Molecular Signals Controlling the Inhibition of Nodulation by Nitrate in Medicago truncatula

Abstract

The presence of nitrogen inhibits legume nodule formation, but the mechanism of this inhibition is poorly understood. We found that 2.5 mM nitrate and above significantly inhibited nodule initiation but not root hair curling in Medicago trunatula. We analyzed protein abundance in M. truncatula roots after treatment with either 0 or 2.5 mM nitrate in the presence or absence of its symbiont Sinorhizobium meliloti after 1, 2 and 5 days following inoculation. Two-dimensional gel electrophoresis combined with mass spectrometry was used to identify 106 differentially accumulated proteins responding to nitrate addition, inoculation or time point. While flavonoid-related proteins were less abundant in the presence of nitrate, addition of Nod gene-inducing flavonoids to the Sinorhizobium culture did not rescue nodulation. Accumulation of auxin in response to rhizobia, which is also controlled by flavonoids, still occurred in the presence of nitrate, but did not localize to a nodule initiation site. Several of the changes included defense- and redox-related proteins, and visualization of reactive oxygen species indicated that their induction in root hairs following Sinorhizobium inoculation was inhibited by nitrate. In summary, the presence of nitrate appears to inhibit nodulation via multiple pathways, including changes to flavonoid metabolism, defense responses and redox changes.

Legume nodulation: The host controls the party

“…This helps conserve resources and energy when nitrogen fixation is not needed, thus maintaining the optimal balance of nitrogen with carbon and other resources (Voisin, Munier‐Jolain, & Salon, ). Low levels of exogenously applied nitrogen (<2 mM) enhance nodulation, likely by promoting plant health without being sufficient to make nitrogen fixation redundant, whereas nodule numbers gradually decrease with the application of increasing nitrogen concentrations (Barbulova, Rogato, D'Apuzzo, Omrane, & Chiurazzi, ; Carroll, McNeil, & Gresshoff, ; Day, Carroll, Delves, & Gresshoff, ; Hussain, Jiang, Broughton, & Gresshoff, ; van Noorden et al, ). Interestingly, the response seems to be contingent on the source of nitrogen, as nitrate and ammonia have a strong inhibitory effect on nodule development, while urea generally does not, with some species‐specific exceptions reported (e.g., cowpea; Dart & Wildon, ; Vigue, Harper, Hageman, & Peters, ).…”

Evaluation of beneficial and inhibitory effects of nitrate on nodulation and nitrogen fixation in common bean (Phaseolus vulgaris)

“…however, root hair curling remained unaffected (van Noorden et al, 2016). The inhibitory effects of nitrate on nodulation involve modifications of flavonoid and defense metabolism, as well as changes to redox (van Noorden et al, 2016).…”

Characterization of nodule-specific, SLAC and MATE membrane transporters in Medicago truncatula

“…MtSLAH1 expression decreased dramatically two days after application of high nitrate, a known repressor of SNF Van Noorden et al, 2016) (Figure 2B). This result indicates a potential symbiotic role of MtSLAH1 during SNF, especially in supporting/maintaining the nodule function or nodule formation.…”

“…MtMATE30 expression was barely detectable at 4 days postinfection (dpi), peaked at 10 dpi (young, mature nodule), and remained steady in the functional nodule at 21 and 28 dpi ( Figure 3B). Transcription of MtMATE30 decreased dramatically (10-fold) two days after the application of nitrate, a known repressor of SNF Van Noorden et al, 2016). This result indicates a potential SNF role of We further examined the MtMATE30 expression in roots infected with S. melliloti mutant strains impaired in specific milestone stages of the symbiosis establishment ( Figure 4B).…”

“…All SLAC channels identified so far have great permeability of nitrate than chloride. Interesting, nitrogen-fixing symbioses are suppressed by nitrate, by inhibiting the existing nodules and promoting the senescence process Van Noorden et al, 2016). The effects of nitrate on the symbiosis are complex and remain unclear.…”