Abstract
Nitrogen fixation by rhizobia is a highly energy-demanding process. Therefore, nodule initiation in legumes is tightly regulated. Environmental nitrate is a potent inhibitor of nodulation. However, the precise mechanism by which this agent (co)regulates the inhibition of nodulation is not fully understood. Here, we demonstrate that in Medicago truncatula the lipo-chitooligosaccharide-induced accumulation of cytokinins is reduced in response to the application of exogenous nitrate. Under permissive nitrate conditions, perception of rhizobia-secreted signalling molecules leads to an increase in the level of four cytokinins (i.e., iP, iPR, tZ, and tZR). However, under high-nitrate conditions, this increase in cytokinins is reduced. The ethylene-insensitive mutant Mtein2/sickle, as well as wild-type plants grown in the presence of the ethylene biosynthesis inhibitor 2-aminoethoxyvinyl glycine (AVG), is resistant to the inhibition of nodulation by nitrate. This demonstrates that ethylene biosynthesis and perception are required to inhibit nodule organogenesis under high-nitrate conditions.
Highlights
Most legumes can engage in a nitrogen-fixing endosymbiosis with diverse bacterial species collectively known as rhizobia [1]
Analysis of cytokinins from M. truncatula was performed by comparing retention times and mass transitions with those of unlabelled standards (Supplementary Table S2), using a Waters XevoTQs mass spectrometer equipped with an electrospray ionization source coupled with an Acquity UPLC system (Waters, Milford, OH, USA)
This was not the case for isopentenyl adenine (iP) and iPR, whose levels did increase upon LCO treatment, but this induction did not vary significantly across the nitrate concentrations (Figure 2B,C, Supplementary Figure S4, Supplementary Table S2). This shows that nitrate interferes with the LCO-induced accumulation of cytokinin in the M. truncatula root susceptible zone. As both induction by LCO signalling of NODULE INCEPTION (NIN) and cytokinin accumulation still occur at high nitrate concentrations, we questioned whether the initiation of pericycle and cortical cell divisions occurs under these conditions
Summary
Most legumes can engage in a nitrogen-fixing endosymbiosis with diverse bacterial species collectively known as rhizobia [1]. There are exceptions [3,4], legume–rhizobium symbiosis is initiated upon perception of rhizobium-secreted lipo-chitooligosaccharide (LCO) signalling molecules [5] Perception of these external signals results in the activation of a suite of transcriptional regulators, including the master regulator of symbiotic signalling NODULE INCEPTION (NIN) [6,7,8,9], and accumulation of the bioactive cytokinins isopentenyl adenine (iP) and trans-zeatin (tZ) [6]. It was shown that the release of ethylene from inoculated alfalfa roots is increased after the addition of nitrates [25] Taken together, this suggests that ethylene could function as a signalling intermediate during inhibition of nodulation by fixed forms of nitrogen. Our data indicate that ethylene biosynthesis and signalling are required for the inhibition of pericycle and cortical cell divisions observed at high nitrate concentrations
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