Specific interaction of the tomato bZIP transcription factor VSF-1 with a non-palindromic DNA sequence that controls vascular gene expression.

Abstract

The grp1.8 gene of French bean (Phaseolus vulgaris) is specifically expressed in vascular tissue and encodes a glycine-rich structural protein (GRP1.8) of the cell wall. Earlier promoter analysis had shown that a 28 bp fragment of the grp1.8 promoter (vs-1) confers vascular expression to heterologous minimal promoters and is bound by the tomato bZIP transcription factor VSF-1. Here, we analysed the interaction of VSF-1 with fragments of the vs-1 element and studied the molecular basis of specific binding both in the DNA sequence of the promoter element as well as in the protein. The minimal binding site of VSF-1 is a 9 bp, non-palindromic sequence with two non-identical half-sites and a central nucleotide which separates them. The amino acid sequence of the VSF-1 DNA-binding basic domain has a Lys at position -10 instead of a conserved Arg found in the other bZIP factors isolated so far. This lysine was found to be required for specific recognition of the non-palindromic binding site: a mutant VSF-1 with a Lys-to-Arg substitution at position -10 bound with higher affinity to a palindromic sequence than the wild-type protein. The minimal binding site of VSF-1 was sufficient and necessary to confer vascular-specific expression to a heterologous promoter in vivo. The vsf-1 promoter also showed vascular-specific expression in transgenic tobacco. The close similarity of these expression patterns suggests that VSF-1 is specifically involved in vascular expression of the grp1.8 gene in plants.