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.
