The Role Of Nitrate Transporter 1.1 In Plant Response To Pb Stress And Its Underling Mechanism

The contamination of lead(Pb)in soil becomes increasingly serious in recent decades with the industrial development.Consumption of Pb in agricultural soils can endanger human health through food chains.Consequently,developing efficient technologies or methods to minimize dietary uptake of Pb from crop foods is desperately urgent.Identification the interactions between plant nutrient and Pb accumulation,and using a biological engineering or genetic breeding approach to reduce Pb levels in crops may be an economical and environment-friendly strategy.Previous studies have observed a close relationship between Pb2+ uptake and nitrogen nutrition in plants,but how nitrogen uptake activities in response to Pb stress and their underling mechanism are still unclear.In this context,we investigated the interaction between NRT-mediated NO3-uptake and Pb2+ exposure in wild-type and NRT-related mutants Arabidopsis plants using physiological and molecular biological methods.The main results are summarized as follows:We first evaluated the effect of Pb2+ exposure on NO3-uptake in the roots of ecotype Columbia-0 of Arabidopsis(Col-0)using NMT.The results showed that Pb2+exposure increased the rate of net NO3-influx in meristematic zone,elongation zone,and maturation zone along the root axis.Then the expression of five NRT genes that are involved in NO3-uptake by roots were examined,and we found that only the expression of NRT1.1 significantly increased after Pb2+ exposure,the expression of the other four NRT genes(NRT1.2,NRT2.1,NRT2.2 and NRT2.4)were not significantly affected.GFP fluorescence was also observed in the roots of pNRT1.1::NRT1.1-GFP transgenic plants,and showed that Pb2+ exposure caused a remarkable increase in NRT 1.1-GFP protein levels.Furthermore,Pb2+ exposure had little effect on the net NO3-influx of the nrt1.1 mutants in three measured segments.These results demonstrate that an induction of NRT1.1 activity was responsible for the increase in root NO3-uptake in the presence of Pb exposure.Then we studied the effects of NRT1.1 function loss on tolerance to Pb stress in Arabidopsisplants grown on agar medium.After Pbro treatment,Uhe root elongation of NRT1.1-null mutants chll.5 and nrt1.1-1 significantly reduced than Col-0 plants,and the Pb levels in roots and shoots of nrt1.1 knockout mutants were much higher than Col-0 plants.Similar results occurred in another NRT1.1-null mutant chll.6.We also analyzed the lipid peroxidation by measuring the amount of malonaldehyde(MDA)in three lines of Arabidopsis plants under Pb2+ stress,and the result showed that the plasma membranes of chl1.5 and nrt1.1-1 mutants were damaged more severely than Col-0 plants.In pNRT1.1::NRT1.1-GFP transgenic plants,the reduction of root elongation and the increase in plant Pb levels owing to NRT1.1 mutation could be rectified by complementation with NRT1.1.We concluded that NRT 1.1-conferred Pb resistance was associated with restraint of Pb2+ entry into plants.The relationship between Pb toxicity and other NRTs were also studied.However,the nrtl.2,nrt2.1,nrt2.2 and nrt2.4 mutants had similar root elongation and Pb levels to their corresponding wild-type Col-0 or Ler plants.These results suggest that NRT 1.1-conferred Pb2+ resistance in plants is a relatively specific manner.Based on the above findings,we investigated the role of NRT1.1 in plants Pb tolerance.We found that the pH in the rooting media of nrt1.1 knockout mutants decreased much more dramatically than that of Col-0 under Pb2+ stress.According to Pb2+ activity at different pH modelling by GEOCHEM-PC,a severer Pb toxicity in nrt1.1 knockout mutants probably resulted from increased Pb2+ availability,which is due to a lower pH in the rhizosphere.We then used MES to buffer the pH in the growth media,the MES treatment almost completely eradicated Pb2+ stress-caused growth inhibition of roots of nrt1.1 mutants,and reduced the differences in both plant Pb accumulation and rhizosphere pH between the Col-0 and two nrt1.1 mutants.The above results indicated that upon Pb2+ exposure,the up-regulation of NRT 1.1 in roots to prevent a decrease in rhizospheric pH may be a mechanism whereby plants enhance their tolerance to Pb2+ toxicity.In addition,in the study of Pb2+ toxicity towards the Col-0,nrt1.1-1,and chl1.5 plants fed with different NO3-to NH4+ ratios(N-A ratios),we found that the co-supply of NO3-and NH4+ was necessary in NRT 1.1-conferred plant Pb2+ resistance.To determine the function of NO3-in NRT 1.1-conferred plant Pb2+ resisitance,we examined the Pb2+ resistance in the chl1.9 mutant,which is defective in terms of NO3-uptake,and nlp7-2 mutants,which shares common features with nrtl.l knockout mutants in loss of functions of many NO3-sensing processes.The results showed that the NO3--uptake process,rather than the NO3--sensing function,was the mechanism behind NRT 1.1-conferred Pb2+resistance.In conclusion,the mechanism of NRT 1.1-conferred plant Pb2+ resisitance is:In the growth media with co-supply of NO3-and NH4+,Pb2+ stress stimulates NRT 1.1-mediated NO3-uptake by roots,NRT1.1 negatively regulates Pb levels in plants by elevating rhizospheric pH through H+ consumption resulting from NO3-uptake.