Abstract
We isolated HvRAF (Hordeum vulgare root abundant factor), a cDNA encoding a novel ethylene response factor (ERF)-type transcription factor, from young seedlings of barley. In addition to the most highly conserved APETALA2/ERF DNA-binding domain, the encoded protein contained an N-terminal MCGGAIL signature sequence, a putative nuclear localization sequence, and a C-terminal acidic transcription activation domain containing a novel mammalian hemopexin domain signature-like sequence. Their homologous sequences were found in AAK92635 from rice and RAP2.2 from Arabidopsis; the ERF proteins most closely related to HvRAF, reflecting their functional importance. RNA blot analyses revealed that HvRAF transcripts were more abundant in roots than in leaves. HvRAF expression was induced in barley seedlings by various treatment regimes such as salicylic acid, ethephon, methyl jasmonate, cellulase, and methyl viologen. In a subcellular localization assay, the HvRAF-GFP fusion protein was targeted to the nucleus. The fusion protein of HvRAF with the GAL4 DNA-binding domain strongly activated transcription in yeast. Various deletion mutants of HvRAF indicated that the transactivating activity was localized to the acidic domain of the C-terminal region, and that the hemopexin domain signature-like sequence was important for the activity. Overexpression of the HvRAF gene in Arabidopsis plants induced the activation of various stress-responsive genes, including PDF1.2, JR3, PR1, PR5, KIN2, and GSH1. Furthermore, the transgenic Arabidopsis plants showed enhanced resistance to Ralstonia solanacearum strain GMI1000, as well as seed germination and root growth tolerance to high salinity. These results collectively indicate that HvRAF is a transcription factor that plays dual regulatory roles in response to biotic and abiotic stresses in plants.