Nitrogen deficiency modulates carbon allocation to promote nodule nitrogen fixation capacity in soybean

Previously, the effect of soil mineral N deficiency on nodule nitrogen fixation capacity (NFC) is unclear. In this study, we found that N deficiency would enhance sucrose allocation to nodules and PEP allocation to bacteroid to promote nodule NFC. Our findings provide new insights into the design of leguminous crops with improved adaptation to fluctuating N levels in the soil. Nitrogen (N) is an essential inorganic nutrient that supports plant growth and development. Plant N acquisition is achieved mainly through the uptake of soil mineral N, while legumes also obtain N through establishing symbiotic relationships with rhizobia. Symbiotic nitrogen fixation (SNF) is a highly energy-consuming process with a carbon (C) consumption of >5 g C g N−1, which costs far more than soil N uptake that generally does not exceed 2.5 g C g N−1.[1-3] Therefore, it is reasonable that sufficient mineral N in the soil would inhibit nodulation and nitrogen fixation, including rhizobial infection, nodule initiation, growth, and nitrogen fixation.[4, 5] In contrast, N deficiency in the soil may enhance nodulation in legumes. Autoregulation of nodulation (AON) is an important strategy for legumes to control nodule number when sufficient rhizobia infection or mineral N exists.[6-8] It was recently reported that N deficiency would enhance the expression of C-terminally encoded peptides (CEPs) in roots to activate the shoot Compact Root Architecture 2 (CRA2) receptor, thereby inhibiting the systemic AON to promote nodule formation in Medicago truncatula.[9-11] However, whether N deficiency also affects nodule nitrogen fixation capacity (NFC) at later stages remains unclear. To examine the effect of N deficiency on nodule NFC, we used nutrient solution without nitrate for the soybean "Williams 82" (W82) plants inoculated with Bradyrhizobium diazoefficiens USDA110 at 10 days post inoculation (DPI). At 25 DPI, as compared with the N-sufficient plants supplied with 2 mM nitrate according to the previous reports,[12-14] the soybean plants with deficient N supply showed similar nodule number and weight, but significantly enhanced nodule nitrogenase activity (Figure 1A), suggesting that N deficiency at late stages can strongly promote nodule NFC in soybean. Moreover, at late stages, N deficiency also inhibited shoot growth, but had no effect on root and nodule development (Figure S1). It was previously reported that N deficiency of the whole plant possibly increases the sucrose content of nodules in M. truncatula.[15] Here we found that the late N deficiency significantly increased nodule sucrose concentration in soybean (Figure 1B). Accordingly, nodule energy state (NES) was elevated after N deficiency (Figure 1E). We also found that the sucrose concentrations in both roots and leaves were significantly reduced under N-deficiency conditions (Figure 1B). However, the sucrose content ratio of underground tissues (including roots and nodules) to leaves was only slightly increased by N deficiency (Figure 1C). Remarkably, the sucrose content ratio of nodule to root was greatly increased under N-deficiency conditions (Figure 1D). The results suggested that the increase in nodule sucrose concentration under N-deficiency conditions is mainly due to more sucrose allocated to nodules from roots, but not due to more sucrose transported to underground tissues from leaves. We have previously found that two nodule-specific mitochondria-localized energy sensors in soybean, GmNAS1 and GmNAP1, can sense the increased NES to reduce GmNFYC10 nuclear accumulation, thereby modulating glycolytic pathway and phosphoenolpyruvate (PEP) allocation to enhance nodule NFC.[16] To investigate whether GmNAS1/GmNAP1 mediates the enhanced nodule NFC under N-deficiency conditions, we examined the nodulation phenotypes of knockout mutants of GmNAS1/GmNAP1 created in our previous study,[16] including cr-nas1, cr-nap1, cr-nas1nap1-1, and cr-nas1nap1-2, under different usable nitrogen conditions. The result suggested that the nodule number and weight of these mutants were similar to the wild type under N-deficiency conditions (Figure S2A,B), and the development of nodule infection zone was also unaffected in these mutants (Figure S2C). However, N deficiency failed to enhance nodule NFC in all of these mutants (Figure 1F), indicating that GmNAS1/GmNAP1 are required for the N-deficiency-enhanced nodule NFC. Moreover, N deficiency also failed to enhance nodule NFC in the cr-nfyc10 mutant (Figure S3), indicating that GmNFYC10 is also necessary for the elevated NFC after N deficiency. Together, these results suggested that the GmNAS1/GmNAP1-GmNFYC10 module regulates the increase in nodule NFC under N-deficiency conditions. Previous study showed that the interaction of GmNAS1/GmNAP1 with GmNFYC10a was inhibited by increased AMP concentration but enhanced by high NES.[16] Considering that NES was elevated after N deficiency (Figure 1E), we tested and found that the interaction of GmNAS1/GmNAP1 with GmNFYC10a was also enhanced under N-deficiency conditions (Figure S4A), while the expression levels of GmNAS1, GmNAP1, and GmNFYC10a were unchanged (Figure S4B). Thus, under N-deficiency conditions, more GmNFYC10a protein was anchored to the mitochondria by GmNAS1/GmNAP1, and less GmNFYC10a protein was localized to the nucleus (Figure 1G). We further observed that the expression of many glycolytic genes activated by GmNFYC10 was downregulated after N deficiency in the wild-type plants (Figure 1H),[16] but not in the knockout mutants of GmNAS1, GmNAP1, and GmNFYC10 (Figure S5), suggesting that the GmNAS1/GmNAP1-GmNFYC10 module attenuates the glycolysis under N-deficiency conditions. Moreover, we observed that the expression of most of these glycolytic genes was higher in the GmNAS1/GmNAP1 knockout mutant nodules than that in the wild-type nodules under N-deficiency conditions (Figure 1I). Considering that half of these glycolytic genes encode pyruvate kinases (PKs),[16] we then examined the PK2a protein content and PK activity. The results showed that N deficiency reduced PK2a protein content and PK activity in W82 nodules, but the decrease was remarkably attenuated in the cr-nas1, cr-nap1, and cr-nas1nap1-1 nodules (Figure S6). Accordingly, the pyruvate contents in the cr-nas1, cr-nap1, and cr-nas1nap1-1 nodules were also greatly higher than that in the wild-type nodules under N-deficiency conditions (Figure 1J). The oxaloacetic acid (OAA) and malate contents in these mutant nodules were lower than those in the wild-type nodules (Figure 1K,L), and the ratio of pyruvate to OAA was dramatically increased in the mutant nodules (Figure 1 M), suggesting that GmNAS1/GmNAP1 play an important role in regulating the competition between pyruvate and OAA production for PEP under N-deficiency conditions.[16] Taken together, these results indicated that GmNAS1/GmNAP1 inhibit GmNFYC10 nuclear accumulation to reduce pyruvate production under N-deficiency conditions, thus enhancing OAA and malate synthesis to improve nodule NFC. As sessile organisms, plants need to adapt to fluctuating nutritional environments to ensure their survival and optimal growth. To cope with mineral N deficiency in the soil, legumes have evolved SNF to acquire N nutrition from the atmosphere. Previous studies reported that N deficiency would promote nodule formation through inhibiting the AON pathway.[9-11] Here, our study showed that N deficiency at later stages can strongly enhance nodule NFC through modulating C allocation in roots and nodules. Under N-sufficiency conditions, more sucrose is allocated to root cells to support mineral N uptake and assimilation (Figure S7A), which is more economical than SNF.[1-3] When the usable nitrogen in the soil is deficient, more sucrose would be allocated to nodules to increase NES. The elevated NES is responded by the GmNAS1/GmNAP1-GmNFYC10 module to lower the expression of PK genes, thereby modulating PEP allocation to promote nodule NFC (Figure S7B). Therefore, under N-deficiency conditions, C allocation in nodules is regulated by the GmNAS1/GmNAP1-GmNFYC10 module, it will be worthy to investigate the underlying mechanism by which N deficiency regulates the C allocation in roots. Our findings reveal how legumes modulate nodule NFC to adapt to mineral N deficiency, and provide novel insights into the design of leguminous crops with improved adaptation to fluctuating usable nitrogen supplies in the soil. Xiaolong Ke and Xuelu Wang designed the experiments; Xiaolong Ke performed most of the experiments; Han Xiao contributed to the vector construction, material collection, and nodulation assays; Yaqi Peng generated the stable transgenic soybean plants; Xue Xia participated in the design of experiments; Xiaolong Ke and Xuelu Wang wrote the manuscript. This work was supported by grants from the National Key Research and Development Program (grant no. 2022YFA0912100 to Xuelu Wang and Xiaolong Ke), the National Natural Science Foundation of China (grant no. 31870257 to Xuelu Wang; grant no. 32200203 to Xiaolong Ke), the Zhongyuan Scholar of Henan Province (grant no. 224000510001 to Xuelu Wang), the Outstanding Talents Fund of Henan University of China (grant no. CX3050A092004 to Xuelu Wang), and the Program for Innovative Research Team in University of Henan Province (grant no. 23IRTSTHN020 to Xuelu Wang and Xiaolong Ke). The authors declare no conflicts of interest. All data are available in the main text or supporting information. For material requests, please contact [email protected]. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.