Browsing by Subject "CIPK"

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    Understanding the role of the Calcineurin B-like (CBL) proteins and the CBL-Interacting Protein Kinases (CIPK) of wheat (Triticum aestivum) in the regulation of its high affinity ammonium transporters
    (2021) Ijato, Toyosi; Ludewig, Uwe
    Ammonium is an important nitrogen source whose potential for toxicity (in plants) has been a limitation to its use as a fertilizer. In this work, the response of wheat to ammonium nutrition and the regulatory mechanism governing its high affinity ammonium transporters was investigated. Wheat was able to utilize sole ammonium in the 1 mM range just like sole nitrate. An elevated ammonium concentration of 10 mM caused ammonium induced toxicity effects. Unexpectedly, the wheat seedlings failed to downregulate TaAMT1;1 and TaAMT1;2 in response to elevated ammonium concentration. Nevertheless, TaAMT1;1 and TaAMT1;2 complement ammonium transport in the mep yeast strain (lacking endogenous ammonium transporters). The Voltage dependent ammonium induced currents of the transporters saturated in a concentration dependent manner with Km AMT1;1 = 76 μM and Km AMT1;2 = 196 μM. The affinity of the transporters for ammonium was voltage dependent and indicated that ammonium passes around 35 % of the membrane electric field before rate limiting deprotonation in both transporters. Furthermore, phospho-proteomics study showed the differential phosphorylation of TaAMT1;1 and TaAMT1;2 specific proteotypic phosphopetides. Interestingly, a mutation that mimics phosphorylation at the conserved threonine T453 (T453D) was able to inactivate the ammonium transport function of the transporters. Additionally TaCBL1, and TaCBL2 were able to independently regulate the activity of TaAMT1;1, while TaCBL1, TaCBL2 and TaCBL6 were able to independently impair the ammonium induced current of TaAMT1;2. Subsequently, TaCBL1, TaCBL2 and TaCBL6 interacted with TaCIPK9 to further reduce the ammonium induce current caused by the single TaCBLs proteins on TaAMT1s. TaCIPK23 and TaCIPK32 were only able to regulate TaAMT1;2 by interacting with both TaCBL2 and TaCBL6. Thus, the robust response of wheat to ammonium N was shown to include the phospho-regulation of its high affinity ammonium transporters in a manner that recruits the activity of its TaCBL and TaCIPK proteins.