Two distinct EIN2 genes cooperatively regulate ethylene signaling in Lotus japonicus

Leguminous plants establish a mutualistic symbiosis with bacteria, collectively referred to as rhizobia. Host plants positively and negatively regulate the symbiotic processes to keep the symbiosis at an appropriate level. Although the plant hormone ethylene is known as a negative regulator of symbiotic processes, the molecular mechanisms of ethylene signaling remain unresolved, especially in the model plant Lotus japonicus. Here, we identified two genes, LjEIN2-1 and LjEIN2-2, from L. japonicus. These genes share moderate similarity in their amino acid sequences, are located on different chromosomes and are composed of different numbers of exons. Suppression of either LjEIN2-1 or LjEIN2-2 expression significantly promoted the root growth of transformed plants on plates containing 1-amino-cyclopropane-carboxylic acid (ACC), the biosynthetic precursor of ethylene. Simultaneous suppression of both LjEIN2-1 and LjEIN2-2 markedly increased the ethylene insensitivity of transgenic roots and resulted in an increased nodulation phenotype. These results indicate that LjEIN2-1 and LjEIN2-2 concertedly regulate ethylene signaling in L. japonicus. We also observed that Nod factor (NF) induced the expression of the ethylene-responsive gene LjACO2, and simultaneous treatment with NF and ACC markedly increases its transcript level compared with either NF or ACC alone. Because LjACO2 encodes ACC oxidase, which is a key enzyme in ethylene biosynthesis, this result suggests the existence of an NF-triggered negative feedback mechanism through ethylene signaling.