Symbiotic nitrogen fixation suppresses root nitrate uptake in Medicago truncatula under nitrate limitation

Abstract

While most plants use nitrate as the main nitrogen source (Gent & Forde, 2017), legumes such as Medicago truncatula can also acquire nitrogen by forming an endosymbiosis with N₂-fixing bacteria called rhizobia, a process referred to as nodulation. In this endosymbiosis, the rhizobia are taken up into nodule cells which provide the low-oxygen environment required for N₂ fixation (Layzell & Hunt, 1990). This energy-intensive process requires a constant supply of carbon from the host to produce ammonia that is directly assimilated in the nodule and then exported to the rest of the plant as amides or ureides, depending on the legume species (Ta et al., 1986; King & Purcell, 2005; Lu et al., 2022). To optimize growth and productivity, legumes need to maintain a balance between nitrogen gains and carbon losses during nodulation (Reid et al., 2011; Nishida & Suzaki, 2018); and this is why under nitrate-sufficient conditions, nodule formation and N₂ fixation are suppressed (Chaulagain & Frugoli, 2021). Recently, it was shown in Lotus japonicus and Medicago truncatula that nitrate suppression of nodulation depends on NRT2.1s and the NIN-like protein (NLP) transcription factors that control their expression (Misawa et al., 2022; Luo et al., 2023). The suppression of nodulation by high nitrate levels presumably reflects the higher cost of symbiotic N₂ fixation over direct N uptake from the soil (Zhao et al., 2024). However, how these two systems interact under low nitrate levels remains unknown.

To investigate this, we examined the effect of nodulation on root nitrate uptake in M. truncatula. Experiments using labeled nitrogen (¹⁵NO₃⁻) revealed a significant reduction of ¹⁵N content in both denodulated roots (i.e. root sections with nodules removed) and shoots of nodulated plants compared to the uninoculated controls at 3 wk postinoculation (wpi) (Fig. 1a). Direct nitrate uptake in plants occurs through high- and low-affinity transport systems which allow plants to adapt to varying soil nitrate levels (Crawford & Glass, 1998). Nitrate uptake from soil is mainly mediated by nitrate transporter 2 (NRT2) /nitrate assimilation related-protein 2 (NAR2) complexes and nitrate transporter 1 (NRT1) / peptide transporter (PTR) family (NPF) members (Gojon et al., 2011; Krapp et al., 2014; Noguero & Lacombe, 2016). We next examined whether NRT2.1, previously identified as a key nitrate uptake transporter in M. truncatula (Luo et al., 2023), might contribute to this process. We found that under low (0.2 and 0.5 mM) nitrate concentrations that are conducive for nodulation, the expression of MtNRT2.1 was significantly downregulated in denodulated roots compared to noninoculated controls 3 wpi with rhizobia (Fig. 1b). By contrast, for plants nodulated under high (5 mM) nitrate, no significant difference was observed in the ¹⁵N content of the shoots or roots, or in the levels of NRT2.1 expression in their denodulated roots, relative to uninoculated controls (Supporting Information Fig. S1a,b). Moreover, we found that this repression did not occur at earlier nodulation time points (Fig. S2). We then tested whether NRT2.1 is important for nitrate uptake under 0.2 mM KNO₃. This revealed that the nrt2.1 mutant shoots and roots absorbed significantly less ¹⁵KNO₃ than in the wild type (WT) (Fig. 1c), similar to what was reported for the mutant when grown under 0.5 and 5 mM KNO₃ (Luo et al., 2023). These findings indicate that nodulation negatively impacts root nitrate uptake by downregulating MtNRT2.1 expression specifically under nitrate limiting conditions.

To gain an insight into how this phenomenon is regulated, we performed RNA-seq analysis on denodulated roots and roots of noninoculated plants grown under 0.2 mM KNO₃. A total of 5105 genes were upregulated, and 3540 genes downregulated in roots of nodulated plants (denodulated roots) compared to control roots. The full list of differentially expressed genes (DEGs) is provided in Table S1. Principal component analysis (PCA) of the RNA-seq results revealed a clear distinction between noninoculated and rhizobia-inoculated root samples for PC1, which explained 61% of the variation (Fig. S3a). A Kyoto Encyclopedia of Genes and Genomes (KEGG) term enrichment analysis revealed that denodulated roots have increased expression of genes related to ‘TCA cycle’, ‘glycolysis/gluconeogenesis’, ‘pyruvate metabolism’, ‘fructose and mannose metabolism’, and ‘starch and sucrose metabolism’, suggesting they have strongly enhanced sugar metabolism, while genes related to ‘nitrogen metabolism’ and ‘circadian rhythm’ were decreased (Fig. S3b). In addition, the results confirmed the repression of NRT2.1 in denodulated roots and revealed similar suppression of NRT2.2 and NAR2.1, which encode components of the high-affinity nitrate uptake complex, NLP4 which is required for nitrate suppression of nodulation, and its homolog NLP5 (Marchive et al., 2013; Xiao et al., 2021; Luo et al., 2023) (Fig. 1d). The ‘nitrogen metabolism’ genes that were downregulated also included the gene encoding the main root isoform of nitrate reductase, NR2, and two glutamate synthase genes, suggesting that nitrate assimilation is reduced in denodulated roots. Notably, members of two transcription factor families, Lateral Organ Boundaries Domain (LBD) proteins and Nitrate-Inducible GARP-type Transcriptional Repressor 1s (NIGT1s), thought to be involved in the repression of NRT2.1 expression in Arabidopsis thaliana and rice (Rubin et al., 2009; Yang et al., 2016; Kiba et al., 2018; Maeda et al., 2018; Zhu et al., 2022), were upregulated in roots of nodulated plants (Fig. 1d, see phylogenetic relationships in Figs S4, S5), indicating their possible role in regulating NRT2.1 expression.

While NIGTs were reported to respond specifically to nitrate (Kiba et al., 2018; Maeda et al., 2018), LBDs have been reported to respond to different forms of fixed nitrogen, including ammonium and glutamine in A. thaliana and rice (Rubin et al., 2009; Zhu et al., 2022). To test whether ammonia could affect NRT2.1 expression, we evaluated its transcript levels 6 h after provision with (NH₄)₂SO₄ in the presence of low concentrations of nitrate. The expression of NRT2.1 was induced by nitrate, and was similarly induced by a combination of ammonium and nitrate, relative to the nitrate-starved controls, and no induction was seen with ammonium (Fig. S6). This is similar to A. thaliana, where ammonium did not downregulate NRT2.1 expression under lower nitrate levels (Nazoa et al., 2003; Krouk et al., 2006), suggesting that ammonia produced in nodules may not be directly involved in the suppression of NRT2.1.

We then investigated glutamine, which was shown to inhibit the expression of NRT2s in Nicotiana plumbaginifolia, A. thaliana, and Hordeum vulgare (Quesada et al., 1997; Krapp et al., 1998; Vidmar et al., 2000) and is a major export product of nodules. We first tested glutamine's effect on nonsymbiotic roots. Consistent with findings from other species, we found that the presence of glutamine strongly reduced the induction of MtNRT2.1 by nitrate (Fig. 1e). Furthermore, the expression of MtNIGT1.1 and MtNIGT1.2 was enhanced both by nitrate and by glutamine (Fig. S7a,b), contrasting with findings in other species where NIGT1 genes were reported to respond exclusively to nitrate (Kiba et al., 2018; Maeda et al., 2018). Interestingly, the MtNIGT1s were induced to a lower extent by the addition of both nitrate and glutamine compared to either treatment alone. Examination of MtLBD19 and MtLBD23 transcript levels revealed that they did not respond to nitrate but were significantly induced by glutamine or by a combination of glutamine and nitrate (Fig. S7c,d). These results are broadly consistent with the potential involvement of nodule produced glutamine acting through LBDs and/or NIGTs to repress NRT2.1-mediated nitrate uptake. To further investigate this possibility, the MtLBDs and MtNIGTs of interest were overexpressed in hairy roots (Fig. S8a,b,d,e). Overexpression of NIGT1.1 and NIGT1.2 suppressed NRT2.1 transcript levels under 0.5 or 5 mM nitrate (Fig. S8c), consistent with their potential importance in suppressing NRT2.1 expression during nodulation, while LBD23 downregulated NRT2.1 expression only under 5 mM nitrate (Fig. S8f).

We then tested whether a short-term glutamine treatment, which repressed NRT2.1 expression, affects nitrate uptake. We found that a 6 h glutamine treatment significantly reduced the uptake of ¹⁵N-labeled nitrate in roots, but not in shoots (Fig. S9a). To test whether a longer-term treatment would have a similar effect, M. truncatula seedlings were treated weekly with 0.5 mM glutamine and 0.2 mM ¹⁵KNO₃ over a 3-wk time period. The plants responded strongly in terms of growth relative to the controls grown on 0.2 mM ¹⁵KNO₃, indicating that M. truncatula can efficiently uptake and utilize glutamine (Fig. S9b). Furthermore, the glutamine supplemented roots exhibited reduced ¹⁵NO₃ uptake both in roots and in shoots (Fig. 1f). These findings support a potential role for nodule assimilates in repressing nitrate uptake by downregulation of MtNRT2.1.

To further investigate the role of nodules in suppressing nitrate uptake in roots, we tested nitrate uptake in two M. truncatula nodulation-defective mutants, nodule inception (nin) and does not fix nitrogen 7 (dnf7). Loss of NIN completely blocks rhizobial infection and nodule formation (Liu & Bisseling, 2020), while the dnf7 mutant, which lacks Nodule Cysteine Rich Peptide 169, forms white nodules incapable of nitrogen fixation due to a defect in symbiosome development (Horváth et al., 2015). Following our earlier experiments, the mutants were grown under nitrogen-starved conditions (0.2 mM KNO₃) with or without rhizobia inoculation, and the expression of MtNRT2.1 was assessed by quantitative real-time PCR at 3 wpi. Our results show that neither nin nor dnf7 mutants exhibited significant changes in MtNRT2.1 transcript levels (Fig. 2a–c). Additionally, rhizobial inoculation did not cause significant changes in the transcript levels of the LBDs or NIGTs of interest in either of the mutants (Fig. S10). In line with these findings, no differences in ¹⁵NO₃ uptake were observed in the shoots and roots of nin and dnf7 seedlings after rhizobia inoculation, in contrast with the WT (Fig. 2d–f). In addition, we used a rhizobial mutant that is unable to fix nitrogen (ΔnifK) to inoculate M. truncatula under low nitrate, and no repression of MtNRT2.1 was found (Fig. S11). In summary, our results using both plant and rhizobium mutants suggest that functional nodules are required for the suppression of direct root nitrate uptake rather than nodule formation per se.

In previous studies, it was shown that under low nitrate conditions nodule formation is reduced in nrt2.1 and nlp1 mutants (Misawa et al., 2022; Luo et al., 2023), which are deficient for nitrate uptake, suggesting that in WT plants, the limited N available is probably invested in forming nodules. This is consistent with our results, which show that NRT2.1 expression is unaffected at the early stages of nodule formation (Fig. S2). However, our results suggest that once productive nodules are established, direct nitrate uptake is suppressed, likely through glutamine or other assimilates. We further found that Lotus japonicus shows a similar repression of LjNRT2.1 in denodulated roots, suggesting that this may be a general feature of legumes (Fig. S12). The finding that nitrate uptake is reduced in nodulated plants is somewhat unexpected, given that on a per molecule basis, the cost of N₂ fixation exceeds that of nitrate reduction to ammonia by 9 kJ (Gutschick, 1978). The prioritization of N fixation over nitrate uptake when nitrate availability is limited suggests that the ‘nitrate scavenging’ strategy, which entails root growth and release of exudates, etc., provides a lower return on investment for the plant than N fixation.

None declared.

FL, AK and JDM designed the research; FL and AK performed the research and analyzed the data; and FL, AK, PX and JDM wrote the manuscript. FL and AK contributed equally to this work.

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