Phosphoenolpyruvate reallocation links nitrogen fixation rates to root nodule energy state

Abstract

Legume-rhizobium symbiosis in root nodules fixes nitrogen to satisfy the plant’s nitrogen demands. The nodules’ demand for energy is thought to determine nitrogen fixation rates. How this energy state is sensed to modulate nitrogen fixation is unknown. Here, we identified two soybean ( Glycine max ) cystathionine β-synthase domain–containing proteins, nodule AMP sensor 1 (GmNAS1) and NAS1-associated protein 1 (GmNAP1). In the high–nodule energy state, GmNAS1 and GmNAP1 form homodimers that interact with the nuclear factor-Y C (NF-YC) subunit (GmNFYC10a) on mitochondria and reduce its nuclear accumulation. Less nuclear GmNFYC10a leads to lower expression of glycolytic genes involved in pyruvate production, which modulates phosphoenolpyruvate allocation to favor nitrogen fixation. Insight into these pathways may help in the design of leguminous crops that have improved carbon use, nitrogen fixation, and growth.

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

“…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.…”

“…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).…”

“…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).…”

“…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).…”

“…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).…”