Cloning and characterization of a cDNA encoding a cobalamin-independent methionine synthase from potato (Solanum tuberosum L.).
A potato cDNA clone, StMS1, that encodes a methionine synthase was isolated. This protein was identified on the basis of both structural and functional evidence. The predicted sequence of the protein encoded by StMS1 shows a high degree of similarity to methionine synthases from other organisms and the expression of StMS1 in bacterial mutant strains restored the mutant's ability to synthesize methionine. Genomic organization and expression analyses suggest that StMS1 is a low-copy gene and is differentially expressed in potato organs. StMS1 expression was found in all tissues, but at elevated levels in flowers, basal levels in sink and source leaves, roots and stolons, and low levels in stems and tubers. RNA expression data were confirmed by western blot analysis 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. These RNA data were not confirmed at the protein level since protein content levels remained constant over the whole day. Feeding experiments of detached leaves revealed that sucrose or sucrose-derived products are responsible for StMS1 induction. This induction can be blocked by treatment with DCMU during the light period. Western analysis revealed that the amount of StMS1 is not affected by either treatment. This experiment confirmed the presence of a day/night rhythm. Methionine synthase expression is regulated by photoassimilates but this seems not to detectably alter protein levels.
Zeh
Michaela
M
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Golm, Germany.
Leggewie
Georg
G
Hoefgen
Rainer
R
Hesse
Holger
H
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000235:genetics* / Q000378:metabolism; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005816:Genetic Complementation Test; D008027:Light; D008969:Molecular Sequence Data; D009154:Mutation; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000201:enzymology / Q000235:genetics*; D014018:Tissue Distribution; D014158:Transcription, Genetic / Q000528:radiation effects; D014805:Vitamin B 12 / Q000502:physiology
D018076:DNA, Complementary; D018744:DNA, Plant; D018749:RNA, Plant; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014805:Vitamin B 12