Target genes for OBP3, a Dof transcription factor, include novel basic helix-loop-helix domain proteins inducible by salicylic acid

Abstract

Overexpression of a salicylic-acid (SA)-inducible Arabidopsis DNA binding with one finger (Dof) transcription factor, called OBF-binding protein 3 (OBP3; AtDof3.6), has previously been shown to result in growth defects. In this study, suppressive subtraction hybridization (SSH) was used to isolate genes induced in an OBP3-overexpression line and several putative clones, called OBP3-responsive genes (ORGs), were isolated. The link with the induced expression levels of these genes and OBP3 overexpression was confirmed by analysing additional OBP3-overexpression lines. ORG1 through ORG4 are novel genes, while ORG5 is an extensin gene, AtExt1. While ORG4 has no similarity with other proteins in the database, ORG1 has weak similarity in different regions of the predicted protein with CDC2 and fibrillin. ORG2 and ORG3 share 80% overall identity in their deduced amino acid sequences and contain a basic helix-loop-helix DNA-binding domain, suggesting that ORG2 and ORG3 may be transcription factors. The expression of the ORG1, ORG2 and ORG3 genes was co-regulated under all conditions examined including upregulation by SA and downregulation by jasmonic acid (JA). Fifteen OBP3-silenced lines were generated to further explore the function of OBP3. Although there were no visible phenotypic changes in any of these lines, the expression of ORG1, ORG2 and ORG3 was reduced. Among the ORG genes, ORG1, ORG2 and ORG3 contained the highest number of potential Dof-binding sites in the promoter region, and their expression was significantly increased within 3 h after induction of OBP3 expression using an inducible promoter system, and closely reflected the expression levels of the exogenous OBP3 protein. The results from the gain-of-function and loss-of-function experiments suggest that the ORG1, ORG2 and ORG3 genes are direct target genes of OBP3.