A transgene for high methionine protein is posttranscriptionally regulated by methionine

β-Zein is one of the seed storage proteins of maize that is high in methionine (Met). In alfalfa, the β-zein gene driven by the CaMV 35S promoter showed an 8-fold lower level of transcript and protein when compared with the level in tobacco transformed with the same gene construct. The reporter gene (GUS) driven by the CaMV 35S promoter showed only a 4-fold difference between alfalfa and tobacco, suggesting that the expression of the β-zein gene is posttranscriptionally regulated in alfalfa. Callus of alfalfa transformants with the β-zein gene construct treated with exogenous Met, showed a significant increase in the β-zein level, suggesting that free Met may be limiting in the synthesis of β-zein in alfalfa. The introduction of the Arabidopsis thaliana cystathionine γ-synthase (AtCγS) gene driven by the CaMV 35S promoter into alfalfa showed a significant increase in the level of free Met and its metabolite, S-methyl methionine (SMM), but not in the bound fraction. Coexpression of AtCγS and β-zein in alfalfa increased the level of β-zein transcript and protein and decreased free Met, which suggests that the β-zein is posttranscriptionally regulated by free Met. The expression of AtCγS in tobacco did not produce a significant increase in free Met or SMM and coexpression of AtCγS and β-zein did not result in changes in the β-zein level. The results demonstrate the efficacy of the synergistic approach of increasing both the sink and the source for increasing the levels of high Met β-zein.