Phosphorus (P) is necessary for nitrogen fixation in the root nodules of soybeans, a symbiotic process whereby plants support bacterial nitrogen fixation to obtain nitrogen needed for plant growth. Nitrogen accumulation, quantity, weight, specific nitrogenase activity (SNA) and acetylene reduction activity (ARA) of root soybean nodules were analyzed, using a broadly targeted metabolomics method incorporating liquid chromatography-mass spectrometry (LC-MS) to study the effects of P level (1, 11, 31, 61 mg/L denoted by P1, P11, P31, P61) on the types and abundance of various metabolites and on the expression of associated metabolic pathways in soybean root nodules. Nitrogen accumulation, quantity, weight, SNA and ARA of root nodules were inhibited by P stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that root nodules responded to P stress by increasing the number of amino acids and derivatives. Down-regulation of ABA, phosphorylcholine, and D-glucose 6-phosphate affected carotenoid biosynthesis, glycerophospholipid metabolism and sugar metabolism which inhibited nodule nitrogen fixation under P stress. More flavonoids were involved in metabolic processes in soybean root nodules under P stress that regulated the nodulation and nitrogen fixation. The pathway ascorbate and aldarate metabolism, and associated metabolites, were involved in the adaptation of the symbiotic soybean root nodule system to P starvation. This work provides a foundation for future investigations of physiological mechanisms that underly phosphorus stress on soybeans.

In agroecosystems, a plant-usable form of nitrogen is mainly generated by legume-based biological nitrogen fixation, a process that requires phosphorus (P) as an essential nutrient. To investigate the physiological mechanism whereby phosphorus influences soybean nodule nitrogen fixation, soybean root nodules were exposed to four phosphate levels: 1 mg/L (P stress), 11 mg/L (P stress), 31 mg/L (Normal P), and 61 mg/L (High P) then proteome analysis of nodules was conducted to identify phosphorus-associated proteome changes. We found that phosphorus stress-induced ribosomal protein structural changes were associated with altered key root nodule protein synthesis profiles. Importantly, up-regulated expression of peroxidase was observed as an important phosphorus stress-induced nitrogen fixation-associated adaptation that supported two nodule-associated activities: scavenging of reactive oxygen species (ROS) and cell wall growth. In addition, phosphorus transporter (PT) and purple acid phosphatase (PAPs) were up-regulated that regulated phosphorus transport and utilization to maintain phosphorus balance and nitrogen fixation function in phosphorus-stressed root nodules.