In plants, phosphate (Pi) homeostasis is regulated by the interaction of PHR transcription factors with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates. Here, we combined different methods t...In plants, phosphate (Pi) homeostasis is regulated by the interaction of PHR transcription factors with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates. Here, we combined different methods to obtain a comprehensive picture of how inositol (pyro)phosphate metabolism is regulated by Pi and dependent on the inositol phosphate kinase ITPK1. We found that inositol pyrophosphates are more responsive to Pi than lower inositol phosphates, a response conserved across kingdoms. With CE-ESI-MS we could separate different InsP7 isomers in Arabidopsis and rice, and identify 4/6-InsP7 and a PP-InsP4 isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP7, 5-InsP7 and InsP8 increase severalfold in shoots after Pi resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP6 compartmentalization. Notably, ITPK1 is critical for Pi-dependent 5-InsP7 and InsP8 synthesis in planta and its activity regulates Pi starvation responses in a PHR-dependent manner. Furthermore, we demonstrate that ITPK1-mediated conversion of InsP6 to 5-InsP7 requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP7 under low ATP. Collectively, our study provides deeper insights into Pi-dependent changes in nutritional and energetic states with the synthesis of regulatory inositol pyrophosphates.展开更多
(Molecular Plant 14,1864-1880;November 12021)We identified a mistake made when calculating the absolute concentrations of inositol(pyro)phosphates derived from CE-ESl-MS measurements reported in the originally publish...(Molecular Plant 14,1864-1880;November 12021)We identified a mistake made when calculating the absolute concentrations of inositol(pyro)phosphates derived from CE-ESl-MS measurements reported in the originally published version of our manuscript.Due to a calculation error when determining concentrations per fresh biomass,the absolute values shown in the manuscript are approximately 30-fold lower than they should be.This mistake affects Figures 2D,3C,4C,and 7D and Figures S4,S11B,S13,and S15A.Since all conclusions made in the article referred to relative differences between genotypes and treatments,they remained unaffected by these mistakes.展开更多
Inositol pyrophosphates(PP-InsPs)are important signaling molecules that regulate diverse cellular processes in eukaryotes,including energy homeostasis,phosphate(Pi)signaling,and phytohormone perception.Yet,in plants,t...Inositol pyrophosphates(PP-InsPs)are important signaling molecules that regulate diverse cellular processes in eukaryotes,including energy homeostasis,phosphate(Pi)signaling,and phytohormone perception.Yet,in plants,the enzymes responsible for their turnover remain largely unknown.Using a non-hydrolysable PP-InsP analog in a pull-down approach,we identified a family of Arabidopsis NUDIX-type hydrolases(NUDTs)that group into two closely related subclades.Through in vitro assays,heterologous expression systems,and higher order gene-edited mutants,we explored the substrate specificities and physiological roles of these hydrolases.Using a combination of strong anion exchange high-performance liquid chromatography(SAX-HPLC),polyacrylamide gel electrophoresis(PAGE),and capillary electrophoresis electrospray ionization mass spectrometry(CE-ESI-MS),we found that their PP-InsP pyrophosphatase activity is enantiomer selective and Mg^(2+)dependent.Specifically,SubcladeⅠNUDTs preferentially hydrolyze 4-InsP_(7),while SubcladeⅡNUDTs target 3-InsP_(7),with minor activity against other PP-InsPs,including5-InsP_(7).In higher order mutants of SubcladeⅡNUDTs,we observed defects in both Piand iron homeostasis,accompanied by increased levels of 1/3-InsP_(7)and 5-InsP_(7),with a markedly larger increase in 1/3-InsP_(7).Ectopic expression of NUDTs from both subclades induced local Pi starvation responses(PSRs),while RNA-seq analysis comparing wild-type(WT)and SubcladeⅡnudt12/13/16 loss-of-function plants indicates additional PSR-independent roles,potentially involving 1/3-InsP_(7) in the regulation of plant defense.Consistently,nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection,indicating a role in bacterial pathogen susceptibility.Expanding beyond SubcladeⅡNUDTs,we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs,and found that such activities are conserved across plants and humans.Additionally,we observed that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to SubcladeⅠNUDTs.Collectively,our findings reveal new roles for NUDTs in PP-InsP signaling,plant nutrient and immune responses,and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.展开更多
基金This work was funded by grants from the Deutsche Forschungsgemein-schaft(HE 8362/1-1,DFG Eigene Stelle,to R.F.H.G.SCHA 1274/4-1,SCHA 1274/5-1,Research Training Group GRK 2064 and Germany's Excellence Strategy,EXC-2070-390732324,PhenoRob to G.S.+1 种基金JE 572/4-1 and Germany's Excellence Strategy,ClBSS-EXC-2189-Project ID 390939984 to H.J.JLA 4541/1-1 postdoctoral research fellowship to D.L.),grants from the Medical Research Council(MRC award MR/T028904/1 to A.S.),and a DBT-IISc Program to D.L.
文摘In plants, phosphate (Pi) homeostasis is regulated by the interaction of PHR transcription factors with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates. Here, we combined different methods to obtain a comprehensive picture of how inositol (pyro)phosphate metabolism is regulated by Pi and dependent on the inositol phosphate kinase ITPK1. We found that inositol pyrophosphates are more responsive to Pi than lower inositol phosphates, a response conserved across kingdoms. With CE-ESI-MS we could separate different InsP7 isomers in Arabidopsis and rice, and identify 4/6-InsP7 and a PP-InsP4 isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP7, 5-InsP7 and InsP8 increase severalfold in shoots after Pi resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP6 compartmentalization. Notably, ITPK1 is critical for Pi-dependent 5-InsP7 and InsP8 synthesis in planta and its activity regulates Pi starvation responses in a PHR-dependent manner. Furthermore, we demonstrate that ITPK1-mediated conversion of InsP6 to 5-InsP7 requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP7 under low ATP. Collectively, our study provides deeper insights into Pi-dependent changes in nutritional and energetic states with the synthesis of regulatory inositol pyrophosphates.
文摘(Molecular Plant 14,1864-1880;November 12021)We identified a mistake made when calculating the absolute concentrations of inositol(pyro)phosphates derived from CE-ESl-MS measurements reported in the originally published version of our manuscript.Due to a calculation error when determining concentrations per fresh biomass,the absolute values shown in the manuscript are approximately 30-fold lower than they should be.This mistake affects Figures 2D,3C,4C,and 7D and Figures S4,S11B,S13,and S15A.Since all conclusions made in the article referred to relative differences between genotypes and treatments,they remained unaffected by these mistakes.
基金funded by grants from the Deutsche Forschungsgemeinschaft(SCHA 1274/4-1,SCHA 1274/5-1,and under Germany's Excellence Strategy,EXC2070–390732324,Pheno Rob to G.S.,JE 572/4-1 and under Germany's Excellence Strategy,CIBSS–EXC-2189–Project ID 390939984 to H.J.J.,LA 1338/18-1 to T.L.,and TRR356/I(491090170),TP-B08 and Project No.451218338 to M.K.R.-L.)by the Marie Sklodowska-Curie Action(Grant Agreement ID 101108767)to S.W.by the Department of Biotechnology,Government of India(Grant No.BT/PR45561/AGIII/103/1386/2023)to S.B.Open Access funding enabled and organized by Projekt DEAL
文摘Inositol pyrophosphates(PP-InsPs)are important signaling molecules that regulate diverse cellular processes in eukaryotes,including energy homeostasis,phosphate(Pi)signaling,and phytohormone perception.Yet,in plants,the enzymes responsible for their turnover remain largely unknown.Using a non-hydrolysable PP-InsP analog in a pull-down approach,we identified a family of Arabidopsis NUDIX-type hydrolases(NUDTs)that group into two closely related subclades.Through in vitro assays,heterologous expression systems,and higher order gene-edited mutants,we explored the substrate specificities and physiological roles of these hydrolases.Using a combination of strong anion exchange high-performance liquid chromatography(SAX-HPLC),polyacrylamide gel electrophoresis(PAGE),and capillary electrophoresis electrospray ionization mass spectrometry(CE-ESI-MS),we found that their PP-InsP pyrophosphatase activity is enantiomer selective and Mg^(2+)dependent.Specifically,SubcladeⅠNUDTs preferentially hydrolyze 4-InsP_(7),while SubcladeⅡNUDTs target 3-InsP_(7),with minor activity against other PP-InsPs,including5-InsP_(7).In higher order mutants of SubcladeⅡNUDTs,we observed defects in both Piand iron homeostasis,accompanied by increased levels of 1/3-InsP_(7)and 5-InsP_(7),with a markedly larger increase in 1/3-InsP_(7).Ectopic expression of NUDTs from both subclades induced local Pi starvation responses(PSRs),while RNA-seq analysis comparing wild-type(WT)and SubcladeⅡnudt12/13/16 loss-of-function plants indicates additional PSR-independent roles,potentially involving 1/3-InsP_(7) in the regulation of plant defense.Consistently,nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection,indicating a role in bacterial pathogen susceptibility.Expanding beyond SubcladeⅡNUDTs,we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs,and found that such activities are conserved across plants and humans.Additionally,we observed that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to SubcladeⅠNUDTs.Collectively,our findings reveal new roles for NUDTs in PP-InsP signaling,plant nutrient and immune responses,and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.
