#METABOLOMICS WORKBENCH lilin_20240428_081352 DATATRACK_ID:4796 STUDY_ID:ST003190 ANALYSIS_ID:AN005238 PROJECT_ID:PR001986 VERSION 1 CREATED_ON May 6, 2024, 7:26 pm #PROJECT PR:PROJECT_TITLE A clade of receptor-like cytoplasmic kinases and 14-3-3 proteins coordinate the PR:PROJECT_TITLE inositol hexaphosphate accumulation PR:PROJECT_TYPE research PR:PROJECT_SUMMARY Inositol hexaphosphate (InsP6) is the major storage form of phosphorus in seeds. PR:PROJECT_SUMMARY Reducing seed InsP6 content is a breeding objective in agriculture, as InsP6 PR:PROJECT_SUMMARY negatively impacts animal nutrition and the environment. Nevertheless, how InsP6 PR:PROJECT_SUMMARY accumulation is regulated remains largely unknown. Here, we identify a clade of PR:PROJECT_SUMMARY receptor-like cytoplasmic kinases (RLCKs), named Inositol Polyphosphate-related PR:PROJECT_SUMMARY Cytoplasmic Kinases 1-6 (IPCK1-IPCK6), deeply involved in InsP6 accumulation. PR:PROJECT_SUMMARY The InsP6 concentration is dramatically reduced in seeds of ipck quadruple PR:PROJECT_SUMMARY (T-4m/C-4m) and quintuple (C-5m) mutants, accompanied with the obviously PR:PROJECT_SUMMARY increase of phosphate (Pi) concentration. The plasma membrane-localized IPCKs PR:PROJECT_SUMMARY recruit IPK1 involved in InsP6 synthesis, and facilitate its binding and PR:PROJECT_SUMMARY activity via phosphorylation of GRF 14-3-3 proteins. IPCKs also recruit IPK2s PR:PROJECT_SUMMARY and PI-PLCs required for InsP4/InsP5 and InsP3 biosynthesis respectively, to PR:PROJECT_SUMMARY form a potential IPCK-GRF-PLC-IPK2-IPK1 complex. Our findings therefore uncover PR:PROJECT_SUMMARY a previously uncharacterized regulatory mechanism of InsP6 accumulation governed PR:PROJECT_SUMMARY by IPCKs, shedding new light on the mechanisms of InsP biosynthesis in PR:PROJECT_SUMMARY eukaryotes. PR:INSTITUTE zhejiang University PR:LAST_NAME XU PR:FIRST_NAME LILIN PR:ADDRESS 866 yuhangtang road, hangzhou, zhejiang, 310027, China PR:EMAIL 1164702127@qq.com PR:PHONE 18667919279 #STUDY ST:STUDY_TITLE A clade of receptor-like cytoplasmic kinases and 14-3-3 proteins coordinate the ST:STUDY_TITLE inositol hexaphosphate accumulation ST:STUDY_SUMMARY Inositol hexaphosphate (InsP6) is the major storage form of phosphorus in seeds. ST:STUDY_SUMMARY Reducing seed InsP6 content is a breeding objective in agriculture, as InsP6 ST:STUDY_SUMMARY negatively impacts animal nutrition and the environment. Nevertheless, how InsP6 ST:STUDY_SUMMARY accumulation is regulated remains largely unknown. Here, we identify a clade of ST:STUDY_SUMMARY receptor-like cytoplasmic kinases (RLCKs), named Inositol Polyphosphate-related ST:STUDY_SUMMARY Cytoplasmic Kinases 1-6 (IPCK1-IPCK6), deeply involved in InsP6 accumulation. ST:STUDY_SUMMARY The InsP6 concentration is dramatically reduced in seeds of ipck quadruple ST:STUDY_SUMMARY (T-4m/C-4m) and quintuple (C-5m) mutants, accompanied with the obviously ST:STUDY_SUMMARY increase of phosphate (Pi) concentration. The plasma membrane-localized IPCKs ST:STUDY_SUMMARY recruit IPK1 involved in InsP6 synthesis, and facilitate its binding and ST:STUDY_SUMMARY activity via phosphorylation of GRF 14-3-3 proteins. IPCKs also recruit IPK2s ST:STUDY_SUMMARY and PI-PLCs required for InsP4/InsP5 and InsP3 biosynthesis respectively, to ST:STUDY_SUMMARY form a potential IPCK-GRF-PLC-IPK2-IPK1 complex. Our findings therefore uncover ST:STUDY_SUMMARY a previously uncharacterized regulatory mechanism of InsP6 accumulation governed ST:STUDY_SUMMARY by IPCKs, shedding new light on the mechanisms of InsP biosynthesis in ST:STUDY_SUMMARY eukaryotes. ST:INSTITUTE Zhejiang University ST:LAST_NAME XU ST:FIRST_NAME LILIN ST:ADDRESS zhejiang university ST:EMAIL 1164702127@qq.com ST:PHONE 18667919279 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Arabidopsis thaliana SU:TAXONOMY_ID 3702 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - WT-IP3-FigS2b Sample source:Plant Seed | Treatment:Control RAW_FILE_NAME(Raw file name)=WT-IP3-FigS2b.xml SUBJECT_SAMPLE_FACTORS - WT-IP4-FigS2b Sample source:Plant Seed | Treatment:Control RAW_FILE_NAME(Raw file name)=WT-IP4-FigS2b.xml SUBJECT_SAMPLE_FACTORS - WT-IP5-FigS2b Sample source:Plant Seed | Treatment:Control RAW_FILE_NAME(Raw file name)=WT-IP5-FigS2b.xml SUBJECT_SAMPLE_FACTORS - WT-IP6-FigS2b Sample source:Plant Seed | Treatment:Control RAW_FILE_NAME(Raw file name)=WT-IP6-FigS2b.xml SUBJECT_SAMPLE_FACTORS - WT-IP7-FigS2b Sample source:Plant Seed | Treatment:Control RAW_FILE_NAME(Raw file name)=WT-IP7-FigS2b.xml SUBJECT_SAMPLE_FACTORS - WT-IP8-FigS2b Sample source:Plant Seed | Treatment:Control RAW_FILE_NAME(Raw file name)=WT-IP8-FigS2b.xml SUBJECT_SAMPLE_FACTORS - WT-IP5-Fig2f Sample source:Plant Seed | Treatment:Control RAW_FILE_NAME(Raw file name)=WT-IP5-Fig2f.xml SUBJECT_SAMPLE_FACTORS - WT-IP6-Fig2f Sample source:Plant Seed | Treatment:Control RAW_FILE_NAME(Raw file name)=WT-IP6-Fig2f.xml SUBJECT_SAMPLE_FACTORS - C-5m-1-IP3-FigS2b Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-1-IP3-FigS2b.xml SUBJECT_SAMPLE_FACTORS - C-5m-1-IP4-FigS2b Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-1-IP4-FigS2b.xml SUBJECT_SAMPLE_FACTORS - C-5m-1-IP5-FigS2b Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-1-IP5-FigS2b.xml SUBJECT_SAMPLE_FACTORS - C-5m-1-IP6-FigS2b Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-1-IP6-FigS2b.xml SUBJECT_SAMPLE_FACTORS - C-5m-1-IP7-FigS2b Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-1-IP7-FigS2b.xml SUBJECT_SAMPLE_FACTORS - C-5m-1-IP8-FigS2b Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-1-IP8-FigS2b.xml SUBJECT_SAMPLE_FACTORS - C-5m-1-IP5-Fig2f Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-1-IP5-Fig2f.xml SUBJECT_SAMPLE_FACTORS - C-5m-1-IP6-Fig2f Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-1-IP6-Fig2f.xml SUBJECT_SAMPLE_FACTORS - C-5m-2-IP3-FigS2b Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-2-IP3-FigS2b.xml SUBJECT_SAMPLE_FACTORS - C-5m-2-IP4-FigS2b Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-2-IP4-FigS2b.xml SUBJECT_SAMPLE_FACTORS - C-5m-2-IP5-FigS2b Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-2-IP5-FigS2b.xml SUBJECT_SAMPLE_FACTORS - C-5m-2-IP6-FigS2b Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-2-IP6-FigS2b.xml SUBJECT_SAMPLE_FACTORS - C-5m-2-IP7-FigS2b Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-2-IP7-FigS2b.xml SUBJECT_SAMPLE_FACTORS - C-5m-2-IP8-FigS2b Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-2-IP8-FigS2b.xml SUBJECT_SAMPLE_FACTORS - C-5m-2-IP5-Fig2f Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-2-IP5-Fig2f.xml SUBJECT_SAMPLE_FACTORS - C-5m-2-IP6-Fig2f Sample source:Plant Seed | Treatment:mutant RAW_FILE_NAME(Raw file name)=C-5m-2-IP6-Fig2f.xml #COLLECTION CO:COLLECTION_SUMMARY 1 g total seedlings grown on agar medium for 15 d or 0.1 g dry seeds were CO:COLLECTION_SUMMARY collected for InsP5/InsP6 detection; For InsP3/InsP4/InsP5/InsP6/InsP7/InsP8 CO:COLLECTION_SUMMARY detection in seeds and seedlings, 10 g of 12-day-old seedlings or 2.4 g of dry CO:COLLECTION_SUMMARY seeds were used for InsPs enrichment with 300 mg of TiO2 beads for each sample. CO:SAMPLE_TYPE Plant #TREATMENT TR:TREATMENT_SUMMARY No treatment, All seedlings are planted on 1/2 MS plates, and the seeds are TR:TREATMENT_SUMMARY healthy and dry. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The enrichment of InsPs with TiO2 beads and SDS-PAGE assay were performed as SP:SAMPLEPREP_SUMMARY described by Wilson et al47. In brief, 1 g total seedlings grown on agar medium SP:SAMPLEPREP_SUMMARY for 15 d or 0.1 g dry seeds were ground in liquid nitrogen, suspended in 5 ml of SP:SAMPLEPREP_SUMMARY 1 M cold perchloric acid, and kept rotating for 15 min at 4 ℃. After SP:SAMPLEPREP_SUMMARY centrifugation at 12,000 g for 10 min at 4 ℃, the supernatant was transferred SP:SAMPLEPREP_SUMMARY into a new tube containing 30 mg of TiO2 beads (5 mm Titansphere; GL Sciences, SP:SAMPLEPREP_SUMMARY Japan) and kept rotating at 4 ℃ for 20 - 30 min. After centrifugation at 5000 SP:SAMPLEPREP_SUMMARY g for 10 min at 4 ℃, the beads were transferred into a new 1.5 ml tube and SP:SAMPLEPREP_SUMMARY washed with pre-cold PA for 3-5 times, then eluted with 500 µl of 10 % ammonia SP:SAMPLEPREP_SUMMARY solution. The eluate was freeze-dried at -50 ℃ and resuspended with 50 µl of SP:SAMPLEPREP_SUMMARY 10 % ammonia solution, and the enriched InsPs was resolved in a 33 % SP:SAMPLEPREP_SUMMARY polyacrylamide / Tris–borate–EDTA (TBE) gel and stained with toluidine blue. SP:SAMPLEPREP_SUMMARY 10 µM synthetic InsP5 (myo-Inositol-1,3,4,5,6-pentaphosphate ammonium salt, SP:SAMPLEPREP_SUMMARY Cayman) and InsP6 (Sichem) were used as the markers and standards. In order to SP:SAMPLEPREP_SUMMARY verify the specific components in the eluate, the total eluate was firstly SP:SAMPLEPREP_SUMMARY freeze-dried, then dissolved with 100 µl 80 % acetonitrile. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY InsPs were detected using Hydrophilic Interaction High Performance Liquid CH:CHROMATOGRAPHY_SUMMARY Chromatography-Tandem Mass Spectrometry on an Agilent 1290 infinity HPLC system CH:CHROMATOGRAPHY_SUMMARY coupled to an Agilent 6460 triple Quad LC-MS/MS using InfinityLab Poroshell 120 CH:CHROMATOGRAPHY_SUMMARY HILIC-Z (2.1 × 100) column (Agilent Technologies, USA). Nitrogen was used as CH:CHROMATOGRAPHY_SUMMARY the sheath gas and drying gas. The nebulizer pressure was set to 45 psi and the CH:CHROMATOGRAPHY_SUMMARY flow rate of drying gas was 5 L / min. The flow rate and temperature of the CH:CHROMATOGRAPHY_SUMMARY sheath gas were 11 L / min and 350℃, respectively. Chromatographic separation CH:CHROMATOGRAPHY_SUMMARY was carried out on a HPLC column (100 × 2.1 mm, 2.7 µm). The column CH:CHROMATOGRAPHY_SUMMARY temperature was 35℃. The mobile phases consisted of (A) ammonium acetate in CH:CHROMATOGRAPHY_SUMMARY distilled water (pH 10.0) and (B) Acetonitrile. The gradient program was as CH:CHROMATOGRAPHY_SUMMARY follows: 0-10 min, 90 % → 55 % of B; 10-12 min, 55 % → 90 % of B; 12-20 min, CH:CHROMATOGRAPHY_SUMMARY 90 % of B. The flow rate was set at 0.3 ml / min, and the injection volume was CH:CHROMATOGRAPHY_SUMMARY 10 μl. Mass spectrometric detection was completed by use of an electrospray CH:CHROMATOGRAPHY_SUMMARY ionization (ESI) source in negative ion multiple-reaction monitoring (MRM) mode. CH:CHROMATOGRAPHY_SUMMARY InsPs were identified based on comparison to known InsPs species. The mass CH:CHROMATOGRAPHY_SUMMARY spectrometry parameters corresponding to different InsPs show as below: InsP3 CH:CHROMATOGRAPHY_SUMMARY (MRM: 419 -> 321, 419 -> 337, Acquisition time is 6-7 min); InsP4 (MRM: 499 -> CH:CHROMATOGRAPHY_SUMMARY 401, 499 -> 417, Acquisition time is 6-7 min); InsP5 (MRM: 579 -> 480.9, 579 -> CH:CHROMATOGRAPHY_SUMMARY 382.8, Acquisition time is 6-7 min); InsP6 (MRM: 659 -> 560.8, 659 -> 577, CH:CHROMATOGRAPHY_SUMMARY Acquisition time is 6-7 min); InsP7 (MRM: 739 -> 575, 739 -> 657, Acquisition CH:CHROMATOGRAPHY_SUMMARY time is 5-6 min); InsP8 (MRM: 819 -> 737, 819 -> 655, Acquisition time is 4-5 CH:CHROMATOGRAPHY_SUMMARY min). According to the regression equation calculated from the standard sample, CH:CHROMATOGRAPHY_SUMMARY substitute the response value of the sample into the equation to convert the CH:CHROMATOGRAPHY_SUMMARY corresponding concentration. For InsP3/InsP4/InsP5/InsP6/InsP7/InsP8 detection CH:CHROMATOGRAPHY_SUMMARY in seeds and seedlings, 10 g of 12-day-old seedlings or 2.4 g of dry seeds were CH:CHROMATOGRAPHY_SUMMARY used for InsPs enrichment with 300 mg of TiO2 beads for each sample. The CH:CHROMATOGRAPHY_SUMMARY enriched substances were analyzed by HPLC-MS/MS. The purchased InsP3 CH:CHROMATOGRAPHY_SUMMARY (1,4,5-InsP3, MedChemExpress), InsP4 (1,3,4,5-InsP4, MedChemExpress), InsP5 and CH:CHROMATOGRAPHY_SUMMARY InsP6, InsP7 (5-InsP7) and InsP8 (1,5-InsP8) from Lei lab25 were used as CH:CHROMATOGRAPHY_SUMMARY standard samples for generating the calibration curves. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um) CH:SOLVENT_A 100% Distilled Water; 10% ammonium acetate (pH 10.0) CH:SOLVENT_B 100% Acetonitrile CH:FLOW_GRADIENT 0-10 min, 90 % → 55 % of B; 10-12 min, 55 % → 90 % of B; 12-20 min, 90 % of CH:FLOW_GRADIENT B CH:FLOW_RATE 0.3 mL/min CH:COLUMN_TEMPERATURE 35℃ #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 5973 MS:INSTRUMENT_TYPE HPLC-MS/MS MS:MS_TYPE Other MS:ION_MODE POSITIVE MS:MS_COMMENTS InsPs were detected using Hydrophilic Interaction High Performance Liquid MS:MS_COMMENTS Chromatography-Tandem Mass Spectrometry on an Agilent 1290 infinity HPLC system MS:MS_COMMENTS coupled to an Agilent 6460 triple Quad LC-MS/MS using InfinityLab Poroshell 120 MS:MS_COMMENTS HILIC-Z (2.1 × 100) column (Agilent Technologies, USA). Nitrogen was used as MS:MS_COMMENTS the sheath gas and drying gas. The nebulizer pressure was set to 45 psi and the MS:MS_COMMENTS flow rate of drying gas was 5 L / min. The flow rate and temperature of the MS:MS_COMMENTS sheath gas were 11 L / min and 350℃, respectively. Chromatographic separation MS:MS_COMMENTS was carried out on a HPLC column (100 × 2.1 mm, 2.7 µm). The column MS:MS_COMMENTS temperature was 35℃. The mobile phases consisted of (A) ammonium acetate in MS:MS_COMMENTS distilled water (pH 10.0) and (B) Acetonitrile. The gradient program was as MS:MS_COMMENTS follows: 0-10 min, 90 % → 55 % of B; 10-12 min, 55 % → 90 % of B; 12-20 min, MS:MS_COMMENTS 90 % of B. The flow rate was set at 0.3 ml / min, and the injection volume was MS:MS_COMMENTS 10 μl. Mass spectrometric detection was completed by use of an electrospray MS:MS_COMMENTS ionization (ESI) source in negative ion multiple-reaction monitoring (MRM) mode. MS:MS_COMMENTS InsPs were identified based on comparison to known InsPs species. The mass MS:MS_COMMENTS spectrometry parameters corresponding to different InsPs show as below: InsP3 MS:MS_COMMENTS (MRM: 419 -> 321, 419 -> 337, Acquisition time is 6-7 min); InsP4 (MRM: 499 -> MS:MS_COMMENTS 401, 499 -> 417, Acquisition time is 6-7 min); InsP5 (MRM: 579 -> 480.9, 579 -> MS:MS_COMMENTS 382.8, Acquisition time is 6-7 min); InsP6 (MRM: 659 -> 560.8, 659 -> 577, MS:MS_COMMENTS Acquisition time is 6-7 min); InsP7 (MRM: 739 -> 575, 739 -> 657, Acquisition MS:MS_COMMENTS time is 5-6 min); InsP8 (MRM: 819 -> 737, 819 -> 655, Acquisition time is 4-5 MS:MS_COMMENTS min). According to the regression equation calculated from the standard sample, MS:MS_COMMENTS substitute the response value of the sample into the equation to convert the MS:MS_COMMENTS corresponding concentration. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS mg/g MS_METABOLITE_DATA_START Samples WT-IP3-FigS2b WT-IP4-FigS2b WT-IP5-FigS2b WT-IP6-FigS2b WT-IP7-FigS2b WT-IP8-FigS2b WT-IP5-Fig2f WT-IP6-Fig2f C-5m-1-IP3-FigS2b C-5m-1-IP4-FigS2b C-5m-1-IP5-FigS2b C-5m-1-IP6-FigS2b C-5m-1-IP7-FigS2b C-5m-1-IP8-FigS2b C-5m-1-IP5-Fig2f C-5m-1-IP6-Fig2f C-5m-2-IP3-FigS2b C-5m-2-IP4-FigS2b C-5m-2-IP5-FigS2b C-5m-2-IP6-FigS2b C-5m-2-IP7-FigS2b C-5m-2-IP8-FigS2b C-5m-2-IP5-Fig2f C-5m-2-IP6-Fig2f Factors Sample source:Plant Seed | Treatment:Control Sample source:Plant Seed | Treatment:Control Sample source:Plant Seed | Treatment:Control Sample source:Plant Seed | Treatment:Control Sample source:Plant Seed | Treatment:Control Sample source:Plant Seed | Treatment:Control Sample source:Plant Seed | Treatment:Control Sample source:Plant Seed | Treatment:Control Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant Sample source:Plant Seed | Treatment:mutant IP3 0.1855 0.1424 0.1527 IP4 0.1657 0.1081 0.1252 IP5 0.3576 3.279806876 0.1624 1.04324096 0.1464 0.882777046 IP6 3.0960 0.003096 1.1800 0.00118 1.0234 0.0010234 IP7 0.0643 0.0613 0.0604 IP8 0.1686 0.0955 0.0912 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name IP3 IP4 IP5 IP6 IP7 IP8 METABOLITES_END #END