Preliminary Isolation And Characterization Of An Ammonium Overly Sensitive Mutant

Nitrogen is an essential element for plant growth and development,and is involved in various metabolic processes and the synthesis of many living substances in plants.Ammonium(NH4+)and nitrate(NO3-),as the most important inorganic nitrogen sources,play an irreplaceable role in the plant life.However,the regulation of nitrogen in plants remains to be further studied.Here,upon screening a library of T-DNA insertion mutants of receptor-like kinase(RLK),we isolated one that is hypersensitive to low nitrogen.This mutant was named rlk-152.Nevertheless,we later found that the sensitive phenotype of rlk-152 was not caused by low nitrate,but instead by ammonium stress.When NH4-is the only or the main nitrogen source,it will cause strong toxicity to plants.We found that the growth of rlk-152 was significantly inhibited under ammonium treatment.Further physiological experiments showed that the content of free ammonium in rlk-152 was significantly higher than that in wild type(WT)under ammonium treatment.Consistent with this,the mutant displayed lower capacity of ammonium assimilation than WT.Moreover,we showed that the expression of NH4+ assimilation related genes(GLN1;1 and GLN1;5)in rlk-152 was significantly lower than that in WT under ammonium treatment.Therefore,it is speculated that RLK-152 should be involved in the metabolism of ammonium,and that loss of RLK-152 function leads to blocked ammonium assimilation,leading to the toxicity of ammonium.Although this gene was indeed mutated in the rlk-152 mutant,further study showed that another T-DNA insertion allele of this gene was indistinct from WT in response to ammonium,suggesting that RLK-152 is not the target gene responsible for the ammonium sensitive phenotype of the rlk-152 mutant.We then tried TAIL-PCR and whole genome sequencing,but failed to identify the target mutation that causes rlk-152 sensitive to ammonium.To this end,we have developed the F1 generation of the hybrid line between the mutant and the Ler WT.After the F2 generation is obtained,the target gene will be further identified by map-based cloning,and the gene function will be analyzed in future.In conclusion,we isolated an Arabidopsis ammonium sensitive mutant,rlk-152,and explained this phenotype from the physiological and molecular levels.