Molecular Mechanism Of An Arabidopsis Translation Initiation Factor EIF4E1 In Regulating Nitrate Signaling

Nitrogen which can affect plant growth and development is one of the main nutrient elements,and plays an extremely important role in crop yield and quality.In agricultural production,increasing nitrogen fertilizer application is the main means to increase crop yield,but due to the low utilization rate of nitrogen fertilizer by crops,a large part of nitrogen which is not absorbed by plants is lost to the environment,causing a series of serious environmental pollution problems such as soil acidification and water eutrophication.Improving crop nitrogen use efficiency is the main way to solve this problem.It is of great significance to explore more nitrogen regulatory genes,enrich and improve the gene network of nitrogen regulation,and analyze the mechanisms of nitrogen absorption and utilization for the realization of high yield and high efficiency of agriculture.In this study,we cloned a new NO₃^-regulatory gene eIF4E1（eukaryotic initiation factor 4E 1）by forward genetics and investigated the functions of eIF4E1 in NO₃^-signaling and metabolic regulation systematically.The main results are as follows:（1）We identified a weak fluorescence mutant,Mut36,using an Arabidopsis NO₃^-regulatory gene screening system constructed by Crawford’s laboratory in this study.The mutated gene eIF4E1,which encoded a translation initiation factor 4E,was verified by the mapbased cloning and genome-wide sequencing.Meanwhile,fluorescence observation of allelic hybridization lines and the eIF4E1 complement line further confirmed that the weak fluorescence phenotype of the Mut36 mutant was indeed caused by the mutation of eIF4E1 gene.Further results showed that the induced expression of NO₃^-response genes in eif4e1 mutants was significantly lower than that of the wild type after NO₃^-treatment.These results indicated that eIF4E1 regulated NO₃^-signaling in plants.（2）We identified the function of eIF4E1 on NO₃^-metabolism,and found that the uptake of 15NO₃^-in eif4e1 mutants was decreased significantly compared to wild type and the expression of NRT1.1（Nitrate transporter 1.1）and NRT1.2 genes responsible for NO₃^-transport was significantly decreased,indicating that eIF4E1 could regulate the uptake of NO₃^-in plants.On the other hand,we found that the nitrate reductase（NR）activity and the amino acid content were increased in eif4e1 mutants compared with the wild type,and the expression of NO₃^-assimilation genes was also significantly increased,indicating that eIF4E1 could regulate NO₃^-assimilation in plants.These results indicated that eIF4E1 regulated NO₃^-metabolism in plants.（3）We analyzed the expression pattern of the eIF4E1 gene by q PCR and GUS staining.The results showed that eIF4E1 was expressed in all tissues,mainly expressed in the vascular of roots,leaves and flower.By analyzing the effects of different nitrogen treatments on the expression of eIF4E1,we found that the expression of eIF4E1 was induced by NO₃^-and NH4+,while inhibited by nitrogen starvation.（4）To further illustrate the roles of eIF4E1 in regulating NO₃^-signaling,RNA-seq and Ribo-seq were performed on the eif4e1 mutant.Through the analysis of differentially expressed genes in wild type and mutant with or without NO₃^-treatment from RNA-seq,we found multiple N-related clusters,such as related to NO₃^-response,related to NO₃^-transport,response to nitrogen compounds,transport of nitrogen-containing compounds,etc.,and we found many genes related to NO₃^-response and metabolism among these cluster,indicating that eIF4E1 could affect the expression of many downstream nitrogen-related genes.Further analysis of the Ribo-seq results showed that the translation of multiple NO₃^-related genes including NRT1.1 was affected between the eif4e1 mutant and the wild type after NO₃^-treatment,and polysome profiling analysis showed that the amount of NO₃^-related genes m RNA being translated in eif4e1-2 mutant after NO₃^-treatment was significantly lower than that of the wild type.These results indicated that eIF4E1 could affect the translation of NO₃^-related genes and participate in NO₃^-signaling regulation at the translation level.（5）We further explored the regulatory relationship between eIF4E1 and NRT1.1,and found that eIF4E1 regulated the expression of NRT1.1.Moreover,the fluorescence and the expression of the NO₃^--inducible genes in the eif4e1-4 chl1-13 mutant were similar to those in the chl1-13 mutant.All these results indicated that eIF4E1 worked upstream of NRT1.1 in the NO₃^-signaling pathway.（6）To identify other factors involved in the eIF4E1-regulated NO₃^-signaling,yeast twohybrid（Y2H）screen using eIF4E1-BD as bait was performed and an eIF4E1-interacting protein GEMIN2 was identified.The interaction between eIF4E1 and GEMIN2 was further confirmed by Y2 H,Arabidopsis protoplast Bi FC,and GST Pull-down experiment.The interaction between eIF4E1 and GEMIN2 was dependent on the 4E-BM motif of the GEMIN2 protein.In order to clarify whether GEMIN2 is involved in NO₃^-primary response,we examined the expression of NO₃^-response genes and found that the inducible expression of the NO₃^-response gene in the gemin2 mutant was significantly decreased compared to wild type after NO₃^-treatment,indicating that GEMIN2 could also participate in NO₃^-signal regulation.Moreover,GEMIN2 and eIF4E1 may function in the same NO₃^-signaling pathway.（7）The investigation of eIF4E1 overexpression lines showed that the overexpression of eIF4E1 could promote the growth and development of plants in Arabidopsis under both low nitrogen（0.3 m M KNO3）and high nitrogen（5 m M KNO3）conditions.The overexpression lines showed promoted primary root length,fresh weight per plant and plant height than the wild type.Further analysis of the seed size,1000-seed weight,yield per plant,and NUE in mature plants showed that these indexes in eIF4E1 overexpression lines were significantly higher than those of wild type,indicating that overexpression of eIF4E1 could significantly improve plant yield and NUE.In this study,a new NO₃^-regulatory gene eIF4E1 was cloned,and the functions and mechanisms of eIF4E1 in NO₃^-signaling regulation and metabolism were clarified.The factors that can regulate the NO₃^-signal at the translation level were discovered for the first time,laying a foundation for future studies on the molecular network regulated by NO₃^-at the translation level.It provided a theoretical basis and support for improving crop nitrogen use efficiency,breeding new varieties with high nitrogen efficiency and sustainable development of the agricultural.