On December 2, 2022, the group of Professor Xuelu Wang of the State Key Laboratory of Crop Stress Adaptation and Improvement published the research result entitled “Phosphoenolpyruvate reallocation links nitrogen fixation rates to root nodule energy state” in the form of a research article. The article revealed the energy sensors of soybean nodules and their new mechanisms regulating symbiotic nitrogen fixation. This is another innovative research result published by the group in top international journals after Professor Xuelu Wang's group published a research paper in Science on October 1, 2021 (This achievement was selected as “Top Ten Scientific and Technological Progress in Chinese Universities” in 2021 and “Major Progress in Chinese Agricultural Science” in 2022), which fully demonstrates the innovation vitality and international leading position of Xuelu Wang's group in related research fields, as well as the outstanding achievements made in promoting the development of biological nitrogen fixation, which is another major breakthrough in the scientific and technological innovation, and construction of “Double First-class” in our university.

Nitrogen is a kind of nutrient necessary for plant growth and development, so agricultural production is highly dependent on industrial nitrogen fertilizers. However, the production of nitrogen fertilizer requires a large amount of fossil energy, and excessive application of nitrogen fertilizer will cause soil compaction degradation and water pollution, affecting the sustainable development of agriculture. Biological nitrogen fixation is the largest natural source of biologically usable nitrogen in nature, and legumes and rhizobia can interact to form a unique organ, the symbiotic nodules. Symbiotic nitrogen fixation in nodules is an important way for nitrogen reduction in the earth's ecosystem to ammonia that can be absorbed and utilized by plants, contributing more than 60% of the nitrogen fixation of terrestrial organisms, which is of great significance for maintaining primary production and carbon sinks in agriculture and natural ecosystems. Therefore, improving the symbiotic nitrogen fixation capacity of legumes, and even developing the symbiotic nitrogen fixation of non-leguminous plants or crops, is of great significance to reduce the dependence on industrial nitrogen fertilizers, develop green and sustainable agriculture, and realize the "dual carbon" strategy. Symbiotic nitrogen fixation is a highly energy-consuming enzyme-catalyzed process, and the carbohydrates fixed by the photosynthesis are the most important carbon and energy sources for symbiotic nitrogen fixation (Figure 1). Therefore, the nitrogen fixation capacity of nodules needs to be coordinated with the carbon source and energy level of legumes to balance the carbon consumption of symbiotic nitrogen fixation and other life processes and ensure the normal growth of legumes in different environments. However, the mechanism by which legumes respond to carbon sources and energy levels to regulate nitrogen fixation in nodules remains a mystery.

Figure 1. The source and allocation of carbon and energy during symbiotic nitrogen fixation

Professor Xuelu Wang's research group discovered new energy sensor proteins GmNAS1 (soybean nodule AMP sensor 1) and GmNAP1 (GmNAS1-associated protein 1) in nodules, which can sense the rising energy state, and then regulate the allocation of glycolysis intermediates in soybean nodules for nitrogen fixation and plant cell self-utilization (Figure 2).

The nitrogen fixation capacity of leguminous nodules is affected by the environment, and these environmental factors often affect the energy state of nodules, suggesting an important relationship between the changes in nodule energy state and nitrogen fixation capacity. The cystathionine β-synthase (CBS) domain is a class of conserved functional domains with the ability to bind adenylate and its derivatives, including AMP, ADP, and ATP, and CBS family proteins have the potential to act as cellular energy sensors, such as AMPK in animals and yeast (Baykov et al., 2011; Gonzalez et al., 2020). To identify possible energy sensors in soybean nodules, the authors screened 71 CBS family proteins and identified GmCBS22 (GmNAS1) and GmCBS14 (GmNAP1) highly expressed in nodules. Genetic analysis showed that the loss of function of GmNAS1 and GmNAP1 did not affect the formation and development of nodules, but completely inhibited the increase in nitrogen fixation capacity after the carbon supply of nodules increased. Further studies have found that GmNAS1 and GmNAP1 monitor the energy status of nodule cells by sensing cellular AMP levels, GmNAS1 can directly bind to AMP to form heterodimers on the mitochondrial membrane with GmNAP1, and when the energy status of nodules rises due to increased carbon supply, the AMP content decreases, prompting the dissociation of GmNAS1-GmNAP1 heterodimer to form GmNAS1-GmNAS1 and GmNAP1-GmNAP1 homodimers. This way of feeling energy is different from animal cells, indicating its unique way of feeling and an important contribution to the field of energy sensing in life sciences.

To elucidate the mechanism by which GmNAS1 and GmNAP1 localized on the mitochondrial membrane regulate nitrogen fixation in nodules, the authors identified a transcription factor NF-YC subunit GmNFYC10a that interacts with GmNAS1 and GmNAP1 by coimmunoprecipitation-mass spectrometry. It was found that GmNAS1-GmNAS1 and GmNAP1-GmNAP1 homodimers were formed by reducing AMP levels interacting with GmNFYC10a and anchoring it to mitochondria when the energy state of nodules rises, thereby reducing GmNFYC10a levels in the nucleus, inhibiting pyruvate kinase (PK) gene expression (Figure 2), and regulating the energy distribution between plant cells and bacterioids.

Figure 2. New energy sensors in nodules regulate PEP allocation to mediate carbon source level and nitrogen fixation capacity

This breakthrough reveals the molecular mechanism of the novel energy sensors GmNAS1/GmNAP1 in soybean nodules regulating the redistribution of nodule carbon sources to adjust the nitrogen fixation capacity of nodules, and shows that animal cells and plant cells use their own molecular mechanisms to sense energy. This mechanism enables leguminous plants to adjust the nitrogen fixation efficiency of nodules in time according to the availability of carbon sources when the growth environment changes, so as to maintain the carbon and nitrogen balance in the plant and adapt to changes in the surrounding environment. This breakthrough achievement provides a paradigm for discovering more energy sensors and establishing their signaling pathways in plants that produce carbon sources independently, which will greatly promote the analysis of the evolution and molecular mechanism of carbon source allocation and metabolic regulation at the cellular and individual levels, and provide important theoretical support for the future design of efficient use of carbon sources in crops themselves or the surrounding environment through synthetic biology methods, improve the ability of symbiotic nitrogen fixation, and provide new ideas for the molecular design of efficient nitrogen-fixing crops.

Professor Xuelu Wang of the State Key Laboratory of Crop Stress Adaptation and Improvement jointly established by Henan University and the Institute for Advanced Study of Interdisciplinary Studies of Henan University is the corresponding author of the paper, and Ke Xiaolong, a postdoctoral fellow of Henan University, is the first author. The research was supported by the National Key Research and Development Program of China, the National Natural Science Foundation of China, the Zhongyuan Scholars Program of Henan Province, and the funding of Henan University.

In addition to the successive breakthroughs published in Science, Xuelu Wang's group has recently also made a series of innovative progress in the genetic and molecular mechanisms of soybean-rhizobia co-evolution (Zhang et al., 2021, Nature Plants), the molecular mechanism of environmental stress regulating the development of symbiotic nodules (He et al., 2021, Molecular Plant), and the cytological basis of rhizobia-induced nuclear endoreduplication (Fan et al., 2022, New Phytologist).

Since joining Henan University in December 2019 to lead the group of "Biological Nitrogen Fixation and Leguminous Biology", Professor Xuelu Wang has taken leguminous crops as the main research object to study the genetic, evolutionary and molecular mechanisms of bacteria-plant interactions, genetic and molecular mechanisms of energy sensing and distribution regulating nodule development and symbiotic nitrogen fixation, molecular design and breeding of leguminous crops, etc. At present, the group has brought together a number of outstanding young teachers from the United States, the United Kingdom, Shanghai and Wuhan to join, among which, one was selected for the national young talent program. The team also has attracted more than 10 outstanding doctoral graduates from Wuhan University, Jilin University, Zhejiang University, National University of Singapore, Nanjing Agricultural University and Huazhong Agricultural University to engage in postdoctoral research. Recently, Professor Xuelu Wang as the chief scientist obtained the national key R&D project "Design and System Optimization of Biological Nitrogen Fixation Circuit with Carbon and Nitrogen Enhancement Efficiency", the team will rapidly promote innovative research in the international frontier scientific field of biological nitrogen fixation and carbon and nitrogen efficiency, and improve the high-level synthetic biology and molecular design breeding platform, actively expand to the field of modern biological breeding, and contribute to national food security and green and sustainable development of agriculture.