XB-ART-57624BMC Biol January 1, 2020; 18 (1): 196.
Feedback inhibition of AMT1 NH4+-transporters mediated by CIPK15 kinase.
BACKGROUND: Ammonium (NH4+), a key nitrogen form, becomes toxic when it accumulates to high levels. Ammonium transporters (AMTs) are the key transporters responsible for NH4+ uptake. AMT activity is under allosteric feedback control, mediated by phosphorylation of a threonine in the cytosolic C-terminus (CCT). However, the kinases responsible for the NH4+-triggered phosphorylation remain unknown. RESULTS: In this study, a functional screen identified protein kinase CBL-Interacting Protein Kinase15 (CIPK15) as a negative regulator of AMT1;1 activity. CIPK15 was able to interact with several AMT1 paralogs at the plasma membrane. Analysis of AmTryoshka, an NH4+ transporter activity sensor for AMT1;3 in yeast, and a two-electrode-voltage-clamp (TEVC) of AMT1;1 in Xenopus oocytes showed that CIPK15 inhibits AMT activity. CIPK15 transcript levels increased when seedlings were exposed to elevated NH4+ levels. Notably, cipk15 knockout mutants showed higher 15NH4+ uptake and accumulated higher amounts of NH4+ compared to the wild-type. Consistently, cipk15 was hypersensitive to both NH4+ and methylammonium but not nitrate (NO3-). CONCLUSION: Taken together, our data indicate that feedback inhibition of AMT1 activity is mediated by the protein kinase CIPK15 via phosphorylation of residues in the CCT to reduce NH4+-accumulation.
PubMed ID: 33317525
PMC ID: PMC7737296
Article link: BMC Biol
Genes referenced: cbl pcyt1a pigy
Article Images: [+] show captions
|Fig. 1. CIPK15 inhibits AMT1 activity in Xenopus oocytes. a, b Co-expression of CIPK15 inhibited NH4+-triggered inward currents of AMT1;1 in Xenopus oocytes. Oocytes were injected with water only, 50 ng cRNA of AMT1;1 only, 50 ng AMT1;1 + 50 ng CIPK15, or 50 ng CIPK15 only, and perfused with NH4Cl at the indicated concentrations (a) or (b) 0.2 mM for current recordings (a) and IV curve (b). Oocytes were voltage clamped at a − 120 mV or b − 40 mV and stepped in − 20-mV increments between − 20 and − 200 mV for 300 ms. b Currents (nA) were background subtracted (difference between currents at + 300 ms in the cRNA-injected AMT1;1 only/AMT1;1 + CIPK15/CIPK15 only and water-injected control of the indicated substrates). The data are the mean ± SE for three experiments. c TVEC traces of oocytes injected with water only, 5 ng cRNA of AMT1;1 only, or 5 ng AMT1;1 + 0.5 ng CIPK15, and perfused with NH4Cl at the indicated concentrations. Similar results were obtained in at least three independent experiments using different batches of oocytes|
|Fig. 2. CIPK15 inhibited AmTryoshka1;3 LS-F138I activity in yeast. a Schematic representation of AmTryoshka1;3 LS-F138I . b CIPK15 reduced NH4+-triggered AmTryoshka1;3 LS-F138I  responses in yeast. Amtryoshka1;3 LS-F138I was co-expressed with control (vector only), CIPK15, and CIPK15m (inactive mutant). Results of normalized fluorescence ratio (normalized to buffer control = 1, λexc 440 nm, ratio = FI510nm/570nm) after addition of NH4Cl as represented by box and whiskers (mean ± SE, n = 8). Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by Prism software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. p, significant change as shown in the figure (two-way ANOVA followed by Tukey’s post-test). PM, plasma membrane|
|Fig. 3. NH4+-triggered CIPK15 mRNA accumulation. qRT-PCR analyses of AMT1;1 and CIPK15 mRNA levels in roots after over 10 h after addition of 1 mM NH4+. Levels were normalized to UBQ10 [mean ± SE for four independent experiments (each experiment n > 50, total n > 200)]. p, significant change for mRNA levels of AMT1;1 and CIPK15 at 1, 2, and 10 h compared to at 0 h (two-way ANOVA followed by Tukey’s post-test)|
|Fig. 4. CIPK15 can interact with AMT1;1. Interaction growth assay (a), β-galactosidase staining (b), and filter (c) assay in yeast; and split-fluorescent protein interaction assay (d) in tobacco leaves for AMT1.1 and CIPK15 protein interactions. a Plasmids expressing AMT1;1 and CIPKs were expressed in yeast. Interaction indicated by growth on SD-Trp -Leu -His. Growth on SD-LT as control (Figure S5). Comparable results were obtained in three independent experiments. b, c Interaction of CIPKs and AMT1;1 in a split-ubiquitin system detected by X-Gal staining and filter assays using full-length AMT1;1-Cub-PLV as bait and NubG, NubI, and NubG-full-length CIPKs as prey. NubI/NubG served as positive (blue color) and negative controls, respectively. d Split-fluorescent protein interaction assay for AMT1;1 and CIPK15. YFP/chlorophyll, merged image of fluorescence and chloroplast. Reconstitution of YFP fluorescence from nYFP-AMT1;1 + CIPK15-cCFP and nYPF-AMT1;1 + cCFP (negative control). Comparable results with different combinations shown in Figure S7|
|Fig. 5. AMT1;1-T460 phosphorylation is reduced in cipk15 mutant plants. Plant seedlings were germinated and grown for 7 days in half-strength MS medium with 5 mM KNO3 as the sole nitrogen source, then starved for 2 days in half-strength MS medium without nitrogen. Seedlings were treated with 1 mM NH4Cl for 1 h, membrane fractions were isolated and probed with anti-AMT1-P antibodies (a) and anti-AMT1;1 antibodies (b) . Ponceau S staining served as a loading control. Quantification of phosphorylation of AMT1-P levels normalized to Ponceau S staining and relative to wild-type shown in c. Corresponding data and replications were obtained in three independent experiments. Data (c) are the mean ± SD for three experiments. p, significant change compared to wild-type as shown in figure (two-way ANOVA followed by Tukey’s post-test)|
|Fig. 6. NH4+ content and transport in cipk15 mutants. Plant seedlings were germinated and grown for 7 days in half-strength MS medium with 5 mM KNO3 as the sole nitrogen source, then all seedlings were starved for 2 days in half-strength MS medium without nitrogen. For NH4+ content analyses, a seedlings were collected after being starved for 2 days (−N), or after with 1 mM NH4Cl (NH4+), and 1 mM KNO3 (NO3−) for 1 h. For 15N-labeled uptake, b seedlings were collected after being starved for 2 days (−N) (1 mM 15NH4Cl was used for 15 mins for 15N-labeling), or after treatment with 1 mM NH4Cl (NH4+), and 1 mM KNO3 (NO3−) for 1 h (1 mM 15NH4Cl was used for last 15 mins for N15-labeling for conditions of NH4+ and NO3−). Each data point represents different experiments, in which seedlings n > 15, total n > 60) in Col-0 and two cipk15 knockout mutants and presented as box and whiskers. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by Prism software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. p, significant change compared to before submergence (two-way ANOVA followed by Tukey’s post-test)|
|Fig. 7. Hypersensitivity of cipk15 mutant plants to NH4+and MeA. Representative images (a) and quantification results of primary root length of plants grown on plates containing 20 mM NHCl4 (b) or 20 mM MeA (c). Primary root length in wild-type (Col-0), qko mutant, and cipk15 mutants on 20 mM NH4Cl (b) or on 20 mM MeA (c) are presented as box and whiskers. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by Prism software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots (means ± SE; n ≥ 15). p, significant change of qko mutant and cipk15 mutants compared to wild-type plants (two-way ANOVA followed by Tukey’s post-test). Scale bar: 0.1 cm|
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