Nitrate interferes with the transcriptional regulation of ACC SYNTHASE 10 to inhibit nodule formation on Medicago truncatula. Ethylene-mediated nitrate inhibition of nodulation in M. truncatula

Nitrogen fixation by rhizobia is a highly energy-demanding process. Therefore, legume nodule initiation is tightly regulated. Environmental nitrate and plant-produced ethylene are both potent inhibitors of nodulation. However, the precise mechanism by which these agents (co)regulate the inhibition of nodulation is not fully understood. Here, we demonstrate that in Medicago truncatula (Medicago) nitrate blocks nodulation -in part- by interfering with the transcriptional regulation of the ethylene biosynthesis gene ACC SYNTHASE 10 (MtACS10). Under non-inoculated conditions, MtACS10 is highly expressed in the inner cell layers of the Medicago root; the position where nodules are initiated. Upon perception of rhizobial signals, MtACS10 expression is reduced, and nodule formation is initiated. However, at high nitrate concentrations, MtACS10 expression remains unchanged, and nodule formation is blocked. RNAi-mediated silencing of MtACS10 relieves this inhibition and allows nodule formation under otherwise inhibitory nitrate conditions. Conversely, overexpression of MtACS10 in the inner layers of the Medicago root strongly reduces nodule initiation already at low nitrogen concentrations. This demonstrates that a low level of MtACS10 expression is required to initiate nodule organogenesis, and that nitrate interferes with the transcriptional regulation of MtACS10 to block nodule formation.