The sulfur-containing amino acid methionine is an essential amino acid in animal nutrition. Apart from its role as a protein constituent and its central function in initiating mRNA translation, methionine indirectly regulates a variety of cellular processes as the precursor of S-adenosyl methionine (SAM), the primary biological methyl group donor. SAM is also the precursor of plant metabolites such as ethylene, polyamines, biotin and the Fe-chelator mugineic acid (Droux et al., 2000; Ma et al., 1995; Sun, 1998). Methionine also serves as a donor for secondary metabolites through S-methyl methionine (SMM; Mudd and Datko, 1990). Because methionine biosynthesis is so central to cell physiology, it is subject to complex regulatory control. Elucidation of the mechanisms underlying this regulation is currently an important challenge.
The amount of methionine in a plant cell is regulated by the level of its catabolism, by the level of the last enzyme of the threonine biosynthesis pathway, threonine synthase, but mainly by the level of the first enzyme of the methionine biosynthesis pathway, cystathionine γ-synthase (CGS; Amir et al., 2002; Avraham and Amir, 2005; Bartlem et al., 2000; Hesse and Hoefgen, 2003; Kim et al., 2002; Lee et al., 2005; Onouchi et al., 2004; Ravanel et al., 1998a,b; Zeh et al., 2001). Studies conducted in Arabidopsis revealed that the activity of CGS, which is located in the chloroplast, is not regulated by classical feedback inhibition (Ravanel et al., 1998a,b). Instead, both the transcript and protein levels of CGS are regulated indirectly by methionine via the metabolite of methionine, SAM (Chiba et al., 1999, 2003; Onouchi et al., 2005). Unlike bacterial CGS enzymes, the mature plant CGS (without its plastid transit peptides) contains an additional region of approximately 100 amino acids in the N-terminus that is not essential for the catalytic activity of this enzyme (Hacham et al., 2002). A subdomain of this region (termed MTO1 for methionine over-accumulation), which is conserved in the CGS proteins of all plant species, is apparently active in downregulation of its own mRNA and mediates the ability of SAM to control CGS levels (Chiba et al., 1999, 2003; Onouchi et al., 2005). A model was proposed in which the regulation occurs during CGS mRNA translation when the nascent polypeptide of CGS and its mRNA are in close proximity (Chiba et al., 1999, 2003; Lambein et al., 2003; Onouchi et al., 2004). Consistent with this model, it was demonstrated recently that SAM induces temporal translation elongation arrest which precedes the formation of a degradation intermediate of CGS mRNA, whose 5′ end is next to the 5′ edge of the stalled ribosome (Onouchi et al., 2005). Thus, a crucial control point for methionine synthesis in the Arabidopsis plant cell is the amount of CGS transcript.
Whether or not this autoregulation mechanism for post-transcriptional regulation of CGS expression exists in other plant species is still an open question. In potatoes (Solanum tuberosum), for example, the transcript level of CGS is not modulated by methionine despite the fact that the MTO1 region in potato CGS is highly conserved compared with that of Arabidopsis (Hesse and Hoefgen, 2003; Kreft et al., 2003; Zeh et al., 2001). This observation suggests that potato is missing some elements required for the post-transcriptional CGS regulation to occur and that the MTO1 region is not sufficient. Therefore, it appears that either other sequences outside the MTO1 region or additional trans-acting elements are involved in this regulation of CGS transcript level.
In this work, we report the existence of another control element besides the MTO1 sequence that affects the transcript level of Arabidopsis CGS. While cloning the CGS transcript either via cDNA libraries or by reverse-transcription PCR, we found that in addition to the characterized transcript there is another CGS product. This second transcript contains an internal 90-nt deletion at the N-terminal end. Ribonuclease protection analysis confirmed the presence of this transcript in different organs of the Arabidopsis plant. Its association with polyribosomes suggests that this form of CGS is translated. We found that transgenic plants overexpressing the 90-nt deleted form of CGS display a significantly higher level of methionine than plants overexpressing full-length CGS. When methionine was applied to these transgenic plants, those harboring the deleted transcript failed to respond to methionine exposure. Taken together, these results suggest that an additional transcript of CGS exists in Arabidopsis that is less responsive to methionine levels.