【摘要】 【目的】硝态氮（NO₃^--N）是多数植物吸收利用的主要氮素形态之一，NO₃^--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NO₃^--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NO₃^--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 NO₃^--N处理下各材料之间的生长无显著差异。在1和10 mmol/L NO₃^--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 NO₃^--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NO₃^--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 NO₃^--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 NO₃^--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 NO₃^--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 NO₃^--N条件下均没有显著差异。此外，在1和10 mmol/L NO₃^--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 NO₃^--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。更多还原

【Abstract】 【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, PIN7and SAUR57 in aha1-9 and aha2-5 were significantly up-regulated at 1 mmol/L and 10 mmol/L NO₃^--N, but not at20 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.更多还原