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.