A single-nucleotide mutation in a gene encoding S-adenosylmethionine synthetase is associated with methionine over-accumulation phenotype in Arabidopsis thaliana.

Abstract

Met-overaccumulating mutants provide a powerful genetic tool for examining both the regulation of the Met biosynthetic pathway and in vivo developmental responses of gene expression to altered Met levels. We have previously reported the identification of two Arabidopsis thaliana Met over-accumulation ( mto ) mutants, mto1-1 and mto2-1 , that carry mutations in the genes encoding cystathionine γ -synthase (CGS) and threonine synthase (TS), respectively. A third mutant, mto3-1 , has recently been reported to carry a mutation in the gene encoding S -adenosylmethionine synthetase 3 (SAMS3). Here, we report the isolation of a new ethionine-resistant A. thaliana mutant that over-accumulates soluble Met approximately 20-fold in young rosettes. The causal mutation was determined to be a single, recessive mutation that was mapped to chromosome 3. Sequence analysis identified a single nucleotide change in the gene encoding SAMS3 that was distinct from the mto3-1 mutation and altered the amino acid sequence of the enzyme active site. This mutation was therefore referred to as mto3-2 . Although Met over-accumulation in the mto3-2 mutant was similar to that in the mto2-1 mutant, CGS mRNA levels did not respond to the mto3-2 mutation and were similar to that in equivalent wild-type plants.

Dynamics of methionine biosynthesis

“…Mutations in SAMS3 prevent flow of excess methionine into the metabolic pathway, however, they still allow normal flux to methionine at the OPH branch point. Combined with the finding that CGS levels at least in mto3-2 are not appreciably affected, despite the 20-fold increase in methionine (Goto et al 2002), this also suggested that SAM and not methionine is likely to be the effector of CGS regulation, which was later established by in vitro experiments .…”

“…In young mto3-1 plants, concentrations of methionine were 244-fold higher than that in wild type and SAM levels were reduced by 35% (Shen et al 2002). On the other hand, methionine concentrations were 20-fold higher in young plants of the weaker mto3-2 allele (Goto et al 2002) and SAM levels were not detectably different (D. Goto, M. Ogi, S. Naito, unpublished data) (Table 1).…”

“…Significant advances in our understanding on the regulation of the methionine biosynthetic pathway in plants were provided through the isolation and characterisation of genetic mutants in which methionine accumulation is altered Chiba et al 1999;Bartlem et al 2000;Goto et al 2002;Shen et al 2002;Onouchi et al 2005), as described in further detail below.…”

“…The mto1 , mto2 (Bartlem et al 2000) and mto3 (Goto et al 2002;Shen et al 2002) mutants contain lesions in genes encoding CGS, TS and SAMS, respectively.…”

“…The mto3-1 mutation alters the ATP binding site within the active site (Shen et al 2002), whereas mto3-2 changes the Asp-167 residue that likely binds the methionine substrate (Goto et al 2002) (Figure 6). Although both mto3-1 and mto3-2 affect the SAMS3 active site, the severity of the phenotypes were different in the two mutations.…”