In vivo and in vitro characterization of protein interactions with the dyad G-box of the Arabidopsis Adh gene

Abstract

Expression of the alcohol dehydrogenase (Adh) and ribulose-1,5-bisphosphate carboxylase small subunit (RbcS) genes of higher plants is cell-type-specific and environmentally inducible. However, the tissues in which these two genes are expressed, their modes of induction, and their protein functions are quite distinct. Adh is expressed in non-green tissue, induced by anaerobiosis, and repressed in leaves. RbcS is only expressed in green tissue. An 8-base pair G-box element (5'-CCACGTGG-3') is associated with light-induced expression of RbcS and chalcone synthase. The same sequence is also present in the 5'-flanking region of Arabidopsis thaliana Adh, and this sequence is associated with a trans-acting factor in vivo. We report here that in vitro Adh G-box binding activity is present in crude whole cell extracts of both cell culture and leaves of Arabidopsis. The authenticity of in vitro Adh G-box binding is supported by in vivo and in vitro dimethylsulfate footprinting. A clear in vivo Adh G-box footprint occurs in cell cultures, but comparable in vivo binding to the Adh G-box does not occur in leaves. Therefore, there does not appear to be a direct correlation between the presence of the G-box factor in a tissue and its binding to the Adh G-box.