Dynamics of Ethylene Production in Response to Compatible Nod Factor

Establishment of symbiotic nitrogen-fixationin legumes is regulated by the plant hormone ethylene, but it has remained unclear whether and how its biosynthesis is regulated by the symbiotic pathway. We established a sensitive ethylenedetection system for Lotus japonicusand found that ethyleneproduction increased as early as 6 hours after inoculation with Mesorhizobium loti. This ethyleneresponse was dependent on Nod factorproduction by compatible rhizobia. Analyses of nodulation mutants showed that perception of Nod factorwas required for ethyleneemission, while downstream transcription factors including CYCLOPS, NIN, and ERN1were not required for this response. Activation of the nodulation signaling pathway in spontaneously nodulating mutants was also sufficient to elevate ethyleneproduction. Ethylenesignaling is controlled by EIN2, which is duplicated in L. japonicus . We obtained a L. japonicus Ljein2a Ljein2b double mutant that exhibits complete ethyleneinsensitivity and confirms that these two genes act redundantly in ethylenesignaling. Consistent with this redundancy, both LjEin2a and LjEin2b are required for negative regulation of nodulation and Ljein2a Ljein2b double mutants are hypernodulating and hyperinfected. We also identified an unexpected role for ethylenein the onset of nitrogen fixation, with the Ljein2a Ljein2b double mutant showing severely reduced nitrogen fixation. These results demonstrate that ethyleneproduction is an early and sustained nodulation response that acts at multiple stages to regulate infection, nodule organogenesis, and nitrogen fixationin L. japonicus .