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Abiotic stress, such as drought and high salinity, activates a network of signaling cascades that lead to the expression of many stress-responsive genes in plants. The Arabidopsis FIERY1 (FRY1) protein is a negative regulator of stress and abscisic acid (ABA) signaling and exhibits both an inositol polyphosphatase and a 3',5'-bisphosphate nucleotidase activity in vitro. The FRY1 nucleotidase degrades the sulfation byproduct 3'-phosphoadenosine-5'-phosphate (PAP), yet its in vivo functions and particularly its roles in stress gene regulation remain unclear. Here we developed a LC-MS/MS method to quantitatively measure PAP levels in plants and investigated the roles of this nucleotidase activity in stress response and plant development. It was found that PAP level was tightly controlled in plants and did not accumulate to any significant level either under normal conditions or under NaCl, LiCl, cold, or ABA treatments. In contrast, high levels of PAP were detected in multiple mutant alleles of FRY1 but not in mutants of other FRY1 family members, indicating that FRY1 is the major enzyme that hydrolyzes PAP in vivo. By genetically reducing PAP levels in fry1 mutants either through overexpression of a yeast PAP nucleotidase or by generating a triple mutant of fry1 apk1 apk2 that is defective in the biosynthesis of the PAP precursor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), we demonstrated that the developmental defects and superinduction of stress-responsive genes in fry1 mutants correlate with PAP accumulation in planta. We also found that the hypersensitive stress gene regulation in fry1 requires ABH1 but not ABI1, two other negative regulators in ABA signaling pathways. Unlike in yeast, however, FRY1 overexpression in Arabidopsis could not enhance salt tolerance. Taken together, our results demonstrate that PAP is critical for stress gene regulation and plant development, yet the FRY1 nucleotidase that catabolizes PAP may not be an in vivo salt toxicity target in Arabidopsis. 相似文献
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The Arabidopsis FIERY1 (FRY1) locus was originally identified as a negative regulator of stress‐responsive gene expression and later shown to be required for suppression of RNA silencing. In this study we discovered that the FRY1 locus also regulates lateral root formation. Compared with the wild type, fry1 mutant seedlings generated significantly fewer lateral roots under normal growth conditions and also exhibited a dramatically reduced sensitivity to auxin in inducing lateral root initiation. Using transgenic plants that overexpress a yeast homolog of FRY1 that possesses only the 3′, 5′‐bisphosphate nucleotidase activity but not the inositol 1‐phosphatase activity, we demonstrated that the lateral root phenotypes in fry1 result from loss of the nucleotidase activity. Furthermore, a T‐DNA insertion mutant of another RNA silencing suppressor, XRN4 (but not XRN2 or XRN3), which is an exoribonuclease that is inhibited by the substrate of the FRY1 3′, 5′‐bisphosphate nucleotidase, exhibits similar lateral root defects. Although fry1 and xrn4 exhibited reduced sensitivity to ethylene, our experiments demonstrated that restoration of ethylene sensitivity in the fry1 mutant is not sufficient to rescue the lateral root phenotypes of fry1. Our results indicate that RNA silencing modulated by FRY1 and XRN4 plays an important role in shaping root architecture. 相似文献
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The SAL1 gene of Arabidopsis, encoding an enzyme with 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities, increases salt tolerance in yeast. 总被引:3,自引:0,他引:3
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A cDNA library in a yeast expression vector was prepared from roots of Arabidopsis exposed to salt and was used to select Li(+)-tolerant yeast transformants. The cDNA SAL1 isolated from one of these transformants encodes a polypeptide of 353 amino acid residues. This protein is homologous to the HAL2 and CysQ phosphatases of yeast and Escherichia coli, respectively. Partial cDNA sequences in the data bases indicate that rice produces a phosphatase highly homologous to SAL1 and that a second gene homologous to SAL1 exists in Arabidopsis. The SAL1 protein expressed in E. coli showed 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities. In yeast, SAL1 restored the ability of a hal2/met22 mutant to grow on sulfate as a sole sulfur source, increased the intracellular Li+ tolerance, and modified Na+ and Li+ effluxes. We propose that the product of SAL1 participates in the sulfur assimilation pathway as well as in the phosphoinositide signaling pathway and that changes in the latter may affect Na+ and Li+ fluxes. 相似文献
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Arabidopsis FIERY1, XRN2, and XRN3 are endogenous RNA silencing suppressors 总被引:1,自引:0,他引:1
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Gy I Gasciolli V Lauressergues D Morel JB Gombert J Proux F Proux C Vaucheret H Mallory AC 《The Plant cell》2007,19(11):3451-3461
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Tol1, a fission yeast phosphomonoesterase, is an in vivo target of lithium, and its deletion leads to sulfite auxotrophy
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Miyamoto R Sugiura R Kamitani S Yada T Lu Y Sio SO Asakura M Matsuhisa A Shuntoh H Kuno T 《Journal of bacteriology》2000,182(13):3619-3625
Lithium is the drug of choice for the treatment of bipolar affective disorder. The identification of an in vivo target of lithium in fission yeast as a model organism may help in the understanding of lithium therapy. For this purpose, we have isolated genes whose overexpression improved cell growth under high LiCl concentrations. Overexpression of tol1(+), one of the isolated genes, increased the tolerance of wild-type yeast cells for LiCl but not for NaCl. tol1(+) encodes a member of the lithium-sensitive phosphomonoesterase protein family, and it exerts dual enzymatic activities, 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase. tol1(+) gene-disrupted cells required high concentrations of sulfite in the medium for growth. Consistently, sulfite repressed the sulfate assimilation pathway in fission yeast. However, tol1(+) gene-disrupted cells could not fully recover from their growth defect and abnormal morphology even when the medium was supplemented with sulfite, suggesting the possible implication of inositol polyphosphate 1-phosphatase activity for cell growth and morphology. Given the remarkable functional conservation of the lithium-sensitive dual-specificity phosphomonoesterase between fission yeast and higher-eukaryotic cells during evolution, it may represent a likely in vivo target of lithium action across many species. 相似文献
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Molecular genetic analysis of cold-regulated gene transcription 总被引:19,自引:0,他引:19
Viswanathan C Zhu JK 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2002,357(1423):877-886
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Identification and isolation of a 75-kDa inositol polyphosphate-5-phosphatase from human platelets 总被引:6,自引:0,他引:6
We have identified, isolated, and characterized a second inositol polyphosphate-5-phosphatase enzyme from the soluble fraction of human platelets. The enzyme hydrolyzes inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) to inositol 1,4-bisphosphate (Ins(1,4)P2) with an apparent Km of 24 microM and a Vmax of 25 mumol of Ins(1,4,5)P3 hydrolyzed/min/mg of protein. The enzyme hydrolyzes inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) at a rate of 1.3 mumol of Ins(1,3,4,5)P4 hydrolyzed/min/mg of protein with an apparent Km of 7.5 microM. The enzyme also hydrolyzes inositol 1,2-cyclic 4,5-trisphosphate (cIns(1:2,4,5)P3) and Ins(4,5)P2. We purified this enzyme 2,200-fold from human platelets. The enzyme has a molecular mass of 75,000 as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration chromatography. The enzyme requires magnesium ions for activity and is not inhibited by calcium ions. The 75-kDa inositol polyphosphate-5-phosphatase enzyme differs from the previously identified platelet inositol polyphosphate-5-phosphatase as follows: molecular size (75 kDa versus 45 kDa), affinity for Ins(1,3,4,5)P4 (Km 7.5 microM versus 0.5 microM), Km for Ins(1,4,5)P3 (24 microM versus 7.5 microM), regulation by protein kinase C, wherein the 45-kDa enzyme is phosphorylated and activated while the 75-kDa enzyme is not. The 75-kDa enzyme is inhibited by lower concentrations of phosphate (IC50 2 mM versus 16 mM for the 45-kDa enzyme) and is less inhibited by Ins(1,4)P2 than is the 45-kDa enzyme. The levels of inositol phosphates that act in calcium signalling are likely to be regulated by the interplay of these two enzymes both found in the same cell. 相似文献
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A mutation in the Arabidopsis DET3 gene uncouples photoregulated leaf development from gene expression and chloroplast biogenesis 总被引:4,自引:2,他引:2
Héctor L. Cabrera y Poch Charles A. Peto Joanne Chory 《The Plant journal : for cell and molecular biology》1993,4(4):671-682
The genetic and phenotypic characterization of a new Arabidopsis mutant, de-etiolated -3, ( det 3), involved in light-regulated seedling development is described. A recessive mutation in the DET 3 gene uncouples light signals from a subset of light-dependent processes. The det 3 mutation causes dark-grown Arabidopsis thaliana seedlings to have short hypocotyls, expanded cotyledons, and differentiated leaves, traits characteristic of light-grown seedlings. Despite these morphological changes, however, the det 3 mutant does not develop chloroplasts or show elevated expression of nuclear- and chloroplast-encoded light-regulated mRNAs. The det 3 mutation thus uncovers a downstream branch of the light transduction pathways that separates leaf development from chloroplast differentiation and light-regulated gene expression. In addition, light-grown det 3 plants have reduced stature and apical dominance, suggesting that DET3 functions during growth in normal light conditions as well. The genetic interactions between mutations in det 1, det 2, and det 3 are described. The phenotypes of doubly mutant strains suggest that there are at least two parallel pathways controlling light-mediated development in Arabidopsis . 相似文献
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T S Ross A B Jefferson C A Mitchell P W Majerus 《The Journal of biological chemistry》1991,266(30):20283-20289
Inositol polyphosphate-5-phosphatase (5-phosphatase) hydrolyzes inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thereby functions as a signal terminating enzyme in cellular calcium ion mobilization. A cDNA encoding human platelet 5-phosphatase has been isolated by screening for beta-galactosidase fusion proteins that bind to inositol 1,3,4,5-tetrakisphosphate. The sensitivity of the screening procedure was enhanced 50- to 100-fold by amplification of "sublibraries" prior to carrying out binding assays. The sequences derived from the "expression clone" were used to screen human erythroleukemia cell line and human megakaryocytic cell line cDNA libraries. We obtained two additional clones which together consist of 2381 base pairs. The amino-terminal amino acid sequence from the 75-kDa 5-phosphatase purified from platelets is identical to amino acids 38-56 predicted from the cDNA. This suggests that the platelet 5-phosphatase is formed by proteolytic processing of a larger precursor. The cDNA predicts that the mature enzyme contains 635 amino acids (Mr 72, 891). Antibodies directed against recombinant TrpE fusion proteins of either an amino-terminal region or a carboxyl-terminal region immunoprecipitate the enzyme activity from a preparation of the 75-kDa form of platelet 5-phosphatase (Type II) but do not precipitate the distinct 47-kDa 5-phosphatase (Type I) also found in platelets. In addition, the recombinant protein expressed in Cos-7 cells has the same 5-phosphatase activity as the platelet 5-phosphatase. 相似文献
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(2')3',5'-Bisphosphate nucleotidase 总被引:2,自引:0,他引:2
(2')3',5'-Bisphosphate nucleotidase has been prepared in electrophoretically homogeneous form from guinea pig liver. The enzyme catalyzes the hydrolysis of the 2'- or 3'-phosphate from the appropriate nucleoside 2',5'- and 3',5'-bisphosphates and is active with 3'-phosphoadenosine 5'-phosphosulfate and with coenzyme A but not with ATP. The 40,000-dalton protein is a monomer that requires Mg2+ for activity. 相似文献
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Active-Site Mutations in the Xrn1p Exoribonuclease of Saccharomyces cerevisiae Reveal a Specific Role in Meiosis
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Jachen A. Solinger Donatella Pascolini Wolf-Dietrich Heyer 《Molecular and cellular biology》1999,19(9):5930-5942
Xrn1p of Saccharomyces cerevisiae is a major cytoplasmic RNA turnover exonuclease which is evolutionarily conserved from yeasts to mammals. Deletion of the XRN1 gene causes pleiotropic phenotypes, which have been interpreted as indirect consequences of the RNA turnover defect. By sequence comparisons, we have identified three loosely defined, common 5'-3' exonuclease motifs. The significance of motif II has been confirmed by mutant analysis with Xrn1p. The amino acid changes D206A and D208A abolish singly or in combination the exonuclease activity in vivo. These mutations show separation of function. They cause identical phenotypes to that of xrn1Delta in vegetative cells but do not exhibit the severe meiotic arrest and the spore lethality phenotype typical for the deletion. In addition, xrn1-D208A does not cause the severe reduction in meiotic popout recombination in a double mutant with dmc1 as does xrn1Delta. Biochemical analysis of the DNA binding, exonuclease, and homologous pairing activity of purified mutant enzyme demonstrated the specific loss of exonuclease activity. However, the mutant enzyme is competent to promote in vitro assembly of tubulin into microtubules. These results define a separable and specific function of Xrn1p in meiosis which appears unrelated to its RNA turnover function in vegetative cells. 相似文献
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DE LAMIRANDE G ALLARD C CANTERO A 《The Journal of biophysical and biochemical cytology》1958,4(4):373-376
The intracellular distribution of 5' nucleotidase was investigated in rat liver by biochemical analysis of cell fractions obtained by differential centrifugation. The enzymatic activity was measured by determination of the inorganic phosphorus liberated from 5' nucleotides. The 5' nucleotidase activity was mainly found in the nuclear and microsomal fractions. An attempt to extract the enzyme from these fractions with Mg(++) ion solutions was unsuccessful. It is concluded that 5' nucleotidase is actually present in the nuclear and microsomal fractions of rat liver cells. 相似文献