The Characterization Of Interaction Between NRG2 And NLP7 And Screening For NRG2 Interacting Proteins In Arabidopsis

Nitrogen is one of the essential macroelements for plants. Nitrogen absorption determines growth and productivity for plants. Plants have evolved complex and fine regulation mechanisms to adapt the variable nitrogen environment in the soil. It is of great importance for developing sustainable agriculture and reducing the environmental pollution to strengthern the research on mechanisms of nitrogen utilization and crop breeding for varieties with high nitrogen use efficiency. Nitrogen can be found in forms of nitrate, ammonium, amino acids, proteins, and other Nitrogen-containing substansces in the soil. Nitrate is the main form absorpted by plants. Four genes families play important roles in the nitrate absorption and transport: nitrate transporter family 1(NRT1), nitrate transporter family 2(NRT2), the chloride channel family(CLC), and slow anion channel-associated homologues(SLAC/SLAH). Besides acting as an essential nutrient element, nitrate also serve as a signaling molecule to break seed dormancy, induce leaf expansion, regulate lateral development and coordinate the expression of the nitrate-related genes. As the development of the bio-technique, more and more attentions are paid on the genes involoved in nitrate regulation, and some nitrate regulatory genes such as NRT1.1, NLP7, CIPK8, CIPK23 have been characterized.NRG2 is a new nitrate regulatory gene identified by our lab using forward genetics. It is localized in nucleus. NRG2 regulates the expression and works upstream of NRT1.1. NLP7 has been identified as a transcription factor involved in nitrate response. Its location is affected by the nitrate condition. In this study, yeast two-hybrid and bimolecular fluorescence complementation analysis was used to investigate the interaction of NRG2 and NLP7. The results showed that NRG2 can interact with NLP7 in the nuclear under nitrate condtions. The interaction site in NRG2 was found to be in the area between the two conserved domains: DUF 630 and DUF 632. To explore more genes involved in NRG2 regulatory network, we used yeast two hybrid to screen for proteins that can interact with NRG2. 22 proteins were isolated and two of them(At1g20100 and At5g61590) were tested if they fuction in the nitrate signaling. The expression of the nitrate inducible genes showed no difference between their mutants and wild type indicating that these two genes may not be involved in nitrate regulation. The screening and characterization of NRG2 interacting proteins will further promote the exploration of novel nitrate regulatory genes and illustration of nitrate signaling network.