Summary of Study ST003190

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001986. The data can be accessed directly via it's Project DOI: 10.21228/M81142 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003190
Study Title	A clade of receptor-like cytoplasmic kinases and 14-3-3 proteins coordinate the inositol hexaphosphate accumulation
Study Summary	Inositol hexaphosphate (InsP6) is the major storage form of phosphorus in seeds. Reducing seed InsP6 content is a breeding objective in agriculture, as InsP6 negatively impacts animal nutrition and the environment. Nevertheless, how InsP6 accumulation is regulated remains largely unknown. Here, we identify a clade of receptor-like cytoplasmic kinases (RLCKs), named Inositol Polyphosphate-related Cytoplasmic Kinases 1-6 (IPCK1-IPCK6), deeply involved in InsP6 accumulation. The InsP6 concentration is dramatically reduced in seeds of ipck quadruple (T-4m/C-4m) and quintuple (C-5m) mutants, accompanied with the obviously increase of phosphate (Pi) concentration. The plasma membrane-localized IPCKs recruit IPK1 involved in InsP6 synthesis, and facilitate its binding and activity via phosphorylation of GRF 14-3-3 proteins. IPCKs also recruit IPK2s and PI-PLCs required for InsP4/InsP5 and InsP3 biosynthesis respectively, to form a potential IPCK-GRF-PLC-IPK2-IPK1 complex. Our findings therefore uncover a previously uncharacterized regulatory mechanism of InsP6 accumulation governed by IPCKs, shedding new light on the mechanisms of InsP biosynthesis in eukaryotes.
Institute	Zhejiang University
Last Name	XU
First Name	LILIN
Address	zhejiang university
Email	1164702127@qq.com
Phone	18667919279
Submit Date	2024-04-28
Raw Data Available	Yes
Raw Data File Type(s)	.xml
Analysis Type Detail	Other
Release Date	2024-05-08
Release Version	1

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Project:

Project ID:	PR001986
Project DOI:	doi: 10.21228/M81142
Project Title:	A clade of receptor-like cytoplasmic kinases and 14-3-3 proteins coordinate the inositol hexaphosphate accumulation
Project Type:	research
Project Summary:	Inositol hexaphosphate (InsP6) is the major storage form of phosphorus in seeds. Reducing seed InsP6 content is a breeding objective in agriculture, as InsP6 negatively impacts animal nutrition and the environment. Nevertheless, how InsP6 accumulation is regulated remains largely unknown. Here, we identify a clade of receptor-like cytoplasmic kinases (RLCKs), named Inositol Polyphosphate-related Cytoplasmic Kinases 1-6 (IPCK1-IPCK6), deeply involved in InsP6 accumulation. The InsP6 concentration is dramatically reduced in seeds of ipck quadruple (T-4m/C-4m) and quintuple (C-5m) mutants, accompanied with the obviously increase of phosphate (Pi) concentration. The plasma membrane-localized IPCKs recruit IPK1 involved in InsP6 synthesis, and facilitate its binding and activity via phosphorylation of GRF 14-3-3 proteins. IPCKs also recruit IPK2s and PI-PLCs required for InsP4/InsP5 and InsP3 biosynthesis respectively, to form a potential IPCK-GRF-PLC-IPK2-IPK1 complex. Our findings therefore uncover a previously uncharacterized regulatory mechanism of InsP6 accumulation governed by IPCKs, shedding new light on the mechanisms of InsP biosynthesis in eukaryotes.
Institute:	zhejiang University
Last Name:	XU
First Name:	LILIN
Address:	866 yuhangtang road, hangzhou, zhejiang, 310027, China
Email:	1164702127@qq.com
Phone:	18667919279

Subject:

Subject ID:	SU003309
Subject Type:	Plant
Subject Species:	Arabidopsis thaliana
Taxonomy ID:	3702

Factors:

Subject type: Plant; Subject species: Arabidopsis thaliana (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Treatment
SA347864	WT-IP5-Fig2f	Plant Seed	Control
SA347865	WT-IP3-FigS2b	Plant Seed	Control
SA347866	WT-IP8-FigS2b	Plant Seed	Control
SA347867	WT-IP6-Fig2f	Plant Seed	Control
SA347868	WT-IP7-FigS2b	Plant Seed	Control
SA347869	WT-IP4-FigS2b	Plant Seed	Control
SA347870	WT-IP5-FigS2b	Plant Seed	Control
SA347871	WT-IP6-FigS2b	Plant Seed	Control
SA347872	C-5m-2-IP7-FigS2b	Plant Seed	mutant
SA347873	C-5m-2-IP6-FigS2b	Plant Seed	mutant
SA347874	C-5m-2-IP5-FigS2b	Plant Seed	mutant
SA347875	C-5m-2-IP5-Fig2f	Plant Seed	mutant
SA347876	C-5m-2-IP4-FigS2b	Plant Seed	mutant
SA347877	C-5m-2-IP6-Fig2f	Plant Seed	mutant
SA347878	C-5m-2-IP8-FigS2b	Plant Seed	mutant
SA347879	C-5m-1-IP7-FigS2b	Plant Seed	mutant
SA347880	C-5m-1-IP5-FigS2b	Plant Seed	mutant
SA347881	C-5m-1-IP4-FigS2b	Plant Seed	mutant
SA347882	C-5m-1-IP3-FigS2b	Plant Seed	mutant
SA347883	C-5m-1-IP6-FigS2b	Plant Seed	mutant
SA347884	C-5m-1-IP8-FigS2b	Plant Seed	mutant
SA347885	C-5m-1-IP6-Fig2f	Plant Seed	mutant
SA347886	C-5m-1-IP5-Fig2f	Plant Seed	mutant
SA347887	C-5m-2-IP3-FigS2b	Plant Seed	mutant

Showing results 1 to 24 of 24

Showing results 1 to 24 of 24

Collection:

Collection ID:	CO003302
Collection Summary:	1 g total seedlings grown on agar medium for 15 d or 0.1 g dry seeds were collected for InsP5/InsP6 detection; For InsP3/InsP4/InsP5/InsP6/InsP7/InsP8 detection in seeds and seedlings, 10 g of 12-day-old seedlings or 2.4 g of dry seeds were used for InsPs enrichment with 300 mg of TiO2 beads for each sample.
Sample Type:	Plant

Treatment:

Treatment ID:	TR003318
Treatment Summary:	No treatment, All seedlings are planted on 1/2 MS plates, and the seeds are healthy and dry.

Sample Preparation:

Sampleprep ID:	SP003316
Sampleprep Summary:	The enrichment of InsPs with TiO2 beads and SDS-PAGE assay were performed as described by Wilson et al47. In brief, 1 g total seedlings grown on agar medium for 15 d or 0.1 g dry seeds were ground in liquid nitrogen, suspended in 5 ml of 1 M cold perchloric acid, and kept rotating for 15 min at 4 ℃. After centrifugation at 12,000 g for 10 min at 4 ℃, the supernatant was transferred into a new tube containing 30 mg of TiO2 beads (5 mm Titansphere; GL Sciences, Japan) and kept rotating at 4 ℃ for 20 - 30 min. After centrifugation at 5000 g for 10 min at 4 ℃, the beads were transferred into a new 1.5 ml tube and washed with pre-cold PA for 3-5 times, then eluted with 500 µl of 10 % ammonia solution. The eluate was freeze-dried at -50 ℃ and resuspended with 50 µl of 10 % ammonia solution, and the enriched InsPs was resolved in a 33 % polyacrylamide / Tris–borate–EDTA (TBE) gel and stained with toluidine blue. 10 µM synthetic InsP5 (myo-Inositol-1,3,4,5,6-pentaphosphate ammonium salt, Cayman) and InsP6 (Sichem) were used as the markers and standards. In order to verify the specific components in the eluate, the total eluate was firstly freeze-dried, then dissolved with 100 µl 80 % acetonitrile.

Sampleprep ID:

SP003316

Sampleprep Summary:

The enrichment of InsPs with TiO2 beads and SDS-PAGE assay were performed as described by Wilson et al47. In brief, 1 g total seedlings grown on agar medium for 15 d or 0.1 g dry seeds were ground in liquid nitrogen, suspended in 5 ml of 1 M cold perchloric acid, and kept rotating for 15 min at 4 ℃. After centrifugation at 12,000 g for 10 min at 4 ℃, the supernatant was transferred into a new tube containing 30 mg of TiO2 beads (5 mm Titansphere; GL Sciences, Japan) and kept rotating at 4 ℃ for 20 - 30 min. After centrifugation at 5000 g for 10 min at 4 ℃, the beads were transferred into a new 1.5 ml tube and washed with pre-cold PA for 3-5 times, then eluted with 500 µl of 10 % ammonia solution. The eluate was freeze-dried at -50 ℃ and resuspended with 50 µl of 10 % ammonia solution, and the enriched InsPs was resolved in a 33 % polyacrylamide / Tris–borate–EDTA (TBE) gel and stained with toluidine blue. 10 µM synthetic InsP5 (myo-Inositol-1,3,4,5,6-pentaphosphate ammonium salt, Cayman) and InsP6 (Sichem) were used as the markers and standards. In order to verify the specific components in the eluate, the total eluate was firstly freeze-dried, then dissolved with 100 µl 80 % acetonitrile.

Combined analysis:

Analysis ID	AN005238
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Agilent 1290 Infinity
Column	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)
MS Type	Other
MS instrument type	HPLC-MS/MS
MS instrument name	Agilent 5973
Ion Mode	POSITIVE
Units	mg/g

Chromatography:

Chromatography ID:	CH003965
Chromatography Summary:	InsPs were detected using Hydrophilic Interaction High Performance Liquid Chromatography-Tandem Mass Spectrometry on an Agilent 1290 infinity HPLC system coupled to an Agilent 6460 triple Quad LC-MS/MS using InfinityLab Poroshell 120 HILIC-Z (2.1 × 100) column (Agilent Technologies, USA). Nitrogen was used as the sheath gas and drying gas. The nebulizer pressure was set to 45 psi and the flow rate of drying gas was 5 L / min. The flow rate and temperature of the sheath gas were 11 L / min and 350℃, respectively. Chromatographic separation was carried out on a HPLC column (100 × 2.1 mm, 2.7 µm). The column temperature was 35℃. The mobile phases consisted of (A) ammonium acetate in distilled water (pH 10.0) and (B) Acetonitrile. The gradient program was as follows: 0-10 min, 90 % → 55 % of B; 10-12 min, 55 % → 90 % of B; 12-20 min, 90 % of B. The flow rate was set at 0.3 ml / min, and the injection volume was 10 μl. Mass spectrometric detection was completed by use of an electrospray ionization (ESI) source in negative ion multiple-reaction monitoring (MRM) mode. InsPs were identified based on comparison to known InsPs species. The mass spectrometry parameters corresponding to different InsPs show as below: InsP3 (MRM: 419 -> 321, 419 -> 337, Acquisition time is 6-7 min); InsP4 (MRM: 499 -> 401, 499 -> 417, Acquisition time is 6-7 min); InsP5 (MRM: 579 -> 480.9, 579 -> 382.8, Acquisition time is 6-7 min); InsP6 (MRM: 659 -> 560.8, 659 -> 577, Acquisition time is 6-7 min); InsP7 (MRM: 739 -> 575, 739 -> 657, Acquisition time is 5-6 min); InsP8 (MRM: 819 -> 737, 819 -> 655, Acquisition time is 4-5 min). According to the regression equation calculated from the standard sample, substitute the response value of the sample into the equation to convert the corresponding concentration. For InsP3/InsP4/InsP5/InsP6/InsP7/InsP8 detection in seeds and seedlings, 10 g of 12-day-old seedlings or 2.4 g of dry seeds were used for InsPs enrichment with 300 mg of TiO2 beads for each sample. The enriched substances were analyzed by HPLC-MS/MS. The purchased InsP3 (1,4,5-InsP3, MedChemExpress), InsP4 (1,3,4,5-InsP4, MedChemExpress), InsP5 and InsP6, InsP7 (5-InsP7) and InsP8 (1,5-InsP8) from Lei lab25 were used as standard samples for generating the calibration curves.
Instrument Name:	Agilent 1290 Infinity
Column Name:	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)
Column Temperature:	35℃
Flow Gradient:	0-10 min, 90 % → 55 % of B; 10-12 min, 55 % → 90 % of B; 12-20 min, 90 % of B
Flow Rate:	0.3 mL/min
Solvent A:	100% Distilled Water; 10% ammonium acetate (pH 10.0)
Solvent B:	100% Acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS004971
Analysis ID:	AN005238
Instrument Name:	Agilent 5973
Instrument Type:	HPLC-MS/MS
MS Type:	Other
MS Comments:	InsPs were detected using Hydrophilic Interaction High Performance Liquid Chromatography-Tandem Mass Spectrometry on an Agilent 1290 infinity HPLC system coupled to an Agilent 6460 triple Quad LC-MS/MS using InfinityLab Poroshell 120 HILIC-Z (2.1 × 100) column (Agilent Technologies, USA). Nitrogen was used as the sheath gas and drying gas. The nebulizer pressure was set to 45 psi and the flow rate of drying gas was 5 L / min. The flow rate and temperature of the sheath gas were 11 L / min and 350℃, respectively. Chromatographic separation was carried out on a HPLC column (100 × 2.1 mm, 2.7 µm). The column temperature was 35℃. The mobile phases consisted of (A) ammonium acetate in distilled water (pH 10.0) and (B) Acetonitrile. The gradient program was as follows: 0-10 min, 90 % → 55 % of B; 10-12 min, 55 % → 90 % of B; 12-20 min, 90 % of B. The flow rate was set at 0.3 ml / min, and the injection volume was 10 μl. Mass spectrometric detection was completed by use of an electrospray ionization (ESI) source in negative ion multiple-reaction monitoring (MRM) mode. InsPs were identified based on comparison to known InsPs species. The mass spectrometry parameters corresponding to different InsPs show as below: InsP3 (MRM: 419 -> 321, 419 -> 337, Acquisition time is 6-7 min); InsP4 (MRM: 499 -> 401, 499 -> 417, Acquisition time is 6-7 min); InsP5 (MRM: 579 -> 480.9, 579 -> 382.8, Acquisition time is 6-7 min); InsP6 (MRM: 659 -> 560.8, 659 -> 577, Acquisition time is 6-7 min); InsP7 (MRM: 739 -> 575, 739 -> 657, Acquisition time is 5-6 min); InsP8 (MRM: 819 -> 737, 819 -> 655, Acquisition time is 4-5 min). According to the regression equation calculated from the standard sample, substitute the response value of the sample into the equation to convert the corresponding concentration.
Ion Mode:	POSITIVE