Browsing by Person "Straub, Tatsiana"

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Ammonium uptake, mediated by ammonium transporters, mitigates manganese toxicity in duckweed, Spirodela polyrhiza
(2023) Kishchenko, Olena; Stepanenko, Anton; Straub, Tatsiana; Zhou, Yuzhen; Neuhäuser, Benjamin; Borisjuk, Nikolai
Nitrogen is an essential nutrient that affects all aspects of the growth, development and metabolic responses of plants. Here we investigated the influence of the two major sources of inorganic nitrogen, nitrate and ammonium, on the toxicity caused by excess of Mn in great duckweed, Spirodela polyrhiza. The revealed alleviating effect of ammonium on Mn-mediated toxicity, was complemented by detailed molecular, biochemical and evolutionary characterization of the species ammonium transporters (AMTs). Four genes encoding AMTs in S. polyrhiza, were classified as SpAMT1;1, SpAMT1;2, SpAMT1;3 and SpAMT2. Functional testing of the expressed proteins in yeast and Xenopus oocytes clearly demonstrated activity of SpAMT1;1 and SpAMT1;3 in transporting ammonium. Transcripts of all SpAMT genes were detected in duckweed fronds grown in cultivation medium, containing a physiological or 50-fold elevated concentration of Mn at the background of nitrogen or a mixture of nitrate and ammonium. Each gene demonstrated an individual expression pattern, revealed by RT-qPCR. Revealing the mitigating effect of ammonium uptake on manganese toxicity in aquatic duckweed S. polyrhiza, the study presents a comprehensive analysis of the transporters involved in the uptake of ammonium, shedding a new light on the interactions between the mechanisms of heavy metal toxicity and the regulation of the plant nitrogen metabolism.
Plant ammonium transporter (AMT) integration in regulatory networks
(2016) Straub, Tatsiana; Ludewig, Uwe
Ammonium is a ubiquitous key nutrient in agricultural soils and the preferred nitrogen source for plants. However, excessive ammonium accumulation represses plant growth and development. Ammonium is taken up by plant cells via high-affinity ammonium transporters (AMTs). Six AMT genes were identified in Arabidopsis, which are separated in two distinct clades, five AMT1s and one AMT2. In the plasma membrane, AMT proteins form homo- and heterotrimers with extra-cytoplasmic N-termini and cytoplasmic C-termini. In addition to transcriptional and post-transcriptional control of AMTs by ammonium, phosphorylation in the C-terminus serves as a rapid allosteric switch of the AMT activity and prevents further internal ammonium accumulation. In a physiological screen, a kinase (CIPK23) was identified, which directly regulates ammonium transport activity under high-NH4+ conditions. Interestingly, CIPK23 is already known to regulate nitrate and potassium uptake in roots. Lesion of the CIPK23 gene significantly increased ammonium uptake, but caused growth inhibition. As expected, cipk23 plants were also limited in potassium accumulation, but high potassium availability failed to rescue the cipk23 phenotype. Furthermore, cipk23 plants were more susceptible to methylammonium (MeA), a non-metabolizable analogue of ammonium. The sensitivity to MeA was lost upon genetic suppression of AMT1 genes in the cipk23 background. The data suggest that CIPK23 directly phosphorylates AMT1s in a complex with CBL1 (calcineurin B-like protein) and thereby regulates transport activity. The expression of the CIPK23 and the CBL1 genes were ammonium-dependent and increased when N-starved plants were resupplied with ammonium. Furthermore, cbl1 mutants had enhanced NH4+ accumulation; this phenocopies the larger ammonium uptake in the cipk23 loss-of-function mutant. In vivo experiments demonstrated bimolecular interaction between CIPK23, AMT1;1, and AMT1;2, but not with AMT2;1, suggesting direct phosphorylation of AMT1-type ammonium transporters by CIPK23. However, Western blot analysis with the cipk23 mutant suggested that the loss of the kinase was not sufficient to completely abolish AMT1;1 and AMT1;2 phosphorylation, indicating several independent pathways to regulate ammonium transport activity in AMT trimers. The data identify complex post-translational regulation of ammonium transporters via the CBL1–CIPK23 pathway, which ensures reduction of AMT1 activity and suppression of ammonium uptake under high external NH4+ concentrations.