Molluscan smooth muscles, such as the bivalve adductor muscles and the mussel anterior byssus retractor muscles (ABRM), exhibit a unique contraction called “catch”. Catch contraction is regulated through twitchin ph...Molluscan smooth muscles, such as the bivalve adductor muscles and the mussel anterior byssus retractor muscles (ABRM), exhibit a unique contraction called “catch”. Catch contraction is regulated through twitchin phosphorylation and dephosphorylation. Twitchin from the ABRM of the Mediterranean mussel, Mytilus galloprovincialis, is phosphorylated by cAMP-dependent protein kinase (PKA), and PKA phosphorylation sites are located in both the N- and C-terminal regions of the twitchin molecule. The D2 site, which is adjacently located to the C-terminus, participates in forming a myosin, actin, and twitchin complex that is thought to contribute towards the maintenance of tension in the catch state. In contrast, although it has been reported to interact with thin-filaments, the molecular function of the region including the D1 site has remained largely unstudied. Three additional PKA consensus sequences were identified near the D1 site;however, it was not known if these sites could be directly phosphorylated by PKA. Here, we performed phosphorylation assays to identify phosphorylation sites near the D1 site using recombinant protein variants (TWD1-SSSS, TWD1-AAAS, TWD1-AASA, TWD1-ASAA, TWD1-SAAA, and TWD1-AAAA). All variants, except TWD1-AAAA (where all phosphorylatable serine residues were replaced by alanines), were phosphorylated by PKA. The four phosphorylation sites were named D1-1, D1-2, D1-3, and D1-4 (the originally identified D1) in order from the N-terminus. Phosphorylation assays using a 1/12.5 weight ratio of PKA to each TWD1 variant revealed that D1-4 was the most rapidly phosphorylated, closely followed by D1-1. However, D1-2 and D1-3 were phosphorylated at a lower level under equivalent conditions and were not phosphorylated when PKA was incubated with each TWD1 variant at a 1/100 weight ratio. Furthermore, we observed that TWD1-SSSS was phosphorylated in a stepwise fashion. These findings contribute towards the elucidation of the function of the twitchin D1 region in the regulatory system of catch contraction.展开更多
The Saccharomyces cerevisiae polyphosphatase PPN1 (uniprot/Q04119) degrades inorganic polyphosphates both by cleaving Pi from the chain end and by fragmenting long-chain polymers into shorter ones. In this study, we h...The Saccharomyces cerevisiae polyphosphatase PPN1 (uniprot/Q04119) degrades inorganic polyphosphates both by cleaving Pi from the chain end and by fragmenting long-chain polymers into shorter ones. In this study, we have found a new activity of this protein: it releases phosphate from dATP. The dATP phosphohydrolase activity of pure PPN1 was ~7-fold lower compared to the exopolyphosphatase activity. This activity was strongly stimulated by Co<sup>2+</sup> ions, as well as by ammonium ions, and inhibited by heparin and pyrophosphate similar to the exopolyphosphatase activity of PPN1. The Km value for dATP was 0.88 ± 0.14 mM. The dATP phosphohydrolase activity in the cells of PPN1-overexpressing yeast strain was several-fold higher than that in the parent strain. The other exopolyphosphatase of S. cerevisiae, PPX1, did not split Pi from dATP.展开更多
Soil phosphorus(P) fractionation, adsorption, and desorption isotherm, and rice yield and P uptake were investigated in flooded tropical rice(Oryza sativa L.) following 42-year fertilizer and manure application. The t...Soil phosphorus(P) fractionation, adsorption, and desorption isotherm, and rice yield and P uptake were investigated in flooded tropical rice(Oryza sativa L.) following 42-year fertilizer and manure application. The treatments included low-input [unfertilized control without N, P, or K(C0N0)], farmyard manure(FYM)(C1N0), NP(C0NP), NPK(C0NPK), FYM + NP(C1NP), and high-input treatment, FYM + NPK(C1NPK). Grain yield was increased significantly by 74%over the control under the combined application of FYM + NPK. However, under low- and high-input treatments, yield as well as P uptake was maintained at constant levels for 35 years.During the same period, high yield levels and P uptake were maintained under the C0 NP, C0 NPK,and C1 NPK treatments. These are unique characteristics of a tropical flooded ecosystem, which is a self-sustaining system for rice production. The Fe–P fraction was highest compared to the Ca–P and Al–P fractions after 42 years of fertilizer application and was significantly higher under FYM + NPK treatment. The P adsorption capacity of soil was highest under the low-input treatment and lowest under long-term balanced fertilization(FYM + NPK). In contrast, P desorption capacity was highest under NPK and lowest in the control treatment. Long-term balanced fertilization in the form of FYM + NPK for 42 years lowered the bonding energy and adsorption capacity for P in soil but increased its desorption potential, increasing P availability to the plant and leading to higher P uptake and yield maintenance.展开更多
Although the phosphate 1（PHO1）gene family has been implicated in inorganic phosphate transport and homeostasis,the underlying mechanism of this gene in the strawberry has not yet been revealed.In the present study,w...Although the phosphate 1（PHO1）gene family has been implicated in inorganic phosphate transport and homeostasis,the underlying mechanism of this gene in the strawberry has not yet been revealed.In the present study,we analyzed the expression of the PHO1;H9 gene in the strawberry（Fragaria×ananassa）,revealing the involvement of this gene in the regulation of phosphorus（P）content.The coding sequence（CDS）of the PHO1;H9 gene,was isolated from the cultivated strawberry‘Sachinoka’and named as Fa PHO1;H9.The full-length CDS of this gene was 2 292 bp,encoding 763 amino acids,and the protein contained both SYG1/Pho81/XPR1（SPX）and ERD1/XPR1/SYG1（EXS）domains,which were involved in phosphate（Pi）signaling.Real-time reverse transcription-polymerase chain reaction（RT-PCR）data suggested that the level of Fa PHO1;H9 expression was consistent with the P content in different organs,except for the petiole.Particularly,its expression level was also correlated with P content in fruits of different developmental stages.The expression of Fa PHO1;H9 was also consistent with P content in leaves under different concentrations of P fertilizer application.Furthermore,transgenic Arabidopsis lines were generated,and the P content in Arabidopsis plants over-expressing Fa PHO1;H9was significantly higher than that in wild-type plants.Therefore,we proposed that Fa PHO1;H9 functions in P transport.展开更多
The title compound N,N＇-bis（5,5-dimethyl-2-phospha-2-thio-1,3-dioxan-2-yl） ethylene diamine （DPTDEDA, C12H26N2O4P2S2） was synthesized by the reaction of neopentyl glycol, phosphorus thio-chloride and 1,2-ethylene...The title compound N,N＇-bis（5,5-dimethyl-2-phospha-2-thio-1,3-dioxan-2-yl） ethylene diamine （DPTDEDA, C12H26N2O4P2S2） was synthesized by the reaction of neopentyl glycol, phosphorus thio-chloride and 1,2-ethylenediamine, and characterized by elemental analysis, IR and ^1H NMR spectra. Its crystal structure was determined by single-crystal X-ray diffraction analysis and the thermal property was analyzed by TG analysis. The crystal structure belongs to monoclinic, space group P21/c, with a = 14.557（16）, b = 11.299（12）, c = 12.163（13）A,β = 98.707（19）^o, Dc = 1.305 g/cm^3, Z = 4, γ = 0.71073A,μ（MoKa） = 0.447 mm^-1, Mr = 388.41, V = 1977（4）A3, F（000） = 824, S = 1.107, the final R = 0.0478 and wR = 0.0810 for 1738 observed reflections （I 〉 2σ（I））. X-ray analysis reveals that the crystal structure is centrosymmetrically distributed through 1,2-ethylenediamine to join two distorted six-membered rings. The weak N-H…S interactions are observed and link the molecules into sheets. TG analysis shows that the title compound has good thermal stability and char-forming capability, which are required for an excellent intumescent fire retardant.展开更多
Bisphenol A bis（diphenyl phosphate） oligomer（BDP） is prepared successfully from the reactants consisting of phosphorus oxyehloride （POCl3）, bisphenol A and phenol with a Friedel-Crafts catalyst. The resultant pr...Bisphenol A bis（diphenyl phosphate） oligomer（BDP） is prepared successfully from the reactants consisting of phosphorus oxyehloride （POCl3）, bisphenol A and phenol with a Friedel-Crafts catalyst. The resultant products were examined with thermtygravimetrie analysis （TGA） and high performance liquid chro- matography（HPLC）. Thermogravimetry data shows that BDP decomposes at 375℃ when 5 % weight lost. Experiments results show that catalyst is preferably AICI3 and the amount of it is preferably 1% relative to bisphenol A by mole. POCl3/bisphenol A mole ratio is preferably about 5：1 to 6：1. Experiments unclosed that a seal apparatus is very important to the properties of product.展开更多
Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Here we introduce a novel FRET-based fluorescence quenching system for assa...Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Here we introduce a novel FRET-based fluorescence quenching system for assaying the activity of alkaline phosphatase (AP) by using a phos-phate-binding tag molecule, Phos-tag {1,3-bis[bis(pyridine-2-ylmethyl)amino]propan-2-olato dizinc(II) complex}, attached to a nonfluorescent 4-{[4-(dimethylamino)phenyl]diazenyl}benzoyl (Dabcyl: λmax 475 nm) dye group. The fluorogenic biomolecule riboflavin 5’-phosphate (FMN: λem 525 nm) was used as an AP substrate. The Dabcyl-labeled Phos-tag specifically captured FMN to form a stable 1:1 complex, resulting in efficient fluorescence quenching. The quenching efficiency was more than 95% for a mixture of 12 μM FMN and 13.5 μM Dabcyl-labeled Phos-tag in aqueous solution at pH 7.4 and 25°C. When FMN was dephosphorylated with AP, riboflavin was released into the solution and fluorescence from the flavin moiety appeared. By using this quenching system, we succeeded in detecting time- and dose-dependent dephosphorylation of FMN by AP under near-physiological conditions.展开更多
Phosphorylation of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-rel...Phosphorylation of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-related protein kinase in Arabidopsis, WD-repeat protein (WDRP) might be CRK5-interact-protein based on Y2H results. Here, we used bimolecular fluorescence complementation (BiFC) further to study and visualize the interaction between CRK5 and WDRP in living cells. Then, we combined Phos-tagTM SDS-PAGE with western blot (WB) analysis, using WDRP antibody and the anti-6×His antibody, to detect phosphorylated WDRP. This approach confirmed that WDRP might be phosphorylated by CRK5 in vitro. Site mutation analysis suggested that serine-70 might be the amino acid phosphorylated by CRK5 in WDRP. Cell extracts isolated from WT, OERK5, and crk5 used to analyze the kinase reaction using recombinant WDRP as substrate. These results demonstrated that WDRP was phosphorylated by cell extracts and that there may be additional kinases that phosphorylate WDRP in Arabidopsis. Phos-tagTM SDS-PAGE thus provides a suitable and convenient method for analysis of phosphorylation in plants.展开更多
OsPho1 in Zhonghua 11(ZH11)was edited using the clustered regularly interspaced short palindromic repeatsassociated endonuclease 9(CRISPR/Cas9)system.Two homozygous T1 mutants(cr-pho1-34 and cr-pho1-37)displayed a cha...OsPho1 in Zhonghua 11(ZH11)was edited using the clustered regularly interspaced short palindromic repeatsassociated endonuclease 9(CRISPR/Cas9)system.Two homozygous T1 mutants(cr-pho1-34 and cr-pho1-37)displayed a chalky endosperm with a white core,which significantly decreased 1000-grain weight.In addition,many rounded starch granules and abnormal amyloplasts were present in the central region of mutant endosperm cells with increased amylose and lipid contents,decreased total protein content,and altered physicochemical properties of starch.The OsPho1 protein is localized in chloroplasts,and quantitative real-time PCR(qRT-PCR)andβ-glucuronidase(GUS)staining indicated that OsPho1 was highly expressed in seeds at 5 d after fertilization(DAF).OsPho1 mutations displayed close relationships with plastidial phosphoglucomutase and ADPGlc pyrophosphorylase based onα-D-glucose-1P at different temperatures.Moreover,the expressions of starch metabolismrelated genes were also altered in the mutant,and the overexpression of OsPho1 may cause grain chalkiness.展开更多
文摘Molluscan smooth muscles, such as the bivalve adductor muscles and the mussel anterior byssus retractor muscles (ABRM), exhibit a unique contraction called “catch”. Catch contraction is regulated through twitchin phosphorylation and dephosphorylation. Twitchin from the ABRM of the Mediterranean mussel, Mytilus galloprovincialis, is phosphorylated by cAMP-dependent protein kinase (PKA), and PKA phosphorylation sites are located in both the N- and C-terminal regions of the twitchin molecule. The D2 site, which is adjacently located to the C-terminus, participates in forming a myosin, actin, and twitchin complex that is thought to contribute towards the maintenance of tension in the catch state. In contrast, although it has been reported to interact with thin-filaments, the molecular function of the region including the D1 site has remained largely unstudied. Three additional PKA consensus sequences were identified near the D1 site;however, it was not known if these sites could be directly phosphorylated by PKA. Here, we performed phosphorylation assays to identify phosphorylation sites near the D1 site using recombinant protein variants (TWD1-SSSS, TWD1-AAAS, TWD1-AASA, TWD1-ASAA, TWD1-SAAA, and TWD1-AAAA). All variants, except TWD1-AAAA (where all phosphorylatable serine residues were replaced by alanines), were phosphorylated by PKA. The four phosphorylation sites were named D1-1, D1-2, D1-3, and D1-4 (the originally identified D1) in order from the N-terminus. Phosphorylation assays using a 1/12.5 weight ratio of PKA to each TWD1 variant revealed that D1-4 was the most rapidly phosphorylated, closely followed by D1-1. However, D1-2 and D1-3 were phosphorylated at a lower level under equivalent conditions and were not phosphorylated when PKA was incubated with each TWD1 variant at a 1/100 weight ratio. Furthermore, we observed that TWD1-SSSS was phosphorylated in a stepwise fashion. These findings contribute towards the elucidation of the function of the twitchin D1 region in the regulatory system of catch contraction.
文摘The Saccharomyces cerevisiae polyphosphatase PPN1 (uniprot/Q04119) degrades inorganic polyphosphates both by cleaving Pi from the chain end and by fragmenting long-chain polymers into shorter ones. In this study, we have found a new activity of this protein: it releases phosphate from dATP. The dATP phosphohydrolase activity of pure PPN1 was ~7-fold lower compared to the exopolyphosphatase activity. This activity was strongly stimulated by Co<sup>2+</sup> ions, as well as by ammonium ions, and inhibited by heparin and pyrophosphate similar to the exopolyphosphatase activity of PPN1. The Km value for dATP was 0.88 ± 0.14 mM. The dATP phosphohydrolase activity in the cells of PPN1-overexpressing yeast strain was several-fold higher than that in the parent strain. The other exopolyphosphatase of S. cerevisiae, PPX1, did not split Pi from dATP.
文摘Soil phosphorus(P) fractionation, adsorption, and desorption isotherm, and rice yield and P uptake were investigated in flooded tropical rice(Oryza sativa L.) following 42-year fertilizer and manure application. The treatments included low-input [unfertilized control without N, P, or K(C0N0)], farmyard manure(FYM)(C1N0), NP(C0NP), NPK(C0NPK), FYM + NP(C1NP), and high-input treatment, FYM + NPK(C1NPK). Grain yield was increased significantly by 74%over the control under the combined application of FYM + NPK. However, under low- and high-input treatments, yield as well as P uptake was maintained at constant levels for 35 years.During the same period, high yield levels and P uptake were maintained under the C0 NP, C0 NPK,and C1 NPK treatments. These are unique characteristics of a tropical flooded ecosystem, which is a self-sustaining system for rice production. The Fe–P fraction was highest compared to the Ca–P and Al–P fractions after 42 years of fertilizer application and was significantly higher under FYM + NPK treatment. The P adsorption capacity of soil was highest under the low-input treatment and lowest under long-term balanced fertilization(FYM + NPK). In contrast, P desorption capacity was highest under NPK and lowest in the control treatment. Long-term balanced fertilization in the form of FYM + NPK for 42 years lowered the bonding energy and adsorption capacity for P in soil but increased its desorption potential, increasing P availability to the plant and leading to higher P uptake and yield maintenance.
基金financially supported by the National Natural Science Foundation of China (31372037)the Program for Excellent Talents in University of Liaoning Province, China (LJQ2014069)
文摘Although the phosphate 1（PHO1）gene family has been implicated in inorganic phosphate transport and homeostasis,the underlying mechanism of this gene in the strawberry has not yet been revealed.In the present study,we analyzed the expression of the PHO1;H9 gene in the strawberry（Fragaria×ananassa）,revealing the involvement of this gene in the regulation of phosphorus（P）content.The coding sequence（CDS）of the PHO1;H9 gene,was isolated from the cultivated strawberry‘Sachinoka’and named as Fa PHO1;H9.The full-length CDS of this gene was 2 292 bp,encoding 763 amino acids,and the protein contained both SYG1/Pho81/XPR1（SPX）and ERD1/XPR1/SYG1（EXS）domains,which were involved in phosphate（Pi）signaling.Real-time reverse transcription-polymerase chain reaction（RT-PCR）data suggested that the level of Fa PHO1;H9 expression was consistent with the P content in different organs,except for the petiole.Particularly,its expression level was also correlated with P content in fruits of different developmental stages.The expression of Fa PHO1;H9 was also consistent with P content in leaves under different concentrations of P fertilizer application.Furthermore,transgenic Arabidopsis lines were generated,and the P content in Arabidopsis plants over-expressing Fa PHO1;H9was significantly higher than that in wild-type plants.Therefore,we proposed that Fa PHO1;H9 functions in P transport.
基金This work was supported by the China Petroleum & Chemical Science and Technology Foundation (No. 205026) the Tianjin Science and Technology Plan Foundation (No. 06TXTJJC14400)
文摘The title compound N,N＇-bis（5,5-dimethyl-2-phospha-2-thio-1,3-dioxan-2-yl） ethylene diamine （DPTDEDA, C12H26N2O4P2S2） was synthesized by the reaction of neopentyl glycol, phosphorus thio-chloride and 1,2-ethylenediamine, and characterized by elemental analysis, IR and ^1H NMR spectra. Its crystal structure was determined by single-crystal X-ray diffraction analysis and the thermal property was analyzed by TG analysis. The crystal structure belongs to monoclinic, space group P21/c, with a = 14.557（16）, b = 11.299（12）, c = 12.163（13）A,β = 98.707（19）^o, Dc = 1.305 g/cm^3, Z = 4, γ = 0.71073A,μ（MoKa） = 0.447 mm^-1, Mr = 388.41, V = 1977（4）A3, F（000） = 824, S = 1.107, the final R = 0.0478 and wR = 0.0810 for 1738 observed reflections （I 〉 2σ（I））. X-ray analysis reveals that the crystal structure is centrosymmetrically distributed through 1,2-ethylenediamine to join two distorted six-membered rings. The weak N-H…S interactions are observed and link the molecules into sheets. TG analysis shows that the title compound has good thermal stability and char-forming capability, which are required for an excellent intumescent fire retardant.
文摘Bisphenol A bis（diphenyl phosphate） oligomer（BDP） is prepared successfully from the reactants consisting of phosphorus oxyehloride （POCl3）, bisphenol A and phenol with a Friedel-Crafts catalyst. The resultant products were examined with thermtygravimetrie analysis （TGA） and high performance liquid chro- matography（HPLC）. Thermogravimetry data shows that BDP decomposes at 375℃ when 5 % weight lost. Experiments results show that catalyst is preferably AICI3 and the amount of it is preferably 1% relative to bisphenol A by mole. POCl3/bisphenol A mole ratio is preferably about 5：1 to 6：1. Experiments unclosed that a seal apparatus is very important to the properties of product.
文摘Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Here we introduce a novel FRET-based fluorescence quenching system for assaying the activity of alkaline phosphatase (AP) by using a phos-phate-binding tag molecule, Phos-tag {1,3-bis[bis(pyridine-2-ylmethyl)amino]propan-2-olato dizinc(II) complex}, attached to a nonfluorescent 4-{[4-(dimethylamino)phenyl]diazenyl}benzoyl (Dabcyl: λmax 475 nm) dye group. The fluorogenic biomolecule riboflavin 5’-phosphate (FMN: λem 525 nm) was used as an AP substrate. The Dabcyl-labeled Phos-tag specifically captured FMN to form a stable 1:1 complex, resulting in efficient fluorescence quenching. The quenching efficiency was more than 95% for a mixture of 12 μM FMN and 13.5 μM Dabcyl-labeled Phos-tag in aqueous solution at pH 7.4 and 25°C. When FMN was dephosphorylated with AP, riboflavin was released into the solution and fluorescence from the flavin moiety appeared. By using this quenching system, we succeeded in detecting time- and dose-dependent dephosphorylation of FMN by AP under near-physiological conditions.
文摘Phosphorylation of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-related protein kinase in Arabidopsis, WD-repeat protein (WDRP) might be CRK5-interact-protein based on Y2H results. Here, we used bimolecular fluorescence complementation (BiFC) further to study and visualize the interaction between CRK5 and WDRP in living cells. Then, we combined Phos-tagTM SDS-PAGE with western blot (WB) analysis, using WDRP antibody and the anti-6×His antibody, to detect phosphorylated WDRP. This approach confirmed that WDRP might be phosphorylated by CRK5 in vitro. Site mutation analysis suggested that serine-70 might be the amino acid phosphorylated by CRK5 in WDRP. Cell extracts isolated from WT, OERK5, and crk5 used to analyze the kinase reaction using recombinant WDRP as substrate. These results demonstrated that WDRP was phosphorylated by cell extracts and that there may be additional kinases that phosphorylate WDRP in Arabidopsis. Phos-tagTM SDS-PAGE thus provides a suitable and convenient method for analysis of phosphorylation in plants.
基金supported by the National Science Foundation of China(Grant No.3197150429).
文摘OsPho1 in Zhonghua 11(ZH11)was edited using the clustered regularly interspaced short palindromic repeatsassociated endonuclease 9(CRISPR/Cas9)system.Two homozygous T1 mutants(cr-pho1-34 and cr-pho1-37)displayed a chalky endosperm with a white core,which significantly decreased 1000-grain weight.In addition,many rounded starch granules and abnormal amyloplasts were present in the central region of mutant endosperm cells with increased amylose and lipid contents,decreased total protein content,and altered physicochemical properties of starch.The OsPho1 protein is localized in chloroplasts,and quantitative real-time PCR(qRT-PCR)andβ-glucuronidase(GUS)staining indicated that OsPho1 was highly expressed in seeds at 5 d after fertilization(DAF).OsPho1 mutations displayed close relationships with plastidial phosphoglucomutase and ADPGlc pyrophosphorylase based onα-D-glucose-1P at different temperatures.Moreover,the expressions of starch metabolismrelated genes were also altered in the mutant,and the overexpression of OsPho1 may cause grain chalkiness.
