Nitrate inhibits nodule organogenesis through inhibition of cytokinin biosynthesis in Lotus japonicus

Legumes balance nitrogen acquisition from soil nitrate with symbiotic nitrogen fixation. Nitrogen fixation requires establishment of a new organ, which is a cytokinin dependent developmental process in the root. We found cytokinin biosynthesis is a central integrator, balancing nitrate signalling with symbiotic acquired nitrogen. Low nitrate conditions provide a permissive state for induction of cytokinin by symbiotic signalling and thus nodule development. In contrast, high nitrate is inhibitory to cytokinin accumulation and nodule establishment in the root zone susceptible to nodule formation. This reduction of symbiotic cytokinin accumulation was further exacerbated in cytokinin biosynthesis mutants, which display hypersensitivity to nitrate inhibition of nodule development, maturation and nitrogen fixation. Consistent with this, cytokinin application can rescue nodulation and nitrogen fixation of biosynthesis mutants in a concentration dependent manner. These inhibitory impacts of nitrate on symbiosis occur in a Nlp1 and Nlp4 dependent manner and contrast with the positive influence of nitrate on cytokinin biosynthesis that occurs in non-symbiotic species. Altogether this shows that legumes, as exemplified by Lotus japonicus, have evolved a different cytokinin response to nitrate compared to non-legumes.One sentence summaryCytokinin biosynthesis is suppressed by nitrate in Lotus japonicus, providing a mechanism for nitrate inhibition of symbiotic nodule organogenesis.

Nitrate inhibition of nodule formation in Medicago truncatula is mediated by ACC SYNTHASE 10

ABSTRACTLegumes form a mutualistic endosymbiosis with nitrogen-fixing rhizobia. These rhizobia are housed intracellularly in specialised lateral root organs, called nodules. Initiation of these nodules is triggered by bacterial derived signalling molecules, lipochitooligosaccharides (LCO). The process of nitrogen fixation is highly energy-demanding and therefore nodule initiation is tightly regulated. Nitrate is a potent inhibitor of nodulation. However, the precise mechanisms by which nitrate inhibits nodulation is poorly understood. Here, we demonstrate that in Medicago truncatula nitrate interferes with the transcriptional regulation of the ethylene biosynthesis gene ACC SYNTHASE 10. ACSs commit the rate limiting step in ethylene biosynthesis and in M. truncatula ACS10 is highly expressed in the zone of the root where nodulation occurs. Our results show that a reduction in ACS10 expression in response to LCO exposure correlates with the ability to form nodules. In addition, RNAi-mediated knockdown of ACS10 confers nodulation ability under otherwise inhibitory nitrate conditions. This discovery sheds new light on how ethylene is involved in the inhibition of nodulation by nitrate, bringing us one step closer to understanding how plants regulate their susceptibility towards rhizobia.