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Doctoral Thesis
2021
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
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
Ijato, Toyosi
Abstract (English)
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.
Abstract (German)
Ammonium ist ein wichtiger Stickstoff Nährstoff für Pflanzen, dessen Toxizität bei zu hohen Konzentrationen seinen Einsatz als Dünger limitiert. In dieser Arbeit wurde die Resonanz von Weizen gegenüber reiner Ammoniumernährung sowie die regulatorischen Mechanismen, der hochaffinen Ammoniumaufnahme untersucht. Während Weizen geringe Konzentrationen von Ammonium ebenso gut nutzen konnte wie geringe Konzentrationen von Nitrat, verursachten erhöhte Ammoniumkonzentrationen (10 mM) toxische Effekte. Bei diesen erhöhten Konzentrationen schafften es die Weizenkeimlinge unerwarteterweise nicht, die Expression ihre hochaffinen Ammoniumtransporter TaAMT1;1 und TaAMT1;2 zu reduzieren. Jedoch waren beide Transportproteine funktionell und konnten das Wachstum eines Hefestamms ohne eigene Ammoniumtransporter auf Ammonium komplementieren. Ammoniuminduzierte elektrische AMT Ströme in Xenopus laevis Oozyten waren spannungsabhängig und sättigten mit Km AMT1;1 = 76 μM und Km AMT1;2 = 196 μM. Diese Affinität der Transporter für Ammonium war ebenfalls spannungsabhängig was implizierte, dass der ratenlimitierende NH4+-Deprotonierung Schritt erst 35 % innerhalb des elektrischen Feldes der Plasmamembran stattfindet. Ein Phospho-Proteomics Ansatz zeigte TaAMT spezifische und ammoniumabhängige Phosphorylierung. Interessanterweise, konnte das Vortäuschen einer Phosphorylierung am konservierten AMT1 C-terminalen Proteinende (T453D) die Aktivität der TaAMT1s in Hefe und Oozyten inaktivieren. Mehrere Calcium bindende TaCBL Proteine waren in der Lage die Aktivität der TaAMT1 direkt zu reduzieren. In Kombination mit der TaCIPK9 Kinase (bei TaAMT1;2 ebenso TaCIPK23/32) war dieser Effekt viel ausgeprägter und führte teilweise zu einer kompletten Inaktivierung der Transporter. Zusammenfassend konnte gezeigt werden, dass die Phosphoregulation der TaAMT1 Transporter durch TaCBL / TaCIPK Komplexe Teil einer schnellen und effektiven Ammoniumantwort in Weizen ist.
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Notes
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Faculty
Faculty of Agricultural Sciences
Institute
Institute of Crop Science
Examination date
2021-03-28
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Language
English
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Classification (DDC)
630 Agriculture
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Sustainable Development Goals
BibTeX
@phdthesis{Ijato2021,
url = {https://hohpublica.uni-hohenheim.de/handle/123456789/6598},
author = {Ijato, Toyosi},
title = {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},
year = {2021},
school = {Universität Hohenheim},
}