The bean grp1.8 gene is specifically expressed in vascular tissue. Monomers and multimers of a 28 bp regulatory element of the grp1.8 promoter (vs-1) specifically activated both the -82 CaMV 35S and the -76/grp1.8 minimal promoters in vascular tissue of transgenic tobacco plants. vs-1 partially overlaps with a negative regulatory element in the grp1.8 promoter that is necessary for restriction of gene expression to vascular tissue. Nuclear extracts from tobacco and tomato cells contain a factor that binds to vs-1 in vitro. To study the molecular basis of xylem-specific expression mediated by the vs-1 promoter element, a gene was isolated from tomato encoding a protein that binds to vs-1 in vitro. This protein, designated VSF-1, contains a bZIP motif close to the C-terminus. Mutated vs-1 elements were no longer bound by VSF-1 and also failed to activate the minimal -82 CaMV 35S promoter in vivo. Transient expression of VSF-1 in protoplasts stimulated vs-1 dependent activation of the -76/grp1.8 minimal promoter. Binding studies and use of a polyclonal antiserum against VSF-1 provided further evidence that vs-1 is a potential in vivo target site, as VSF-1 was a part of the observed complex formed between vs-1 and nuclear protein extract. vs-1 does not contain the 5'-ACGT-3' core sequence that is part of known plant bZIP protein binding sites or another palindromic sequence. Based on the unusual binding specificity and a characteristic amino acid sequence in the bZIP domain we propose that VSF-1 and the partially homologous PosF21, a bZIP protein from Arabidopsis, belong to a new family of plant bZIP proteins.

The bean grp1.8 full-length promoter is specifically active in vascular tissue during normal development of tobacco. Deletion of a negative regulatory element resulted in ectopic activity of the promoter in cortical cells of hypocotyls, roots and stems. A 169 bp fragment (-205 to -36) of the grp1.8 promoter conferred vascular-specific expression to CaMV 35S minimal promoters whereas a 141 bp fragment (-205 to -64) strongly activated these minimal promoters both in vascular and cortical cells. These experiments defined a new regulatory element (VSE) that is essential for vascular-specific expression and is located between -64 and -36. The 141 bp grp1.8 promoter sequence had enhancer-like properties as it was active in both orientations. A 24 bp sequence (bp -119 to -96, corresponding to the SE1 regulatory element) enhanced expression from several minimal promoters strongly but unspecifically, whereas a 26 bp sequence (-98 to -73, corresponding to the RSE regulatory element) induced vascular-specific expression. Thus, the grp1.8 promoter is regulated by a combinatorial mechanism that can integrate the action of different, non-additively acting regulatory elements into vascular-specific expression.