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Type: Thesis
Title: Elucidation of the Ammonium Major Facilitator (AMF) Family in Plants
Author: Situmorang, Apriadi
Issue Date: 2018
School/Discipline: School of Agriculture, Food and Wine
Abstract: The discovery of the Ammonium Major Facilitator (AMF) family in plants and yeast by overexpression of the soybean transcription factor GmbHLHm1 in yeast has opened up new insights into the transport of NH4+ in eukaryotic systems. Using both yeast and Xenopus oocyte expression systems, ScAMF1 and a plant homolog (GmAMF3) were shown to transport NH4+ and the toxic ammonium analogue methylammonium (MA). The AMF family is conserved in most plants including Arabidopsis thaliana where three homologs exist, these being AtAMF1 (At2g22730), AtAMF2 (At5g64500) and AtAMF3 (At5g65687). All three AMF genes are expressed throughout the plant with noticeable expression in senescing shoot tissues. Transient expression in Nicotiana benthamiana leaves indicated AtAMF1, AtAMF2, and AtAMF3 proteins are located on the ER, tonoplast and plasma membrane, respectively. Functional testing in an ammonium transport deficient yeast strain (31019b) did not result in activities which rescued growth of yeast cells grown on low ammonium concentrations. However, like GmAMF1;3 and ScAMF1, AtAMF2 was capable of inducing an increased sensitivity to the toxic ammonium analogue methylammonium. These alternative protein locations and activities may reflect a distinct function of each protein in cellular NH₄+ transport and homeostasis. A disruption of both the high (Trk1) and low (Trk2) affinity K+ transport proteins in the yeast strain CY162 (trk1D; trk2D) was found to inhibit growth on high concentrations of NH₄+, a process potentially linked to the efflux of amino acids out of yeast cells. The overexpression of the tonoplast-localised AMF protein, AtAMF2, was shown to rescue CY162 when grown at high NH₄+ concentrations while continuing to induce a toxic phenotype to high concentrations of MA. The data suggests that AtAMF2 may participate in the release of acid-trapped NH4+ from the vacuole into the cytoplasm, a process required to supply nutrients to support cellular growth in NH4+ grown but amino acid-starved cells. In contrast, when MA is supplied, vacuole-localised MA+ is released by AtAMF2 into the cytoplasm inducing a toxicity phenotype. This process was enhanced when cells were supplied with only proline or limited concentrations of amino acids. Collectively these data suggest the tonoplast-localised AtAMF2 is a functional NH₄+ efflux protein that can support cellular growth when limited by available nitrogen (NH₄+ and or amino acid) resources. Individual amf T-DNA knockouts in Arabidopsis were identified and used to create multiple amf mutations through selected crosses. The amf1 mutant displayed an increased rate of unidirectional 15NH₄+ influx into Arabidopsis roots that was not present in either amf2 or amf3. The growth of mutants with amf1 background (amf1, amf1amf2 or amf1amf3) on low NO₃- (0.05 mM) and adequate K+ (3.75 mM) were found to be sensitive to 20 mM MA. In the presence of low or high NO₃- (0.05 and 7.5 mM, respectively) and low K+ (<0.1 mM), root growth in the amf2amf3 mutant was significantly inhibited by 2 mM NH₄+, a phenotype that could be rescued with the provision of external K+. These data demonstrated that AtAMF1 might have a dominant role in NH₄+ toxicity tolerance when supplied low concentrations of NO₃- while AtAMF2 and AtAMF3 aid in the management of NH₄+ toxicity in a K+-dependent manner. Collectively with experiments in yeast, the in-planta experiments confirm the importance of K+ availability in mitigating NH4+ toxicity in Arabidopsis, a process which appears to involve members of the AMF family.
Advisor: Kaiser, Brent N.
Tyerman, Stephen D.
Shelden, Megan C.
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2019
Keywords: Ammonium transporter
ammonium toxicity
low potassium
ammonium efflux
complementation assay
31019b yeast strain
CY162 yeast strain
amino acid excretion
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
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