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

Abstract

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 rescues 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 species that do not form symbiotic root nodules. Altogether this shows that legumes, as exemplified by Lotus japonicus, have evolved a different cytokinin response to nitrate compared to non-legumes.

Fig. 1. Nitrate inhibits induction of symbiotic cytokinin biosynthesis.

A The root zone which was susceptible to nodule initiation at time of inoculation was used for qRT-PCR and cytokinin analysis. B–E Relative transcript abundance of selected cytokinin biosynthesis genes by qRT-PCR 1 and 2 days post inoculation (dpi) with M. loti in the absence and presence of 5 mM KNO₃. Ubiquitin is used as a reference gene. E, F Cytokinin-free base content (iP and tZ) analysed in mock or 2 days post inoculation with M. loti in the absence and presence of 5 mM KNO₃. Bars show mean ± SE for the n value shown on each bar. Significant differences among different conditions are indicated by letters (p < 0.05) as determined by ANOVA and Tukey post-hoc testing for B–E. Pairwise P-values as determined by Tukey testing are available in the Source Data file. Significant differences between nitrate presence and absence conditions as determined by Wilcoxon rank-sum test is indicated by the annotated P values in F, G.

Fig. 2. ipt3 and ipt4 mutants show reduced root iP content in high nitrate conditions.

A, B Cytokinin iP (A) and tZ (B) free base content analysed in mock or 2 days post inoculation with M. loti in the absence and presence of 5 mM KNO₃ using the same root tissue as illustrated in Fig. 1A. Bars show mean ± SE for the n value shown on each bar. Significant differences between nitrate presence and absence conditions as determined by Wilcoxon rank-sum test are indicated by the annotated P values.

Fig. 3. Ipt3 and Ipt4 are required for resistance to nitrate inhibition of nodulation.

A Images of nodules developed in the absence and presence of 5 mM KNO₃ at 14 dpi with M. loti on the indicated host genotypes. Scale bar = 500 μM. B–D Development of total (B), mature (C) and the proportion of mature nodules (D) in the absence and presence of 5 mM KNO₃ at 14 dpi with M. loti. E Nitrogenase activity assessed by Acetylene Reduction Assay (ARA) at 21 dpi with M. loti in the absence and presence of 5 mM KNO₃. Box plots show Min, Q1, Median, Q3, Max and outlier values in nodulation assays (B–D). Significant differences among different genotypes and nutrient conditions are indicated by letters (p < 0.05) as determined by ANOVA and Tukey post-hoc testing with pairwise P-values available in the Source Data file. For ARA (E), bars show mean ± SE and significant differences between Gifu and mutants are indicated by the annotated P values as determined by Student’s t test. The n values of each group are shown.

Fig. 4. Ipt3 and Ipt4 contribute to maintenance of nodule initiation in the presence of nitrate.

A Nodule primordia number in the absence and presence of 5 mM KNO₃ at 7dpi with M. loti. B Infection thread density in the absence and presence of 5 mM KNO₃ at 7dpi with M. loti. Bars show mean ± SE with significant differences between genotypes and nutrient conditions indicated by letters (p < 0.05) as determined by ANOVA and Tukey post-hoc analysis. Pairwise P-values are available in the Source Data file. The n values of each group are shown.

Fig. 5. Nitrate inhibition of nodule development can be rescued by cytokinin application.

A Images of nodules developed in the presence of 5 mM KNO₃ with mock, 10^-9 or 10^-8 M BA at 14 dpi with M. loti on the indicated host genotypes. Scale bar = 500 μM. B–D Development of total (B), mature (C) and the proportion of mature nodules (D) in the presence of 5 mM KNO₃ with mock, 10^-9 or 10^-8 M BA at 14 dpi with M. loti. E Nitrogenase activity assessed by ARA at 21 dpi with M. loti in the presence of 5 mM KNO₃ with mock, 10^-9 or 10^-8 M BA. Box plots show Min, Q1, Median, Q3, Max and outlier values in nodulation assays (B–D). For ARA (E), bars show mean ± SE. Significant differences among different genotypes and concentration of BA are indicated by letters (p < 0.05) as determined by ANOVA and Tukey post-hoc testing. Pairwise P-values are available in the Source Data file. The n values of each group are shown.

Fig. 6. Nitrate restriction of cytokinin biosynthesis and signalling requires Nlp1 and Nlp4.

Relative transcript abundance of Ipt2 (A), Log1 (B), Log4 (C), RR5 (D) and RR9 (E) by qRT-PCR 1 dpi with M. loti in the absence and presence of 5 mM KNO₃ in the indicated genotypes. Ubiquitin is used as a reference gene. F Cytokinin iP free base content in the indicated genotypes at 2 dpi with M. loti. Bars show mean ± SE for the indicated n values. Significant differences between nitrate presence and absence conditions is indicated with annotated P values as determined by Student’s t test in A–E and Wilcoxon rank-sum test in F.

Fig. 7. Nitrate signalling mediated by Nlp1 and Nlp4 acts upstream of cytokinin biosynthesis to restrict symbiotic signalling.

A Images of nodules developed in the absence and presence of 5 mM KNO₃ at 14 dpi with M. loti on the indicated host genotypes. Scale bar = 1 mm. B–D Development of total (B), mature (C), and proportion of mature nodules (D) in the absence and presence of 5 mM KNO₃ at 14 dpi with M. loti. E Nitrogenase activity assessed by ARA at 21 dpi with M. loti in the absence and presence of 5 mM KNO₃. F Formation of spontaneous nodules in response to cytokinin (BA). Box plots show Min, Q1, Median, Q3, Max and outlier values in nodulation assays (B–D, F). Bars show mean ± SE. Significant differences among different genotypes and nutrient conditions are indicated by letters (p < 0.05) as determined by ANOVA and Tukey post-hoc testing with pairwise P-values available in the Source Data file. The n values are shown below the respective groups.

Fig. 8. Nitrate inhibits symbiotic signalling via Nlp1 and Nlp4.

A A heatmap of NF signalling gene expression at different times following nitrate exposure. B–E Relative transcript abundance of Nfr1 (B), Nfr5 (C), Nsp2 (D), Ern1 (E) and Nin (F) by qRT-PCR 1 dpi with M. loti in the absence and presence of 5 mM KNO₃ in the indicated plant genotypes. Ubiquitin is used as a reference gene. Bars show mean ± SE for for the indicated n values. Significant differences between nitrate presence and absence conditions is annotated with P values as determined by Student’s t test. Gene IDs and expression values are shown in Supplementary Table 4.

Fig. 9. Proposed model for regulation of nodule organogenesis by nitrate.

In low nitrate, NLP signalling contributes to regulation of symbiotic genes, which stimulate cytokinin biosynthesis, ensuring robust nodule maturation and onset of nitrogen fixation. In high nitrate, NLP-dependent nitrate signalling inhibits expression of symbiotic genes and symbiotic cytokinin biosynthesis that is crucial for nodule organogenesis. Nodulation is thus balanced between a permissive low-nitrate state where high cytokinin levels can accumulate, or a restrictive state with low root cytokinin.