RESUMEN

The internal conductance for CO(2) diffusion (g(i)) and CO(2) assimilation rate were measured and the related anatomical characteristics were investigated in transgenic rice leaves that overexpressed barley aquaporin HvPIP2;1. This study was performed to test the hypothesis that aquaporin facilitates CO(2) diffusion within leaves. The g(i) value was estimated for intact leaves by concurrent measurements of gas exchange and carbon isotope ratio. The leaves of the transgenic rice plants that expressed the highest levels of Aq-anti-HvPIP2;1 showed a 40% increase in g(i) as compared to g(i) in the leaves of wild-type rice plants. The increase in g(i) was accompanied by a 14% increase in CO(2) assimilation rate and a 27% increase in stomatal conductance (g(s)). The transgenic plants that had low levels of Aq-anti-HvPIP2;1 showed decreases in g(i) and CO(2) assimilation rate. In the plants with high levels of Aq-anti-HvPIP2;1, mesophyll cell size decreased and the cell walls of the epidermis and mesophyll cells thickened, indicating that the leaves had become xeromorphic. Although such anatomical changes could partially offset the increase in g(i) by the aquaporin, the increase in aquaporin content overcame such adverse effects.

Asunto(s)

Acuaporinas/metabolismo , Dióxido de Carbono/metabolismo , Hordeum/metabolismo , Oryza/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Acuaporinas/genética , Respiración de la Célula/genética , Pared Celular/genética , Pared Celular/metabolismo , Pared Celular/ultraestructura , Cloroplastos/genética , Cloroplastos/metabolismo , Difusión , Regulación de la Expresión Génica de las Plantas/genética , Hordeum/genética , Oryza/citología , Oryza/genética , Fotosíntesis/genética , Epidermis de la Planta/citología , Epidermis de la Planta/genética , Epidermis de la Planta/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Regulación hacia Arriba/genética

RESUMEN

A novel SmtB/ArsR family metalloregulator, denoted BxmR, has been identified and characterized from the cyanobacterium Oscillatoria brevis. Genetic and biochemical evidence reveals that BxmR represses the expression of both bxa1, encoding a CPx-ATPase metal transporter, as well as a divergently transcribed operon encoding bxmR and bmtA, a heavy metal sequestering metallothionein. Derepression of the expression of all three genes is mediated by both monovalent (Ag(I) and Cu(I)) and divalent (Zn(II) and Cd(II)) heavy metal ions, a novel property among SmtB/ArsR metal sensors. Electrophoretic gel mobility shift experiments reveal that apoBxmR forms multiple resolvable complexes with oligonucleotides containing a single 12-2-12 inverted repeat derived from one of the two operator/promoter regions with similar apparent affinities. Preincubation with either monovalent or divalent metal ions induces disassembly of both the BxmR-bxa1 and BxmR-bxmR/bmtA operator/promoter complexes. Interestingly, the temporal regulation of expression of bxa1 and bmtA mRNAs is different in O. brevis with bxa1 induced first upon heavy metal treatment, followed by bmtA/bxmR. A dynamic interplay among Bxa1, BmtA, and BxmR is proposed that maintains metal homeostasis in O. brevis by balancing the relative rates of metal storage and efflux of multiple heavy metal ions.

Asunto(s)

Adenosina Trifosfatasas/química , Proteínas Bacterianas , Cadmio/química , Cobre/química , Cianobacterias/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Proteínas Represoras/metabolismo , Proteínas Represoras/fisiología , Plata/química , Transactivadores/metabolismo , Zinc/química , Secuencia de Bases , Sitios de Unión , Transporte Biológico , Western Blotting , ADN/química , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Iones , Metalotioneína/metabolismo , Metales/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Represoras/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transcripción Genética , Zinc/metabolismo

RESUMEN

The plant plasma membrane H(+)-ATPase is a proton pump which plays a central role in physiological functions such as nutrient uptake and intracellular pH regulation. This pump belongs to the P(3)-type ATPase family and creates an electrochemical gradient across the plasma membrane. The generation of this gradient has a major role in providing the energy for secondary active transport across the plasma membrane. The activity of the proton pump is regulated by the transcriptional and post-translational levels and by membrane environmental factors such as membrane lipids. Several reviews have appeared during the last few years concerning the regulatory mechanism at transcriptional and post-translational levels. The plasma membrane H(+)-ATPase requires lipids for activity. This lipid dependency suggests a possible mode of regulation of the H(+)-ATPase via modification of its lipid environment. This review focuses on the regulation of plasma membrane H(+)-ATPase by membrane lipids surrounding H(+)-ATPase molecules.

Asunto(s)

Membrana Celular/enzimología , Oryza/enzimología , ATPasas de Translocación de Protón/metabolismo , Secuencia de Aminoácidos , Membrana Celular/fisiología , Secuencia Conservada , Electroquímica , Activación Enzimática/efectos de los fármacos , Liposomas/metabolismo , Modelos Moleculares , Oryza/genética , Fosfolípidos/farmacología , Conformación Proteica , ATPasas de Translocación de Protón/clasificación , ATPasas de Translocación de Protón/genética

RESUMEN

A metallothionein (BmtA) and a CPx-ATPase (Bxa1) have been identified and characterized from the cyanobacterium Oscillatoria brevis. Both bmtA and bxa1 expression can be markedly induced in vivo by Zn(2+) or Cd(2+). Over-expression of bmtA or bxa1 in Escherichia coli enhances Zn(2+) and Cd(2+) tolerance in the transformant. Dynamic studies on the expression of two genes showed that the maximum expression of bxa1 induced by Zn(2+) and Cd(2+) was much quicker than that of bmtA, suggesting distinct physiological roles of metallothionein and CPx-ATPase in the handling of surplus metal.

Asunto(s)

Adenosina Trifosfatasas/fisiología , Proteínas Bacterianas/fisiología , Cianobacterias/efectos de los fármacos , Metalotioneína/fisiología , Metales Pesados/farmacología , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Cadmio/farmacología , Cianobacterias/enzimología , Cianobacterias/genética , Cianobacterias/metabolismo , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Cinética , Metalotioneína/genética , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , Homología de Secuencia de Aminoácido , Zinc/farmacología

RESUMEN

Barley HvPIP2;1 is a plasma membrane aquaporin and its expression was down-regulated after salt stress in barley [Katsuhara et al. (2002) Plant Cell Physiol. 43: 885]. We produced and analyzed transgenic rice plants over-expressing barley HvPIP2;1 in the present study. Over-expression of HvPIP2;1 increased (1) radial hydraulic conductivity of roots (Lp(r)) to 140%, and (2) the mass ratio of shoot to root up to 150%. In these transgenic rice plants under salt stress of 100 mM NaCl, growth reduction was greater than in non-transgenic plants. A decrease in shoot water content (from 79% to 61%) and reduction of root mass or shoot mass (both less than 40% of non-stressed plants) were observed in transgenic plants under salt stress for 2 weeks. These results indicated that over-expression of HvPIP2;1 makes rice plants sensitive to 100 mM NaCl. The possible involvement of aquaporins in salt tolerance is discussed.

Asunto(s)

Acuaporinas/metabolismo , Hordeum/genética , Oryza/crecimiento & desarrollo , Proteínas de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Brotes de la Planta/crecimiento & desarrollo , Cloruro de Sodio/farmacología , Adaptación Fisiológica/genética , Adaptación Fisiológica/fisiología , Acuaporinas/efectos de los fármacos , Acuaporinas/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Hordeum/efectos de los fármacos , Hordeum/crecimiento & desarrollo , Datos de Secuencia Molecular , Oryza/efectos de los fármacos , Oryza/genética , Presión Osmótica/efectos de los fármacos , Proteínas de Plantas/efectos de los fármacos , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/genética , Plantas Modificadas Genéticamente

RESUMEN

The tonoplast of Tradescantia virginiana L. was prepared from leaf cells and then solubilized with deoxycholate (DOC) and n-octyl-beta-D-glucoside (n-OG). Three major polypeptides (68, 60, 16 kDa) and several other minor components were isolated. These polypeptides were reconstituted in soybean phospholipids (asolectin). The H(+) pump activity was investigated with the reconstituted system as well as with the tonoplast. In both cases, the quinacrine-fluorescence quenching was observed in the presence of ATP-Mg(2+), indicating the H(+) pumping. The H(+) pump activity was inhibited by gramicidin D, a channel-forming ionophore, and by KNO(3), an inhibitor specific to tonoplast-type (V-type) H(+)-ATPase.

Asunto(s)

Proteínas de la Membrana/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Tradescantia/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/química , Concentración de Iones de Hidrógeno , Liposomas/química , Proteínas de la Membrana/química , Peso Molecular , Péptidos/química , Hojas de la Planta/química , Proteínas de Plantas/química , ATPasas de Translocación de Protón/aislamiento & purificación , Tradescantia/química

RESUMEN

The fusogenic activity of plant Golgi membranes was studied in a cell-free system by assaying lipid mixing and content leakages of fluorescence probes. Golgi membranes from mung bean (Vigna radiata L.) hypocotyl cells fused to liposomes in the absence of any cytosolic proteins and nucleotides. It was demonstrated that the fusion was mediated by integral membrane protein(s), and was influenced by divalent cations (mm). Mg(2+), Ca(2+), and Mn(2+) ions enhanced the lipid mixing by reducing repulsive forces between membranes. In the content leakage assay, Mg(2+) ions also showed a stimulative effect. However, other divalent cations were inhibitory. It is suggested that the fusion system of Golgi membranes comprises at least two components: one that mediates the formation of fusion intermediates prior to pore opening, and one that mediates the subsequent processes. The latter must be sensitive to divalent cations at millimolar concentrations. The fusion of Golgi and biological membranes was induced by divalent cations. We speculated about the biological role of the fusion system studied here.

Asunto(s)

Cationes , Fabaceae/metabolismo , Aparato de Golgi/metabolismo , Plantas/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Cinética , Metabolismo de los Lípidos , Liposomas/metabolismo , Magnesio/metabolismo , Sulfato de Magnesio/farmacología , Manganeso/metabolismo , Factores de Tiempo

RESUMEN

Tonoplast H(+)-ATPase purified from cultured rice cells (Oryza sativa L. var. Boro) was reconstituted into asolectin liposomes containing steryl glucoside (SG) or acyl steryl glucoside (ASG), and the effects of SG and ASG on proton pumping, ATP-hydrolysis activity and proton permeability of the proteoliposome membranes were investigated. In the proteoliposomes containing 10 mol% SG, proton pumping and ATP-hydrolysis activity were increased to around 140% of those in SG-free proteoliposomes. In the proteoliposomes containing ASG, proton pumping and ATP-hydrolysis activity were decreased to one-tenth of those in ASG-free proteoliposomes at 15 mol% ASG; however, activity increased again slightly in the range between 20 and 40 mol% ASG. The change in proton pumping across the proteoliposome membrane is not due to a change of proteoliposome size nor to the location of the catalytic site of the tonoplast H(+)-ATPase in the proteoliposomes. SG and ASG also reduced the passive proton permeability of the proteoliposomes. These results show that SG and ASG modulate proton pumping across the tonoplast toward stimulation and depression, respectively, and they reduce the passive proton permeability of the tonoplast.

Asunto(s)

Glucolípidos/metabolismo , Membranas Intracelulares/metabolismo , Oryza/metabolismo , Bombas de Protones/metabolismo , ATPasas de Translocación de Protón/metabolismo , Transporte Biológico/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Liposomas/metabolismo , Sulfato de Magnesio/farmacología , Nigericina/farmacología , ATPasas de Translocación de Protón/aislamiento & purificación

RESUMEN

We identified three genes homologous to water channels in the plasma membrane type subfamily from roots of barley seedlings. These genes were designated HvPIP2;1, HvPIP1;3, and HvPIP1;5 after comparison to Arabidopsis aquaporins. Competitive reverse transcription (RT)-PCR was applied in order to distinguish and to quantify their transcripts. The HvPIP2;1 transcript was the most abundant among the three in roots. Salt stress (200 mM NaCl) down-regulated HvPIP2;1 (transcript and protein), but had almost no effect on the expressions of HvPIP1;3, or HvPIP1;5. Approximately equal amounts of the transcripts of the three were detected in shoots, and salt stress enhanced the expression of HvPIP2;1 but not of HvPIP1;3, or HvPIP1;5. HvPIP2;1 protein was confirmed to be localized in the plasma membrane. Functional expression of HvPIP2;1 in Xenopus oocytes confirmed that HvPIP2;1 encoded an aquaporin that transports water. This water permeability was reduced by HgCl(2), which is a typical water channel inhibitor. This activity was not modified by some inhibitors against protein kinase and protein phosphatase.

Asunto(s)

Acuaporinas/genética , Proteínas de Arabidopsis , Hordeum/genética , Raíces de Plantas/genética , Adaptación Fisiológica/genética , Adaptación Fisiológica/fisiología , Algoritmos , Secuencia de Aminoácidos , Animales , Acuaporinas/antagonistas & inhibidores , Acuaporinas/fisiología , Carbazoles/farmacología , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , ADN Complementario/genética , ADN Complementario/metabolismo , Femenino , Regulación de la Expresión Génica de las Plantas , Hordeum/efectos de los fármacos , Hordeum/fisiología , Alcaloides Indólicos , Canales Iónicos/genética , Cloruro de Mercurio/farmacología , Datos de Secuencia Molecular , Ácido Ocadaico/farmacología , Oocitos/fisiología , Presión Osmótica/efectos de los fármacos , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/fisiología , Inhibidores de Proteínas Quinasas , Homología de Secuencia de Aminoácido , Cloruro de Sodio/farmacología , Agua/farmacología , Agua/fisiología , Xenopus laevis

RESUMEN

A novel gene related to heavy-metal transport was cloned and identified from the filamentous cyanobacterium Oscillatoria brevis. Sequence analysis of the gene (the Bxa1 gene) showed that its product possessed high homology with heavy-metal transport CPx-ATPases. The CPC motif, which is proposed to form putative cation transduction channel, was found in the sixth transmembrane helix. However, instead of the CXXC motif that is present in the N termini of most metal transport CPx-ATPases, Bxa1 contains a unique Cys-Cys (CC) sequence element and histidine-rich motifs as a putative metal binding site. Northern blotting and real-time quantitative reverse transcription-PCR showed that expression of Bxa1 mRNA was induced in vivo by both monovalent (Cu(+) and Ag(+)) and divalent (Zn(2+) and Cd(2+)) heavy-metal ions at similar levels. Experiments on heavy-metal tolerance in Escherichia coli with recombinant Bxa1 demonstrated that Bxa1 conferred resistance to both monovalent and divalent heavy metals. This is the first report of a CPx-ATPase responsive to both monovalent and divalent heavy metals.

Asunto(s)

Adenosina Trifosfatasas/biosíntesis , Adenosina Trifosfatasas/genética , Cianobacterias/enzimología , Farmacorresistencia Bacteriana Múltiple , Metales Pesados/farmacología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/aislamiento & purificación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Transporte Biológico , Cianobacterias/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Histidina/química , Metales Pesados/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Transcripción Genética

RESUMEN

The large scale asymmetry in surface (poly)peptides of the plasma membrane (PM) of mung bean (Vigna radiata L.) hypocotyl cells was investigated by protease and 1 M KCl treatments of PM vesicles obtained by an aqueous two-phase partition technique. Proteases only slightly reduced the protein content of right-side-out PM vesicles and the treatment with 1 M KCl resulted in the dissociation of only a few peripheral proteins from the outer surface of right-side-out PM vesicles, indicating that few surface peptides including peripheral proteins existed on the outer surface. From experiments of the re-partitioning of endomembrane vesicles removed from surface peptides, it was found that the surface peptide content is a factor determining the partitioning, and the hypothesis that sterols are asymmetrically distributed across higher plant PM was proposed. We speculate that asymmetrical properties between the outer and the inner surfaces of plant PM, especially in partitioning in the two-phase system, derive from the asymmetry of the bulk of surface peptides and PM sterols. The comparatively low hydrophilicity of the outer surface of the PM would be important for the partitioning of right-side-out PM vesicles in the upper phase of the two-phase system.

Asunto(s)

Fabaceae , Hipocótilo/química , Proteínas de la Membrana/química , Proteínas de Plantas/química , Membrana Celular/química , Electroforesis en Gel de Poliacrilamida , Endopeptidasas , Potenciales de la Membrana , Fragmentos de Péptidos/química , Cloruro de Potasio , Propiedades de Superficie , Tripsina