Effects of plasma membrane H+-ATPase on nitrate uptake and utilization in Arabidopsis
[Objectives]The transmembrane transport of nitrate(NO3-)is a major pathway of N uptake and utilization for most plants.The pathway requires coupled proton(H+)cotransport,and the proton motive force is provided by the plasma membrane(PM)H+-ATPase.In this study,we examined the involvement of PM H+-ATPase AHA1 and AHA2 in NO3--N uptake,with the aim of revealing the molecular mechanism of NO3--N acquisition in Arabidopsis root.[Methods]Wild-type Col-0,PM H+-ATPase single mutant aha1-9,aha2-5 and complementation lines AHA1/aha1-9,AHA2/aha2-5 were used as experimental materials.All the seedlings were grown on mediums with NO3-concentrations of 1,10,and 20 mmol/L for 10 days,respectively.Then the root growth and biomass of seedlings were investigated,and the protein and phosphorylation level of root PM H+-ATPase were determined.The expression of genes involved in NO3--N and auxin response and transport(NRT1.1,NRT2.1,NRT2.2,NRT2.4 and NLP7;ARF11,IAA6,PIN7 and SAUR57)were detected.[Results]There was no significant difference in the growth of all genotype seedlings under 20 mmol/L NO3--N treatment.While the growth of aha1-9 and aha2-5 were inhibited compared with Col-0 at 1 mmol/L and 10 mmol/L NO3--N,the root biomass decreased by 55%,31%,45%and 29%,shoot biomass decreased by 55%,27%,39%and 25%,root length reduced by 38%,11%,22%and 13%,the number of lateral roots reduced by 55%,33%,47%and 38%,respectively.In addition,the differences of above indices of aha1-9 with the wild type were significantly lower than those of aha2-5 at 1 mmol/L NO3--N.And the seedling growth of complementation lines AHA1/aha1-9 and AHA2/aha2-5 were similar to Col-0 under all the NO3--N concentrations.By separating cell membranes in root,we found that protein levels of PM H+-ATPase in aha1-9 and aha2-5 decreased by 68%,19%,36%and 53%,and phosphorylation levels decreased by 83%,43%,16%and 42%at 1 mmol/L and 10 mmol/L NO3--N,respectively.Interestingly,protein levels in aha1-9 and aha2-5 were not different from those of the wild type at 20 mmol/L NO3-N,but phosphorylation levels were significantly reduced.Relative to the wild type,the expression of NRT1.1,NRT2.1,NRT2.2 and NLP7 in aha1-9 and aha2-5 roots were significantly down-regulated at 1 mmol/L NO3--N,not significantly changed at 10 mmol/L and 20 mmol/L NO3--N.The expression of ARF11,IAA6,PIN7 and SAUR57 in aha1-9 and aha2-5 were significantly up-regulated at 1 mmol/L and 10 mmol/L NO3--N,but not at 20 mmol/L NO3--N.[Conclusions]Under low NO3--N conditions,knockout of the PM H+-ATPase not only reduces its own protein synthesis and phosphorylation levels,but also influences the expression of nitrate transporters and auxin transporters,resulting in the inhibition of plant growth.
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