Studies On The Physiological Mechanism Of Nitrate-dependent Alleviation Of Ammonium Toxicity In Arabidopsis Thaliana

Nitrogen?N?is one of the most important mineral nutrients required for plant growth and development.N is directly involved in the synthesis of nucleic acid,protein,chlorophyll and many small compounds.There are various forms of nitrogen available to plants,among them,ammonium?NH₄⁺?and nitrate?NO₃^-?are the main inorganic nitrogen sources used by plants.However,when NH₄⁺acts as the sole or dominant nitrogen source,it can cause a strong toxicity to plants,known as ammonium toxicity.In contrast,when NO₃^-serves as the sole nitrogen source,it causes no toxicity to plants.Moreover,within a certain concentration range,the higher the concentration of nitrate is,the more vigorous the growth of plants will be.Studies have shown that a small amount of NO₃^-can significantly mitigate ammonium toxicity,but the mechanism has not been elucidated.In this study,through systematic physiological and biochemical experiments,it is proved that one of the important causes of ammonium toxicity is the strong acidification of rhizosphere.When different concentrations of NO₃^-were applied gradually under ammonium stress condition,the acidification process of the medium was significantly inhibited,indicating that NO₃^-alleviates ammonium toxicity via regulating the medium pH.Further studies showed that NO₃^-uptake was significantly enhanced under high NH₄⁺/low NO₃^-and low pH condition,implying that nitrate transporters are involved.Phenotypic analysis showed that nrt1.1-1 and chl1-5,the two functional null mutants of NRT1.1,were extremely sensitive to high NH₄⁺/low NO₃^-stress condition,displaying shortened primary root and significantly reduced biomass and chlorophyll content compared with Col-0,nrt2.1 and nrt3.1 plants,suggesting that NRT1.1 is directly involved in nitrate-dependent alleviation of ammonium toxicity.Subsequently,NRT1.1 was involved in the process of ammonium detoxification by preventing rhizosphere acidification,which was specifically dependent on its transporter activity.Previous study has shown that the channel SLAH3 is also involved in nitrate-dependent alleviation of ammonium toxicity.Is SLAH3 involved in above process by regulating the medium pH?In this study,it was found that although both Col-0 and slah3-5 mutants could significantly acidify medium under ammonium stress condition,the acidification process of slah3-5 mutants was significantly faster than that of Col-0 plants.At the same time,when only a small amount of NO₃^-was present and the medium pH was 4.5,slah3-5 plants showed a more obvious growth inhibition phenotype compared with Col-0,indicating that SLAH3 participation in the process of ammonium detoxification by regulating the medium pH.Interestingly,NRT1.1 and SLAH3 have opposite functions in mediating NO₃^-transmembrane flux,while their phenotypes under high NH₄⁺/low NO₃^-stress condition were remarkably similar,both showing strong ammonium toxicity symptoms.Moreover,the phenotypes of nrt1.1-1slah3-5 double mutants were not significantly different from those of nrt1.1-1 and slah3-5 single mutants,suggesting that NRT1.1 and SLAH3may be located in the same signaling pathway.Therefore,the author concluded that when plants are suffered from high NH₄⁺/low NO₃^-stress condition,excessive NH₄⁺absorption results in significant acidification of rhizosphere,and then plants perceive this stress signal and activates NRT1.1 to accelerate the symport of extracellular NO₃^-/H⁺,thereby reducing extracellular H⁺concentration and alleviating ammonium toxicity.However,the ability of plants to inhibit root acidification is limited due to the low concentration of extracellular NO₃^-.At this time,SLAH3 is activated to mediate the efflux of NO₃^-,and maintain a certain concentration of extracellular NO₃^-,allowing NRT1.1 transport continuously to enable the continuous transmembrane flux of NO₃^-,leading to ammonium detoxification.Under the conditions of N2,A10N2 and A10N2+MES,the expression of NRT1.1showed the characteristic of“low-high-low”.Therefore,transcriptome analysis was performed under the above three conditions to screen some pH-dependent ammonium stress induced gene?PAG?.Based on the results of transcriptome analyses,the expression of some genes was indeed significantly induced by high NH₄⁺/low NO₃^-stress condition,and this induction was dependent on medium pH,which was very similar to the expression of NRT1.1.In this study,we preliminarily screened some candidate genes that might be involved in plant ammonium detoxification.A transcription factor,PAG-TF2,was showed to activate the promoter of NRT1.1.However,whether it specifically regulates NRT1.1 expression under high NH₄⁺/low NO₃^-stress condition remains to be further studied.