Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

Gaugler, Philipp; Gaugler, Verena; Kamleitner, Marília; Schaaf, Gabriel

doi:10.3791/61495

Cited by 5 publications

(11 citation statements)

References 32 publications

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“…For P i resupply, 620 μM KH 2 PO 4 was added to the medium and the plants were grown for another 6 h before harvest. Afterward, seedlings were washed two times with ultrapure water and frozen in liquid N 2 and the InsPs were extracted as described previously ( Azevedo and Saiardi, 2006 ) and resolved exactly as described in Gaugler et al. (2020) .…”

Section: Methodsmentioning

confidence: 99%

ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis

et al. 2021

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In plants, phosphate (P i ) homeostasis is regulated by the interaction of PHR transcription factors with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates. In this study, we combined different methods to obtain a comprehensive picture of how inositol (pyro)phosphate metabolism is regulated by P i and dependent on the inositol phosphate kinase ITPK1. We found that inositol pyrophosphates are more responsive to P i than lower inositol phosphates, a response conserved across kingdoms. Using the capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) we could separate different InsP 7 isomers in Arabidopsis and rice, and identify 4/6-InsP 7 and a PP-InsP 4 isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP 7 , 5-InsP 7 , and InsP 8 increase several fold in shoots after P i resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP 6 compartmentalization. Notably, ITPK1 is critical for P i -dependent 5-InsP 7 and InsP 8 synthesis in planta and its activity regulates P i starvation responses in a PHRdependent manner. Furthermore, we demonstrated that ITPK1-mediated conversion of InsP 6 to 5-InsP 7 requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP 7 under low ATP. Collectively, our study provides new insights into P i -dependent changes in nutritional and energetic states with the synthesis of regulatory inositol pyrophosphates.

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Section: Methodsmentioning

confidence: 99%

ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis

et al. 2021

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“…Seedlings were allowed to label for 6 days, then washed twice in dH 2 O and frozen in liquid N 2 . Extraction of InsPs was done as described ( Azevedo and Saiardi, 2006 ; Laha et al, 2021 ), and extracts were resolved by SAX–HPLC using a Partisphere SAX 4.6 × 125 mm column (Whatman) at a flow rate of 0.5 mL min −1 with a shallow gradient formed by buffers A (1-mM EDTA) and B [1-mM EDTA and 1.3-M (NH 4 ) 2 HPO 4 , pH 3.8, with H 3 PO 4 ] ( Laha et al, 2015 ; Gaugler et al, 2020 ).…”

Section: Methodsmentioning

confidence: 99%

INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1-dependent inositol polyphosphates regulate auxin responses in Arabidopsis

et al. 2022

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The combinatorial phosphorylation of myo-inositol results in the generation of different inositol phosphates (InsPs), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6 is also an important precursor of the higher phosphorylated inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8, which are characterized by a diphosphate moiety and are also ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in animals and yeast, their biosynthesis and functions in plants has remained largely elusive because plant genomes do not encode canonical InsP6 kinases. Recent work has shown that Arabidopsis (Arabidopsis thaliana) INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1 (ITPK1) and ITPK2 display in vitro InsP6 kinase activity and that, in planta, ITPK1 stimulates 5-InsP7 and InsP8 synthesis and regulates phosphate starvation responses. Here we report a critical role of ITPK1 in auxin-related processes that is independent of the ITPK1-controlled regulation of phosphate starvation responses. Those processes include primary root elongation, root hair development, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity to exogenously applied auxin. We found that the recombinant auxin receptor complex, consisting of the F-Box protein TRANSPORT INHIBITOR RESPONSE1 (TIR1), ARABIDOPSIS SKP1 HOMOLOGUE 1 (ASK1) and the transcriptional repressor INDOLE-3-ACETIC ACID INDUCIBLE 7 (IAA7), binds to anionic inositol polyphosphates with high affinity. We further identified a physical interaction between ITPK1 and TIR1, suggesting a localized production of 5-InsP7, or another ITPK1-dependent InsP/PP-InsP isomer, to activate the auxin receptor complex. Finally, we demonstrate that ITPK1 and ITPK2 function redundantly to control auxin responses, as deduced from the auxin-insensitive phenotypes of itpk1 itpk2 double mutant plants. Our findings expand the mechanistic understanding of auxin perception and suggest that distinct inositol polyphosphates generated near auxin receptors help to fine-tune auxin sensitivity in plants.

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“…They were then diluted 1:200 in 2 mL of fresh medium supplemented with 6 μCi mL –1 [ 3 H]- myo -inositol (30–80 Ci mmol –1 ; Biotrend; ART-0261-5) and grown overnight at 28 °C in a spinning wheel. After centrifugation and washing of the cell pellet, inositol polyphosphates were extracted and analyzed as described before. ,, …”

Section: Methodsmentioning

confidence: 99%

Arabidopsis PFA-DSP-Type Phosphohydrolases Target Specific Inositol Pyrophosphate Messengers

et al. 2022

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Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo-inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling, and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologues display phosphohydrolase activity with a high specificity for the 5-β-phosphate of inositol pyrophosphates and only minor activity against the β-phosphates of 4-InsP 7 and 6-InsP 7 . We further show that heterologous expression of Arabidopsis PFA-DSP1-5 rescues wortmannin sensitivity and deranged inositol pyrophosphate homeostasis caused by the deficiency of the PFA-DSPtype inositol pyrophosphate phosphohydrolase Siw14 in yeast. Heterologous expression in Nicotiana benthamiana leaves provided evidence that Arabidopsis PFA-DSP1 also displays 5-β-phosphate-specific inositol pyrophosphate phosphohydrolase activity in planta. Our findings lay the biochemical basis and provide the genetic tools to uncover the roles of inositol pyrophosphates in plant physiology and plant development.

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Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

Cited by 5 publications

References 32 publications

ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis

ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis

INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1-dependent inositol polyphosphates regulate auxin responses in Arabidopsis

Arabidopsis PFA-DSP-Type Phosphohydrolases Target Specific Inositol Pyrophosphate Messengers

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