Cross talk between the KNOX and ethylene pathways is mediated by intron-binding transcription factors in Barley

In the barley (Hordeum vulgare) Hooded (Kap) mutant, the duplication of a 305-bp intron sequence leads to the overexpression of the Barley knox3 (Bkn3) gene, resulting in the development of an extra flower in the spikelet. We used a one-hybrid screen to identify four proteins that bind the intron-located regulatory element (Kap intron-binding proteins). Three of these, Barley Ethylene Response Factor1 (BERF1), Barley Ethylene Insensitive Like1 (BEIL1), and Barley Growth Regulating Factor1 (BGRF1), were characterized and their in vitro DNA-binding capacities verified. Given the homology of BERF1 and BEIL1 to ethylene signaling proteins, we investigated if these factors might play a dual role in intron-mediated regulation and ethylene response. In transgenic rice (Oryza sativa), constitutive expression of the corresponding genes produced phenotypic alterations consistent with perturbations in ethylene levels and variations in the expression of a key gene of ethylene biosynthesis. In barley, ethylene treatment results in partial suppression of the Kap phenotype, accompanied by up-regulation of BERF1 and BEIL1 expression, followed by down-regulation of Bkn3 mRNA levels. In rice protoplasts, BEIL1 activates the expression of a reporter gene driven by the 305-bp intron element, while BERF1 can counteract this activation. Thus, BEIL1 and BERF1, likely in association with other Kap intron-binding proteins, should mediate the fine-tuning of Bkn3 expression by ethylene. We propose a hypothesis for the cross talk between the KNOX and ethylene pathways.