拟南芥细胞膜质子泵对硝酸盐吸收利用的影响

Effects of plasma membrane H+-ATPase on nitrate uptake and utilization in Arabidopsis

[目的]硝态氮(NO_(3)^(-)-N)是多数植物吸收利用的主要氮素形态之一,NO_(3)^(-)-N的跨膜运输需要耦合质子共转运,质子运转需要细胞膜上的质子泵提供质子驱动力。本研究通过分析拟南芥细胞膜质子泵AHA1和AHA2对硝态氮吸收的信号网络,以明确硝态氮吸收过程中的分子调控机制。[方法]以野生型Col-0、质子泵基因突变体aha1-9、aha2-5以及恢复系AHA1/aha1-9、AHA2/aha2-5为试验材料,在不同NO_(3)^(-)-N浓度(1、10和20mmol/L)培养基上培养10天,观察其表型,记录根系生长以及生物量变化,测定根系细胞膜质子泵蛋白及其磷酸化水平变化,检测根系中参与NO_(3)^(-)-N响应与转运相关基因(NRT1.1、NRT2.1、NRT2.2、NRT2.4和NLP7)和生长素响应与转运相关基因(ARF11、IAA6、PIN7和SAUR57)的相对表达量。[结果]20 mmol/L NO_(3)^(-)-N处理下各材料之间的生长无显著差异。在1和10 mmol/L NO_(3)^(-)-N条件下,与野生型相比,aha1-9和aha2-5的主根长度、侧根数量、根部和地上部生物量均显著降低。1 mmol/L NO_(3)^(-)-N时,aha1-9的上述指标与野生型的差异显著大于aha2-5。恢复系AHA1/aha1-9、AHA2/aha2-5在各个NO_(3)^(-)-N供应水平下均与野生型差异不显著。通过分离根系细胞膜发现,在1、10 mmol/L NO_(3)^(-)-N条件下,相比野生型,aha1-9和aha2-5的细胞膜质子泵蛋白水平分别降低了68%、19%和36%、53%,质子泵蛋白磷酸化水平分别降低了83%、43%和16%、42%;在20 mmol/L NO_(3)^(-)-N条件下,aha1-9和aha2-5的质子泵蛋白水平与野生型差异不显著,但磷酸化水平显著降低。通过RT-qPCR测定发现,在1 mmol/L NO_(3)^(-)-N条件下,aha1-9、aha2-5根系中的NRT1.1、NRT2.1、NRT2.2、NLP7表达量相比野生型显著下调,10和20 mmol/L NO_(3)^(-)-N条件下均没有显著差异。此外,在1和10 mmol/L NO_(3)^(-)-N条件下aha1-9、aha2-5中的ARF11、IAA6、PIN7和SAUR57表达量显著上调,然而在20 mmol/L NO_(3)^(-)-N条件下其表达量没有显著差异。[结论]低氮条件下,敲除细胞膜质子泵基因不仅降低其自身蛋白的合成和磷酸化水平,同时也影响硝酸盐转运蛋白基因和生长素相关基因的表达,进而抑制植物的生长。

[Objectives]The transmembrane transport of nitrate(NO_(3)^(-))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 NO_(3)^(-)-N uptake,with the aim of revealing the molecular mechanism of NO_(3)^(-)-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 NO_(3)^(-)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 NO_(3)^(-)-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 NO_(3)^(-)-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 NO_(3)^(-)-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 NO_(3)^(-)-N.And the seedling growth of complementation lines AHA1/aha1-9 and AHA2/aha2-5 were similar to Col-0 under all the NO_(3)^(-)-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 NO_(3)^(-)-N,respectively.Interestingly,protein levels in aha1-9 and aha2-5 were not different from those of the wild type at 20 mmol/L NO_(3)^(-)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 NO_(3)^(-)-N,not significantly changed at 10 mmol/L and 20 mmol/L NO_(3)^(-)-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 NO_(3)^(-)-N,but not at 20 mmol/L NO_(3)^(-)-N.[Conclusions]Under low NO_(3)^(-)-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.