Screening And Identification Of Arabidopsis Nitrate Regulatory Mutants

Nitrogen is the most important macronutrients for plant growth and development. It is involved in the synthesis of proteins, nucleic acids, chloroplast and various nitrogenous substances. The widespread application of nitrogen fertilizer has promoted global agricultural production, but the nitrogen that can not be completely absorbed by the plants is lost into the environment and has caused serious consequences. These problems have becoming more severe and it is urgent to be solved. Therefore, it’s of great importance for reducing the pollution and realizing the sustainable development of agriculture to strengthen the study of nitrogen, breed new variety with high nitrogen use efficiency and improve the nitrogen use efficiency of crops.Nitrate is the main form of nitrogen taken by plants, it is absorbed by the high-/low-affinity transport systems from the soil, and then transported to roots and leaves, Once inside the plants, it is assimilated to amino acids or amides by nitrate reductase, nitrite reductase, glutamate synthetase and glutamine synthase. Nitrate is not only a nutrient, but also a biological signal molecule. Plants can regulate many physiological processes like root development in response to nitrate signal to adapt to complex environment. Nitrate regulatory genes play important roles in this process. Elucidating the nitrate regulatory gene network is the foundation and the key to improve the nitrogen use efficiency. During the last several years, several nitrate regulatory genes have been identified and characterized, such as CHL1, NLP7, CIPK8/23and LBD37/38/39.Crawford Laboratory discovered a nitrate cis-element through years of efforts. They connected it with a single enhancer fragment and yellow fluorescent protein gene, and developed a nitrate responsive promoter (NRP), which was used to design a nitrate regulatory mutant screening system. By using this system, two Arabidopsis mutants have been isolated. The mutated gene of each mutant has been cloned successfully, and further characterization has proved that both genes are involved in the nitrate regulation.In this study, we isolated one mutant by using the above mutant screen system and identified a novel nitrate regulatory gene:(1) The NRP-containing transgenic plant (SS204-9) seeds were mutagenized by ethyl methane sulfonate. M2population was screened for mutants with strong fluorescence on ammonium succinate media and two mutants (C9, E7) were isolated.(2) The mutation site of C9and E7was narrowed down to the front of chromosome IV by map-based cloning. Combining with the whole genome sequencing result, the mutation was localized to the gene AtBNRGl.(3) Both mutants were crossed and the F1plant roots showed strong YFP signal on ammonium succinate media (nitrate free), indicating that C9and E7are allelic mutants.(4) The nitrate content in both mutants was higher than in wild-type, suggesting that AtBNRGl is involved in nitrate accumulation of plants.(5) The expression of nitrate responsive genes (NIA1, NIR) in mutants were higher than wild-type, suggesting that AtBNRG1is a nitrate regulatory gene.(6) The fluorescence of mutants was stronger than wild-type in different nitrogen conditions (potassium nitrate, ammonium nitrate and ammonium succinate), suggesting that AtBNRGl may be a repressor of nitrate responsive genes.(7) Mutant plants were grown on different nitrogen conditions (potassium nitrate, ammonium nitrate and ammonium succinate) and the root system was studied. Compared with wild-type plants, the primary root length is shorter and lateral root density is higher in the mutants. These results suggest that AtBNRGl may modulate the root growth and development.