Spatiotemporal dynamics of ethylene biosynthesis shape infection and nodule initiation in Medicago truncatula

Ethylene is a well-established negative regulator of nodulation, yet how ethylene biosynthesis and perception are spatially coordinated during early symbiotic signalling remains unresolved. Here, we investigate the dynamics of ethylene responses in Medicago truncatula using transcriptomics, promoter-reporter analyses, loss-of-function approaches and a synthetic reporter. We show that the activity of the ethylene-responsive EBSn reporter shifts from inner root tissues under non-symbiotic conditions to the outer cortex and epidermis following rhizobial inoculation, revealing a spatial reprogramming of ethylene signalling. Among the eight Medicago 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE (ACS) genes, upon rhizobia application MtACS3 is induced in outer root cell layers, while MtACS10 is repressed in the inner cortex and pericycle, mirroring the shift in ethylene perception. Functional analysis demonstrates that MtACS10 restricts nodule initiation, whereas MtACS3 modulates infection thread number, prevents nodule clustering, and contributes to radial positioning of nodule primordia. Rhizobial induced ectopic ACS expression in the root interior counteracts MtACS10 repression and blocks nodulation, highlighting the requirement for spatially confined downregulation of ethylene biosynthesis. Together, these findings establish a framework in which localized shift in ethylene biosynthesis, mediated by distinct Medicago ACS genes, balances infection and organogenesis while co-defining the spatial limits of the root susceptible zone.