann, M Zeh, L Willmitzer, R Höfgen (2001) Approaches towards understanding methionine biosynthesis in higher plants. Amino Acids 20, 3, 281-289S Maimann, R Höfgen and H Hesse (2001) Enhanced cystathionine b-lyase activity in transgenic potato plants does not force metabolite flow towards methionine. Planta, 214, 163-170

S Maimann, C Wagner, O Kreft, M Zeh, L Willmitzer, R Höfgen and H Hesse (2000) Transgenic potato plants reveal the indispensable role of cystathionine b-lyase in plant growth and development. Plant J. 23, 747-758

AP Casazza, A Basner, R Höfgen, and H Hesse (2000) Expression of threonine synthase from Solanum tuberosum L. is not metabolically regulated by photosynthesis-related signals or by nitrogenous compounds. Plant Sci., 157, 43-50

H Hesse, A Basner, L Willmitzer, R Höfgen (1999) Cloning and Characterisation of a cDNA (Accession No. AF082892) Encoding a Second Cystathionine Gamma-Synthase in Potato (Solanum tuberosum L.). (PGR99-161) Plant Physiol. 121: 1053

K Riedel, C Mangelsdorf, W Streber, L Willmitzer, R Höfgen, H Hesse. 1999 Cloning and characterization of cystathionine gamma-synthase from Solanum tuberosum L. Plant biol. 1, 638-644

H Hesse, J Lipke, T Altmann, and R Höfgen. 1999. Molecular cloning and expression analysis of mitochondrial and plastidic isoforms of cysteine synthase (O-Acetylserine(thiol)lyase) from Arabidopsis thaliana. Amino Acids. 16(113-131)

H Hesse and R Höfgen. 1998. Isolation of cDNAs encoding cytosolic (Accs. No. AF044172) and plastidic (Accs. No. AF044173) cysteine synthase isoforms from Solanum tuberosum. Plant Physiol. 116(1604)

H Hesse, J Lipke, T Altmann, and R Höfgen. 1997. Expression analysis and subcellular localisation of cysteine synthase isoforms from Arabidopsis thaliana. In: Sulfur Metabolism in Higher Plants. WJ Cram, LJ De Kok, I Stulen, C Brunold, H Rennenberg, Eds. Buckhuys Publishers, Leiden. (227-230)