Manganese(Mn)is an essential micronutrient for all living organisms.However,excess Mn supply that can occur in acid or waterlogged soils has toxic effects on plant physiology and development.Although a variety of Mn t...Manganese(Mn)is an essential micronutrient for all living organisms.However,excess Mn supply that can occur in acid or waterlogged soils has toxic effects on plant physiology and development.Although a variety of Mn transporter families have been characterized,we have only a rudimentary understanding of how these transporters are regulated to uphold and adjust Mn homeostasis in plants.Here,we demonstrate that two calcineurin-B-like proteins,CBL2/3,and their interacting kinases,CIPK3/9/26,are key regulators of plant Mn homeostasis.Arabidopsis mutants lacking CBL2 and 3 or their interacting protein kinases CIPK3/9/26 exhibit remarkably high Mn tolerance.Intriguingly,CIPK3/9/26 interact with and phosphorylate the tonoplast-localized Mn and iron(Fe)transporter MTP8 primarily at Ser35,which is conserved among MTP8 proteins from various species.Mn transport complementation assays in yeast combined with multiple physiological assays indicate that CBL-CIPK-mediated phosphorylation of MTP8 negatively regulates its transport activity from the cytoplasm to the vacuole.Moreover,we show that sequential phosphorylation of MTP8,initially at Ser31/32 by the calcium-dependent protein kinase CPK5 and subsequently at Ser35 by CIPK26,provides an activation/deactivation fine-tuning mechanism for differential regulation of Mn transport.Collectively,our findings define a two-tiered calcium-controlled mechanism for dynamic regulation of Mn homeostasis under conditions of fluctuating Mn supply.展开更多
Manganese(Mn)is an essential micronutrient in plants.However,excessive Mn absorption in acidic and waterlogged soils can lead to Mn toxicity.Despite their essential roles in Mn homeostasis,transcriptional and post-tra...Manganese(Mn)is an essential micronutrient in plants.However,excessive Mn absorption in acidic and waterlogged soils can lead to Mn toxicity.Despite their essential roles in Mn homeostasis,transcriptional and post-transcriptional modifications of Mn transporters remain poorly understood.Here,we demonstrated that high-Mn stress induces an obvious Ca^(2+) signature in Arabidopsis.We identified four calcium-dependent protein kinases,CPK4/5/6/11,that interact with the tonoplast-localized Mn and iron(Fe)transporter MTP8 in vitro and in vivo.The cpk4/5/6/11 quadruple mutant displayed a dramatic high-Mn-sensitive phenotype similar to that of the mtp8 mutant.CPKs phosphorylated the N-terminal domain of MTP8 primarily at the Ser31 and Ser32 residues.Transport assays combined with multiple physiological experiments on phospho-dead variant MTP8^(S31/32A)and phospho-mimetic variant MTP8^(S31/32D)plants under different Mn and Fe conditions suggested that Ser31 and Ser32 are crucial for MTP8 function.In addition,genetic analysis showed that CPKs functioned upstream of MTP8.In summary,we identified a tonoplast-associated calcium signaling cascade that orchestrates Mn homeostasis and links Mn toxicity,Ca^(2+) signaling,and Mn transporters.These findings provide new insight into Mn homeostasis mechanisms and Ca^(2+) signaling pathways in plants,providing potential targets for engineering heavy metal toxicity-tolerant plants.展开更多
基金This research was funded by a grant from the National Natural Science Foundation of China(31770289 to C.W.)Northwest A&F University(Z111021604 to C.W.)+3 种基金the National Natural Science Foundation of China(31900236 to Z.Z.)supported in part by the open funds of China Postdoctoral Science Foundation(2018M643740 to Z.Z.)the Natural Science Basic Research Plan in Shaanxi Province of China(program no.2019JQ-150)the State Key Laboratory of Plant Physiology and Biochemistry(SKLPPBKF2101 to C.W.).
文摘Manganese(Mn)is an essential micronutrient for all living organisms.However,excess Mn supply that can occur in acid or waterlogged soils has toxic effects on plant physiology and development.Although a variety of Mn transporter families have been characterized,we have only a rudimentary understanding of how these transporters are regulated to uphold and adjust Mn homeostasis in plants.Here,we demonstrate that two calcineurin-B-like proteins,CBL2/3,and their interacting kinases,CIPK3/9/26,are key regulators of plant Mn homeostasis.Arabidopsis mutants lacking CBL2 and 3 or their interacting protein kinases CIPK3/9/26 exhibit remarkably high Mn tolerance.Intriguingly,CIPK3/9/26 interact with and phosphorylate the tonoplast-localized Mn and iron(Fe)transporter MTP8 primarily at Ser35,which is conserved among MTP8 proteins from various species.Mn transport complementation assays in yeast combined with multiple physiological assays indicate that CBL-CIPK-mediated phosphorylation of MTP8 negatively regulates its transport activity from the cytoplasm to the vacuole.Moreover,we show that sequential phosphorylation of MTP8,initially at Ser31/32 by the calcium-dependent protein kinase CPK5 and subsequently at Ser35 by CIPK26,provides an activation/deactivation fine-tuning mechanism for differential regulation of Mn transport.Collectively,our findings define a two-tiered calcium-controlled mechanism for dynamic regulation of Mn homeostasis under conditions of fluctuating Mn supply.
基金funded by grants from the National Natural Science Foundation of China(31770289 to C.W.)Northwest A&F University(Z111021604 to C.W.)+1 种基金the China Postdoctoral Science Foundation(Z109021802 to Z.Z.)partially supported by the open funds of the State Key Laboratory of Plant Physiology and Biochemistry'(SKLPPBKF1901 to Z.Z.).
文摘Manganese(Mn)is an essential micronutrient in plants.However,excessive Mn absorption in acidic and waterlogged soils can lead to Mn toxicity.Despite their essential roles in Mn homeostasis,transcriptional and post-transcriptional modifications of Mn transporters remain poorly understood.Here,we demonstrated that high-Mn stress induces an obvious Ca^(2+) signature in Arabidopsis.We identified four calcium-dependent protein kinases,CPK4/5/6/11,that interact with the tonoplast-localized Mn and iron(Fe)transporter MTP8 in vitro and in vivo.The cpk4/5/6/11 quadruple mutant displayed a dramatic high-Mn-sensitive phenotype similar to that of the mtp8 mutant.CPKs phosphorylated the N-terminal domain of MTP8 primarily at the Ser31 and Ser32 residues.Transport assays combined with multiple physiological experiments on phospho-dead variant MTP8^(S31/32A)and phospho-mimetic variant MTP8^(S31/32D)plants under different Mn and Fe conditions suggested that Ser31 and Ser32 are crucial for MTP8 function.In addition,genetic analysis showed that CPKs functioned upstream of MTP8.In summary,we identified a tonoplast-associated calcium signaling cascade that orchestrates Mn homeostasis and links Mn toxicity,Ca^(2+) signaling,and Mn transporters.These findings provide new insight into Mn homeostasis mechanisms and Ca^(2+) signaling pathways in plants,providing potential targets for engineering heavy metal toxicity-tolerant plants.
