Function Of Ammonium Transporter ZmAMTs For Ammonium Uptake And Xylem Loading In Maize Roots

Nitrogen is one of the macroelements that plants need.Food production depends on the use of large amounts of nitrogen fertilizers.Ammonium is one of the nitrogen sources that can be directly absorbed by the plant,and also is a key intermediate of nitrogen metabolism.Studies in plant Arabidopsis found that ammonium transporters(AMT/MEP/Rh)mediate the uptake of ammonium and transportation to the shoot,while that in important food-effect maize have not been studied systematically.This work aims to determine whether the ZmAMT1;1a gene mediates the uptake of ammonium in maize and whether the ammonium transporter ZmAMT2;1 gene is involved in xylem loading and reveal their regulatory mechanisms,then elucidate the molecular mechanism of ammonium uptake and xylem loading.The main results can be summarized into two parts:1.ZmAMT1;1a mediated ammonium uptake in maize rootsOur previous study found that the ZMAMT1;1a gene encodes a high-affinity ammonium transporter and its expression level is significantly related to the ammonium absorption capacity of the root system.In this study,the ZmAMT1;1a over-expression transgenic maize plants showed an increase in the ammonium uptake capacity of roots as revealed by 15N-labeled ammonium influx assay and further contributed to increases in the total biomass and nitrogen uptake under low ammonium nutrition(0.04 mM).The results showed that the enhanced expression of ZmAMT1;1a gene increased the ammonium uptake capacity of roots under low ammonium conditions.However,under the condition of 1 mM ammonium,the expression level of ZmAMT1;1a protein under high ammonium conditions was higher than that under low ammonium condition in both wild-type and transgenic plants,but the ammonium uptake capacity of roots was lower than that of low ammonium conditions,especially in transgenic plants.Therefore,the increase in the expression level of ZmAMT1;1 a protein does not translate into an enhanced ammonium acquisition,suggesting a possible post-translational down-regulation of ZmAMT1;1 a by high ammonium.The above results show that ammonium transporter ZmAMT1;1 a can mediate ammonium uptake in maize roots and is regulated by post-translational levels.2.ZmAMT2;1 involved in ammonium xylem loadingBy analyzing the expression pattern of ammonium transporter gene ZmAMT2;1,it was found that the expression of this gene was induced by nitrogen deficiency and senescence.Histological localization of the promoter and subcellular localization of the GFP fusion showed that ZmAMT2;1 was localized on the plasma membrane of the vascular system cells in the maize roots.The ammonium concentration of the xylem sap of wild-type W22 plants increased significantly after nitrate cultured maize transferred to ammonium,but the ammonium concentration of the ZmAMT2;1 gene insertion line did not increase significantly.Resupplying with 15NH4+after nitrogen starvation,the¹⁵N accumulation in the shoots of the Mu mutant was significantly lower than in the wild-type.These results indicated that the ZmAMT2;1 gene may mediate the ammonium xylem loading process in maize roots,thereby ensuring the efficient transportation of ammonium to the shoots.In addition;by heterologous expression of ZmAMT2;1 and related muteins in yeast mutants,it was found that phosphorylation of phosphorylation sites(T454 and T480)in the non-conserved region of the C-terminus can activate the ammonium transport activity of the protein.The above results indicated that the ammonium transporter ZmAMT2;1 may be involved in the long-term transportation of ammonium to shoots,and there is a specific phosphorylation activation mechanism at the protein level.