RESUMO
This study aimed to investigate for the first time, the profile of Physarum microplasmodial phosphatase (PPH) activity toward the phosphorylated light chain of Physarum myosin II (PLCM) at pH 7.6, the velocity of cytoplasmic streaming, and PPH expression in spherule formation during dark starvation (DS). In this study, we cloned the full-length cDNA of PPH using polymerase chain reaction, based on the N-terminal amino acid sequence of the purified enzyme. The cDNA contained an open reading frame (ORF) of 1245 bp, corresponding to 415 amino acids. We confirmed that a rapid increase in PPH activity toward PLCM and a rapid decrease in cytoplasmic streaming velocity precede spherule formation by Physarum microplasmodia. The profiles of increase in PPH activity toward PLCM, PPH expression, and PPH accumulation during DS were correlated with spherule formation in the Physarum microplasmodia. Moreover, application of the wheat germ cell-free expression system resulted in the successful production of recombinant PPH and in the expression of phosphatase activity toward PLCM. These results suggest that PPH is involved in the cessation of cytoplasmic streaming in Physarum microplasmodia during DS.
Assuntos
Corrente Citoplasmática/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Physarum/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Miosina Tipo II/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Physarum plasmodium lives as a slimy mass of protoplast in the dark fragments into small multinucleated microplasmodia (mPL) in a liquid medium. When mPL are exposed to several unfavorable environments, they transform into "spherules" with a cell wall. Using a synchronous spherule-induction system for mPL, we examined the effect of 2,6-dichlorobenzonitrile on the synthesis of cellulose in mPL, by observing mPL under a fluorescence microscope, and isolated cellulose from mPL to identify them morphologically under scanning electron microscopy. Moreover, we examined in vivo labeling to determine when cellulose synthesis is activated in step 2. We found that the nourishment medium in step 2 was essential for mPL prior to spherulation and that the conversion starts at 48 h in step 2 of our system. From the experiments using Updegraff reagent for the sedimentation of cellulose in the cell wall fraction from mPL, we propose that cellulose produced in mPL is likely noncrystalline cellulose. We conclude that mPL of multinucleated protoplasts without the cell wall structure synthesize cellulose under constitutive condition and accumulate abundantly noncrystalline cellulose, in preparation for unfavorable environments that may occur in the future in which mPL must initiate the program to form the cell wall of spherules.
Assuntos
Celulose/metabolismo , Physarum/metabolismo , Animais , Parede Celular/metabolismo , Physarum/citologiaRESUMO
The incorporation of xyloglucan oligosaccharide (XXXG) into the walls of suspension-cultured tobacco cells accelerated cell expansion followed by cell division, changed cell shape from cylindrical to spherical, decreased cell size, and caused cell aggregation. Fluorescent XXXG added to the culture medium was found to be incorporated into the surface of the entire wall, where strong incorporation occurred not only on the surface, but also in the interface walls between cells during cell division. Cell expansion was always greater in the transverse direction than in the longitudinal direction and then, immediately, expansion led to cell division in the presence of XXXG; this process might result in the high level of cell aggregation seen in cultured tobacco cells. We concluded that the integration of this oligosaccharide into the walls could accelerate not only cell expansion, but also cell division in cultured cells.
Assuntos
Glucanos/farmacologia , Nicotiana/citologia , Oligossacarídeos/farmacologia , Xilanos/farmacologia , Divisão Celular/efeitos dos fármacos , Crescimento Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia em Gel , Nicotiana/efeitos dos fármacosRESUMO
It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as alpha-xylosidase and beta-glucosidase. The dephosphorylation and phosphorylation of recombinant alpha-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of alpha-xylosidase and beta-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls.
Assuntos
Fosfatase Ácida/metabolismo , Parede Celular/metabolismo , Glicoproteínas/metabolismo , Nicotiana/enzimologia , Proteínas de Plantas/metabolismo , Células Cultivadas , Glucanos/metabolismo , Dados de Sequência Molecular , Fosforilação , Proteoma/metabolismo , Xilanos/metabolismo , Xilosidases/metabolismo , beta-Glucosidase/metabolismoRESUMO
A phosphatase was purified through a combination of ion-exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340-fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The Km of the enzyme for PMLC was 10 microM, and the V(max) was 1.17 nkat/mg of protein. Ca(2+) (10 microM) inhibited the activity of the enzyme, and Mg(2+) (8.5 microM) activated the dephosphorylation of PMLC. Mn(2+) (1.6 microM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.
Assuntos
Miosina Tipo II/metabolismo , Monoéster Fosfórico Hidrolases/química , Physarum polycephalum/enzimologia , Proteínas de Protozoários/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Monoéster Fosfórico Hidrolases/isolamento & purificação , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Especificidade por SubstratoRESUMO
Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The aim of this work was to further investigate the function of one of these phosphatases in tobacco (Nicotiana tabacum), NtPAP12, using transgenic cells overexpressing the enzyme. The transgenic cells exhibited a higher level of phosphatase activity in their walls. The corresponding protoplasts regenerating a cell wall exhibited a higher rate of beta-glucan synthesis and cellulose deposition was increased in the walls of the transgenic cells. A higher level of plasma membrane glucan synthase activities was also measured in detergent extracts of membrane fractions from the transgenic line, while no activation of Golgi-bound glycan synthases was detected. Enzymatic hydrolysis and methylation analysis were performed on the products synthesized in vitro by the plasma membrane enzymes from the wild-type and transgenic lines extracted with digitonin and incubated with radioactive UDP-glucose. The data showed that the glucans consisted of callose and cellulose and that the amount of each glucan synthesized by the enzyme preparation from the transgenic cells was significantly higher than in the case of the wild-type cells. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis.
Assuntos
Fosfatase Ácida/metabolismo , Parede Celular/enzimologia , Glucosiltransferases/metabolismo , Glicoproteínas/metabolismo , Nicotiana/citologia , Nicotiana/enzimologia , Membrana Celular/enzimologia , Ativação Enzimática , Espaço Extracelular/metabolismo , Glucanos/metabolismo , Complexo de Golgi/enzimologia , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Polissacarídeos/metabolismo , Ligação Proteica , Transporte Proteico , Protoplastos/enzimologia , Regeneração , Fatores de Tempo , Nicotiana/genéticaRESUMO
Purple acid phosphatase isolated from the walls of tobacco cells appears to be a 220kDa homotetramer composed of 60kDa subunits, which is purple in color and which contains iron as its only metal ion. Although the phosphatase did not require dithiothreitol for activity and was not inhibited by phenylarsine oxide, the enzyme showed a higher catalytic efficiency (k(cat)/K(m)) for phosphotyrosine-containing peptides than for other substrates including p-nitrophenyl-phosphate and ATP. The phosphatase formed as a 120kDa dimer in the cytoplasm and as a 220kDa tetramer in the walls, where Brefeldin A blocked its secretion during wall regeneration. According to our double-immunofluorescence labeling results, the enzyme might be translocated through the Golgi apparatus to the walls at the interphase and to the cell plate during cytokinesis.
Assuntos
Fosfatase Ácida/isolamento & purificação , Fosfatase Ácida/metabolismo , Parede Celular/metabolismo , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Nicotiana/enzimologia , Proteínas de Plantas/metabolismo , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ferro/metabolismo , Peso Molecular , Proteínas de Plantas/isolamento & purificação , Especificidade por SubstratoRESUMO
Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.
Assuntos
Fosfatase Ácida/genética , Parede Celular/enzimologia , Genes de Plantas , Glicoproteínas/genética , Nicotiana/genética , Fosfatase Ácida/química , Sequência de Aminoácidos , Células Cultivadas , DNA Complementar/isolamento & purificação , Regulação da Expressão Gênica de Plantas , Glicoproteínas/química , Isoenzimas/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Filogenia , Protoplastos/metabolismo , RNA Mensageiro/análise , Alinhamento de Sequência , Fatores de TempoRESUMO
The behavior of phosphatase isoforms under dark-starvation from plasmodium of Physarum polycephalum were investigated to determine their possible roles in sclerotium formation. Two and a half days after dark-starvation, approximately 95% of plasmodia plates formed sclerotia. Specific phosphatase activity increased markedly up to ca. two-fold within the first day of starvation, after which the enzymatic activity decreased rapidly to a level less than the initial level within 2 days of the starvation period. Among the two isoforms of enzyme detected just before sclerotization under dark-starvation conditions, the enzymatic activity of the major isoform (Rm value of 0.6) decreased gradually within 1.5 days of starvation, then linearly to less than 20% of that at the beginning of the observation. Those of other major isoform (Rm value of 0.7) increased up to ca. two-fold within the first day of starvation, then decreased linearly to levels less than that of the first 2 days of the starvation period. Behavior of this isoform strongly suggests that it initiates the formation of sclerotium under dark-starvation conditions.