Molecular analysis of three maize 22 kDa auxin‐binding protein genes — transient promoter expression and regulatory regions

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Abstract
The site I 22 kDa auxin‐binding proteins from maize are encoded by a small gene family comprising at least five members. Here the cloning and molecular analysis of the Zm‐ERabp1, Zm‐ERabp4, and Zm‐ERabp5 genes is presented. All three encode 22–23 kDa proteins displaying a transit peptide, a C‐terminal KDEL sequence, as well as glycosylation and auxin‐binding sites. The Zm‐ERabp4 and Zm‐ERabp5 genes are very similar. The Zm‐ERabp1 gene encodes a related protein, but its promoter, leader and signal peptide are very different. Northern analysis using gene‐specific oligonucleotide probes indicates that Zm‐ERabp4 is expressed in leaves and coleoptiles but weakly in roots, whereas Zm‐ERabp5 expression is barely detectable in these tissues. RNA‐PCR indicated that all three genes are none the less expressed in many tissues. Primer‐extension analysis revealed an unusually long (320 bases) Zm‐ERabp1 leader containing an 80 codon ORF which, if expressed, would encode a positively charged protein with some similarity to transcription factors. In a transient promoter—reporter gene expression system using maize leaf protoplasts the Zm‐ERabp1 promoter is more active than the Zm‐ERabp4 and Zm‐ERabp5 promoters. Promoter deletion analysis of Zm‐ERabp1 has identified a negative regulatory sequence in a region from −364 bp and −130 bp, deletion of which results in about twofold higher expression. This region contains both enhancer‐ and G‐box‐related sequences. Deletion of −129 bp to +64 bp, which contains the TATA box and transcription start, results in a large decrease in expression.

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