Mechanisms For Ammonium Transporters (BcAMT1s) To Regulate Ammonium Absorption Under The Mixture Of Ammonium And Nitrate In Choysum

Ammonium(NH₄⁺)and nitrate(NO₃^-)are the major inorganic nitrogen sources for plants;they have important roles in the growth development,yield and quality of crops.Compared with sole NH₄⁺or NO₃^-,the mixed nutrition has shown to accelerate the growth of crops by improving nitrogen absorption and nitrogen use efficiency.It shows that NH₄⁺may be involved in the processes of nitrogen absorption and nitrate accumulation.Ammonium transporter(AMT),which is an intramembrane protein mediated the active transport of ammonium,plays an important role in the absorption and transportation of ammonium.However,the physiological and molecular mechanisms by which AMT regulates nitrogen uptake are unclear in most crops.Choysum(Brassica campestris L.ssp.chinensis var.utilis Tsen et Lee),a subspecies variant of Chinese cabbage,is a prominent vegetable in South China due to the taste and nutrient content of its flower stalks,and is the one of the largest growing area and yield of vegetables in South China.It has been cultivated widely in China.Owing to absorbing mainly NO₃^-in usual cultivation,nitrate content is accumulated easily in the products of choysum.Compared with sole NO₃^-,the mixture of NH₄⁺and NO₃^-enhances significantly the yield and nutrient qualities of choysum,with reducing the nitrate content.It is necessary to further research the mechanisms of BcAMT1s regulationg nitrogen uptake under the mixture nitrition.Therefore,under the different nitrogen treatments,the absorption characteristics and interactions of NH₄⁺and NO₃^-in choysum were analyzed by the non-invasive microtest technology(NMT);and the expression levels of BcAMT1s in choysum were measured by quantitative RT-PCR(q RT-PCR).Furthermore,the effects of overexpressing BcAMT1s on the absorption of nitrogen and the the the activities of BcAMT1s promoters in different nitrogen treatments were studied in the transgenic Arabidopsis.The main results are as follows:1.Using NMT technology,the net flux rates of NH₄⁺and NO₃^-were analyzed in response to different nitrogen treatments in choysum roots.It showed that the net influx rates of NH₄⁺were higher than the ones of NO₃^-at both 0.25 and 1 m M concentration.Compared with sole NH₄⁺or NO₃^-,the mixture nitrition increased the net influx rate and the amount of total nitrogen which accelerated the growth of choysum.Furthermore,NH₄⁺and NO₃^-have complex interactions with each other:NO₃^-had a positive effect on the net NH₄⁺influx,while NH₄⁺had a negative influence on the net NO₃^-influx.2.The expressions of BcAMT1s in choysum had different responses to the different nitrogen forms.The expressions of BcAMT1.1,BcAMT1.3,and BcAMT1.5 were induced by nitrogen deficiency,and seriously inhibited by adding NH₄⁺,whereas BcAMT1.2 expression was induced by adding NH₄⁺and enhanced obviously by adding NO₃^-.Therefore,BcAMT1.2 was differed from other BcAMT1s in response to nitrogen.3.BcAMT1.1,BcAMT1.2,BcAMT1.3 and BcAMT1.5 were transmembrane proteins which could transport NH₄⁺by the heterologous expression in yeast.They have different responses to the nitrogen treatments by transient expression in choysum.Both the fluorescence intensity of BcAMT1.1-GFP and BcAMT1.3-GFP were stronger in nitrogen deficiency than in 0.5 m M NH₄⁺;whereas the ones of BcAMT1.2-GFP were stronger in 0.5m M NH₄⁺.The fluorescence signal of BcAMT1.5-GFP was not obviously affected by different nitrogen treatments.Therefore,BcAMT1s may have different biological functions in the absorption and transportation of ammonium.4.Either in sole NH₄⁺or in mixture of NH₄⁺and NO₃^-,all the lines of BcAMT1s overexpression in Arabidopsis significantly accelerated the growth of plants over wildtype,and increased net influx rates of NH₄⁺.In leaves,all transgenic lines also increased the expression levels of nitrogen assimilation genes,such as encoded glutamine synthase genes(At GLN1.1,At GLN1.2,and At GLN2),glutamate dehydrogenase gene(At GDH2),and glutamate synthase gene(At GLT1).So there was no excessive NH₄⁺accumulation in plants.In roots,the lines of overexpressing BcAMT1.1 and BcAMT1.2 also significantly increased the expression of At GLN1.2,while the ones of BcAMT1.3 and BcAMT1.5 had no significant influence and decreased the expression levels of At GLN2 and At GLT1.Except for BcAMT1.5-overexpressed lines,other transgenic lines significantly increased the net efflux rates of NO₃^-which may lead to reduce the content of NO₃^-.5.The results of bimolecular fluorescence complementation system showed that BcAMT1.1,BcAMT1.2 could interact with protein kinase CIPK23(CBL-interacting serine/threonine-protein kinase 23),but BcAMT1.3 and BcAMT1.5 didn't.Protein kinase CIPK23 may be involved in the process of nitrogen uptake by regulated BcAMTs.6.The sequences of BcAMT1.1,BcAMT1.2,and BcAMT1.5 promoter were isolated from choysum,and identified by using the heterologous expression in Arabidopsis.The results showed that the lines of transgenic BcAMT1s_pro had different responses to the nitrogen treatments.The GUS stains of BcAMT1.1_pro,BcAMT1.3_pro and BcAMT1.5_pro were stronger in nitrogen starvation than that of supplying NH₄⁺,but the response of BcAMT1.2_pro was opposite.The results were consistent with the ones of the transient expression of BcAMT1s-GFP in choysum.Based on the results of the sequence analysis and literatures,we speculated that the DOFCOREZM acting element(AAAG)may be the key element of BcAMT1s promoter,which can bind to DOF transcription factor and regulate the absorption of NH₄⁺.In sum,NH₄⁺and NO₃^-interacted with each other under the mixture of NH₄⁺and NO₃^-,which NO₃^-accelerated the absorption of NH₄⁺in choysum,while NH₄⁺inhibited the absorption of NO₃^-.BcAMT1s plays important roles in the absorption and transportation of NH₄⁺,which BcAMT1.1,BcAMT1.2,and BcAMT1.5 may be mainly responsible for the absorption of NH₄⁺while BcAMT1.2 may be mainly responsible for the transportation.In addition,protein kinase CIPK23 may be involved in BcAMTs-mediated nitrogen absorption of choysum.