The Triple Response Assay and Its Use to Characterize Ethylene Mutants in Arabidopsis

Exposure of plants to ethylene results in drastic morphological changes. Seedlings germinated in the dark in the presence of saturating concentrations of ethylene display a characteristic phenotype known as the triple response. This phenotype is robust and easy to score. In Arabidopsis the triple response is usually evaluated at 3 days post germination in seedlings grown in the dark in rich media supplemented with 10 μM of the ethylene precursor ACC in air or in unsupplemented media in the presence of 10 ppm ethylene. The triple response in Arabidopsis consists of shortening and thickening of hypocotyls and roots and exaggeration of the curvature of apical hooks. The search for Arabidopsis mutants that fail to show this phenotype in ethylene or, vice versa, display the triple response in the absence of exogenously supplied hormone has allowed the identification of the key components of the ethylene biosynthesis and signaling pathways. Herein, we describe a simple protocol for assaying the triple response in Arabidopsis. The method can also be employed in many other dicot species, with minor modifications to account for species-specific differences in germination. We also compiled a comprehensive table of ethylene-related mutants of Arabidopsis, including many lines with auxin-related defects, as wild-type levels of auxin biosynthesis, transport, signaling, and response are necessary for the normal response of plants to ethylene.