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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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.