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1.
The effect of an in vivo and in vitro treatment with cadmium on transport activities of root plasma membrane enriched vesicles was studied in oat (Avena sativa L. cv. Argentina) plants. Addition of 100 mumol/L CdSO4 to nutrient solution decreases both proton transport activity and ATPase activity to the same level. In vitro experiments show that cadmium seems to have a differential inhibiting effect on proton transport activity and ATPase activity, the most pronounced one on ATP-dependent H(+)-accumulation, suggesting that cadmium would interfere with membrane permeability properties. This is indeed the case. The results demonstrate that cadmium decreases passive permeability to protons. 相似文献
2.
The plant vacuole has long been suspected of being a site for accumulation of Ni in plant roots, but testing this hypothesis
directly by vacuole isolation is technically difficult and has not been reported. Here, we have attempted to determine if
Ni can be transported into isolated oat (Avena sativa L.) root tonoplast vesicles as an alternative approach toward understanding the importance of the vacuole in Ni accumulation
in roots. We found that, in contrast to Ca and Cd, Ni did not affect the proton gradient of vesicles (MgATP energized or artificially
created), and further, that Cd/H antiport activity was not affected by the presence of Ni. Nickel was associated with vesicles,
but relative rates of accumulation/association of metals with vesicles were Ca > Cd Ni. Protonophores and the potential Ni ligands citrate and histidine, and nucleoside triphosphates or PPi did not stimulate
Ni association with vesicles. Comparison of Ni versus Ca and Cd associated with vesicles using various membrane perturbants
indicated that while Ca and Cd are rapidly and principally antiported to the vesicle sap, Ni is only slowly associated with
the membrane in a not-easily dissociated condition. Our results indicate the absence of an Ni/H antiport or Ni-nucleotide-dependent
pump in oat root tonoplasts, and support the contention that the vacuole is not a major compartment for Ni accumulation in
oat roots.
Received: 2 June 1997 / Accepted: 17 July 1997 相似文献
3.
Regeneration of fertile green plants from isolated oat microspores is reported for the first time. Factors critical for microspore
growth and regeneration include cold pre-treatment, pH of culture medium and the use of conditioned culture medium. It was
found that cold pre-treatment at 4°C in the dark for a minimum of 6 weeks was necessary to consistently achieve microspore
growth into multicellular structures (MCS). Longer pre-treatments of up to 9 weeks were tested and found to be positively
correlated with the number of MCS produced. Microspore culture medium with pH 8.0 produced significantly more MCS larger than
eight cells in size than media with pH 5.8. The use of medium conditioned by actively growing barley microspores significantly
increased the numbers of MCS larger than eight cells in size compared to non-conditioned media. Plants were regenerated only
from cultures using conditioned medium. A total of 2 green plants and 15 albinos were regenerated. Of the green plants, one
had the haploid chromosome complement (n = 3x = 21) and the other had the parental hexaploid chromosome complement (2n = 6x = 42) which may be due to spontaneous chromosome doubling. The hexaploid plant set seed naturally and the haploid plant set
seed after its chromosome complement was doubled with colchicine. 相似文献
4.
Hans Peter Getz 《Planta》1991,185(2):261-268
Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl2 in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H+-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K
m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom. 相似文献
5.
Steven Fabijanski Gregory J. Matlashewski Illimar Altosaar 《Plant molecular biology》1985,4(4):205-210
Polyadenylated mRNA from developing oat (Avena sativa L.) seeds was isolated and analyzed. Prominent mRNA species of 18S, 15S and 12S were observed; the 18S mRNA was judged to
be esentially free of ribosomal RNA by hybridization analysis. Size fractionation andin vitro translation of this mRNA was performed. SDS, IEF-SDS gel electrophoresis and immunoprecipitation were used to analyze the
translation products. It is shown that globulin mRNA (18S) accounts for roughly 30% of the total mRNA in developing seeds,
the 12S and 15S mRNAs accounting for the remainder. The 18S mRNA directs the synthesis of a series of distinct but related
polypeptides, suggesting that some of the heterogeneity seen in the oat globulins is at the amino acid sequence level. 相似文献
6.
The pH gradient and the electric potential across the tonoplast in mechanically isolated beetroot vacuoles has been studied by following the uptake of [14C]methylamine and [14C]triphenyl-methylphosphoniumchloride. In response to Mg-ATP, the vacuolar interior is acidified by 0.8 units. This strong acidification is accompanied by a slight hyperpolarization of the membrane potential, which is probably caused by a proton diffusion potential. In preparations where only a small acidification (0.4 units) occurred, the membrane potential was depolarized by the addition of Mg-ATP. Different monovalent cations and anions were tested concerning their effect on the pH gradient and ATPase activity in proton-conducting tonoplasts. Chloride stimulation and NO
3
-
inhibition were clearly present. The observed decline of the pH gradient upon the addition of Na+ salts is probably caused by an Na+/H+ antiport system.Abbreviations and symbol CCCP
carbonylcyanide-m-chlorophenylhydrazone
- Mes
2(N-morpholino)ethanesulfonic acid
- TPMP+
triphenylmethylphosphoniumchloride
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol
-
membrane potential
Dedicated to Professor A. Betz on the occasion of his 65th birthday 相似文献
7.
Oat polysomes direct the synthesisin vitro of a large number of products, the majority of which are the salt-soluble globulins (1,3,10,11,21). Total RNA or poly A+ RNA isolated from these polysomes directs the synthesis of the same number and types of products; however, the amount of
globulins synthesized no longer represents the major products; rather, there is a decreased level of globulins and an increased
amount of the other products synthesizedin vitro (6, 18). These results imply that the translational control can dictate final product levels. Reconstruction experiments
using oat poly A+ mRNA and polysomal factors that are made free of endogenous RNA by nuclease digestion demonstrate that these factors do influence
the translational specificity of oat globulin mRNA relative to other mRNAs. It is suggested that translational control is
partially responsible for the levels of globulin in the mature grain. 相似文献
8.
Emilia Ballesteros Eduardo Blumwald Juan Pedro Donaire rés Belver 《Physiologia plantarum》1997,99(2):328-334
Na+ transport across the tonoplast and its accumulation in the vacuoles is of crucial importance for plant adaptation to salinity. Mild and severe salt stress increased both ATP- and PPi-dependent H+ transport in tonoplast vesicles from sunflower seedling roots, suggesting the possibility that a Na+/H+ antiport system could be operating in such vesicles under salt conditions (E. Ballesteros et al. 1996. Physiol. Plant. 97: 259–268). During a mild salt stress, Na+ was mainly accumulated in the roots. Under a more severe salt treatment, Na+ was equally distributed in shoots and roots. In contrast to what was observed with Na+, all the salt treatments reduced the shoot K+ content. Dissipation by Na+ of the H+ gradient generated by the tonoplast H+-ATPase, monitored as fluorescence quenching of acridine orange, was used to measure Na+/H+ exchange across tonoplast-enriched vesicles isolated by sucrose gradient centrifugation from sunflower (Helianthus annuus L.) roots treated for 3 days with different NaCl regimes. Salt treatments induced a Na+/H+ exchange activity, which displayed saturation kinetics for Na+ added to the assay medium. This activity was partially inhibited by 125 μM amiloride, a competitive inhibitor of Na+/H+ antiports. No Na+/H+ exchange was detected in vesicles from control roots. The activity was specific for Na+. since K+ added to the assay medium slightly dissipated H+ gradients and displayed non-saturating kinetics for all salt treatments. Apparent Km for Na+/H+ exchange in tonoplast vesicles from 150 mM NaCl-treated roots was lower than that of 75 mM NaCl-treated roots, Vmax remaining unchanged. The results suggest that the existence of a specific Na+/H+ exchange activity in tonoplast-enriched vesicle fractions, induced by salt stress, could represent an adaptative response in sunflower plants, moderately tolerant to salinity. 相似文献
9.
Membrane vesicles were isolated from mesophyll cells of Mesembryanthemum crystallinum in the C3 state and in the crassulacean acid metabolism (CAM) state. The distribution of ATP-hydrolysis and H+-transport activities, and the activities of hydroxypyruvate reductase and Antimycin-insensitive cytochrome-c-reductase on continuous sucrose gradients was studied. For isolations carried out routinely a discontinuous sucrose gradient (24%/37%/50%) was used. Nitrate-sensitive ATP-hydrolysis and H+-transport activities increased several-fold during the transition from C3 photosynthesis to CAM. Nitrate-sensitive ATPase showed a substrate preference for ATP with an apparent Km (MgATP2-) of 0.19–0.37 mM. In both C3 and CAM states the ATPase showed a concentration-dependent stimulation by the anions chloride and malate. However, the pH optima of the two states were different: the ATPase of C3-
M. crystallinum had an optimum of pH 7.4 and that of CAM-M. crystallinum an optimum of pH 8.4. The optical probe oxonol-VI was used to demonstrate the formation of MgATP2--dependent electric-potential gradients in tonoplast vesicles.Abbreviations Bistris-Pronane
1,3-bis [tris(hydroxymethyl)-methylaminol propane
- CAM
Crassulacean acid metabolism
- DIDS
4,4-dilsothiocyano-2,2-stilbene disulfonic acid:
- DTT
dithiothreitol
- ER
endoplasmic reticulum
- Hepes
4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid
- HPR
hydroxypyruvate reductase
- IDPase
inosine 5-diphosphatase
- OX-VI
oxonol VI
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
10.
The effects of anions on inorganicpyrophosphate-dependent H+-transport in isolated tonoplast vesicles from oat (Avena sativa L.) roots were determined. Both fluorescent and radioactive probes were used to measure formation of pH gradients and membrane potential in the vesicles. Pyrophosphate hydrolysis by the H+-translocating pyrophosphatase was unaffected by anions. Nonetheless, some anions (Cl-, Br- and NO3-) stimulated H+-transport while others (malate,
and iminodiacetate) did not. These differential effects were abolished when the membrane potential was clamped at zero mV using potassium and valinomycin. Stimulation of H+-transport by Cl- showed saturation kinetics whereas that by NO3- consisted of both a saturable component and a linear phase. For Cl- and NO3-, the saturable phase had a K
m
of about 2 mol·m-3. The anions that stimulated H+-transport also dissipated the membrane potential (.) generated by the pyrophosphatase. It is suggested that the stimulatory anions cross the tonoplast in response to the positive generated by the pyrophosphatase, causing dissipation of and stimulation of pH, as expected by the chemiosmotic hypothesis. The work is discussed in relation to recent studies of the effects of anions on ATP-dependent H+-transport at the tonoplast, and its relevance to anion accumulation in the vacuole in vivo is considered.Abbreviations and symools BTP
1,3-bis[tris(hydroxymethyl)-methylamino]-propane
- EGTA
ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid
- Hepes
4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid
- IDA
iminodiacetate
-
membrane potential
- pH
pH gradient
- PPase
inorganic pyrophosphatase
- PPi
morganic pyrophosphate 相似文献
11.
N. Mendu H. Rines C. D. Silflow 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1993,86(1):135-140
Summary The allohexaploid nature of Avena sativa L. (2n=6x=42) and the availability of aneuploid lines was exploited in designing a strategy for mapping beta-tubulin sequences in the oat genome. Evidence for a minimum of eight beta-tubulin genes was obtained by Southern-blot analysis. Three betatubulin sequences were localized to chromosomes using DNA from monosomic and nullisomic lines in the variety Sun II. One sequence was localized to the chromosome missing in nullisome I. Two other sequences were mapped to satellite chromosome 2, the chromosome that is missing in nullisome VIII and to which one ribosomal RNA gene cluster had previously been mapped. Restriction fragments carrying these two beta-tubulin genomic sequences and the cluster of ribosomal RNA sequences were missing in DNA from nullisomics VIII, IX and X, suggesting that all three nullisome classes are deficient for an identical chromosomal segment that includes these three loci. This study demonstrates how molecular analyses can be used to characterize aneuploid stocks and to better define their genetic constitution.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or the University of Minnesota and does not imply its approval to the exclusion of other products or vendors that may be suitable 相似文献
12.
A fluorescence method for the direct measurement of Cl- transport in isolated tonoplast vesicles is described. This technique utilises the Cl--sensitive fluorescent compound, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). This is a water-soluble compound with excitation and emission wavelengths of 350 and 440 nm, respectively. Its fluorescence is quenched by Cl-, Br-, I-, SCN-, NO
2
-
and tetraphenylborate but not by NO
3
-
, SO
4
2-
, iminodiacetate or malate. These effects are independent of pH. This compound was loaded into tonoplast vesicles from red beet (Beta vulgaris L.) storage roots or from barley (Hordeum vulgare L.) roots by incubation at 37° C and the external probe was then removed by repeated centrifugation of the vesicles in SPQ-free medium. In this way a large proportion of the observed fluorescence signal was from the interior of the vesicles, and its quenching could be used to monitor, quantitatively, and in real time, the intravesicular Cl- concentration. In this paper we describe some of the problems encountered in using this probe to measure Cl- transport in tonoplast vesicles, how these were overcome and some characteristics of Cl- transport at the tonoplast as measured by the probe.Abbreviations and symbols BTP
1,3-bis[tris(hydroxymethyl)-methylamino-propane
- DTT
dithiothreitol
-
membrane potential
- pH
pH gradient
- PPase
inorganic pyrophosphatase
- PPi
inorganic pyrophosphate
- SPQ
6-methoxy-1-(3-sulfonatopropyl)quinolinium
- Tricine
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 相似文献
13.
William Parker Todd A. Wells Susanne Meza-Keuthen In-Soo Kim Pill-Soon Song 《Journal of Protein Chemistry》1995,14(2):53-57
Recently, Mummertet al. [Nature
363, 644–648 (1993)] isolated a proposed TCP1-related chaperone. Here we report several findings concerning the protein which they sequenced. Two similar N-terminal sequences were obtained from this abundant 60-kDa protein. Internal sequences were also acquired by protease digestion. Initially it was believed the protein was able to completely inhibit citrate synthase aggregation, but later purifications demonstrated that the 60-kDa polypeptide lacked both chaperone activity and the previously reported kinase activity [Grimmet al., Planta
178, 199–206 (1989)]. It is now our belief that this protein is neither a chaperone nor a kinase. 相似文献
14.
15.
16.
The rate of senescence and the two-dimensional pattern of soluble proteins from detached oat leaves senescing in either darkness or light were analyzed, and compared to those of leaves in which senescence was delayed by application of the cytokinin benzyladenine or enhanced through the action of abscisic acid.Senescence of detached leaves in light did not differ significantly from senescence in attached leaves on intact plants. In darkness, protein was lost at a higher rate than in light, but several individual proteins showed relative increases. Notably, proteins previously characterized as high-molecular-weight proteins and senescence-associated proteins (Klerk et al., 1992) increased. Changes observed during incubation in light or darkness appeared to be related to this condition rather than the rate or progress of senescence. Cytokinins delayed and abscisic acid accelerated the changes in protein pattern compared to water. Beside changes previously identified in leaves senescing on the plant, detached leaves show alterations that reflect their condition of incubation rather than their developmental progress.Abbreviations 2D-PAG
two-dimensional polyacrylamide gel electrophoresis
- ABA
abscisic acid
- BA
N6-benzyladenine
- BSA
bovine serum albumin
- EDTA
ethylenediamine tetraacetic acid
- IEF
isoelectric focusing
- Rubisco
ribulosebisphosphate carboxylase/oxygenase
- SDS
sodium dodecyl sulfate
- Tris
tris (hydroxymethyl) aminomethane 相似文献
17.
The membrane potential in vacuoles isolated from storage roots of red beet (Beta vulgaris L.) has been studied by following changes in the fluorescence of the dye 3,3-diethylthiodicarbocyanine iodide, and by determining the uptake of the lipophilic triphenylmethylphosphonium cation. The vacuoles have a membrane potential, internal negative, which is estimated to be around-60 mV. These potentials become less negative by nearly 10 mV on addition of ATP. This ATP-dependent depolarisation is inhibited by the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and by the ATPase inhibitors, N,N-dicyclohexylcarbodiimide and trimethyltin chloride, but it is largely insensitive to sodium orthovanadate. Fusicoccin had no significant effect on the isolated vacuoles, but its addition to excised tissue caused a hyperpolarisation of the cells measured using a microelectrode.Abbreviations DCCD
N,N-dicyclohexylcarbodiimide
- DiS-C2-(5)
3,3-diethylthiodicarbocyanine iodide
- FCCP
carbonylcyanide p-trifluoromethoxyphenylhydrazone
- TPMP+
triphenylmethylphosphonium ion 相似文献
18.
Barbara Deumling 《Plant Systematics and Evolution》1978,129(4):261-267
3H-RNA, complementary to repetitive DNA of wheat, rye, barley, and oat, was hybridizedin situ to root tip or pollen mother cells of the species mentioned. The cRNAs hybridized best with the DNA in cell nuclei of the species from which they were prepared. Cross hybridization with cells of the other related species resulted in a significant but diminished labelling. Wheat, rye, and barley hybridized better to each other than to oat, andvice versa, in agreement with the usual taxonomical classification. Over the interphase nuclei the label was distributed unevenly; not all regions of dense chromatin were labelled, and little label was found over the nucleoli. On chromosomes, the repetitive DNA was located somewhere along the chromosome arms or near the centromers in wheat, barley, and oat. Only in rye, most of the label was located near the telomers, probably over the large heterochromatin areas. 相似文献
19.
Right-side-out and sealed plasmalemma vesicles were isolated from roots of spring wheat (Triticum aestivum L. cv. Drabant) and oat (Avena sativa L. cv. Brighton) by two-phase partition in a medium containing sucrose (0.25 mol l-1). Oat root plasmalemma vesicles were discovered to contain a strongly fluorescent compound with an emission maximum at 418 nm. The surface potential of the membranes was monitored by 9-aminoacridine fluorescence and the effect of protein concentration, mannitol versus sucrose, absence of osmoticum, concentrations of salt, and titrations with chelators investigated. It is concluded that i) protein concentrations of less than 50 g ml-1 for oat and 100 g ml-1 for wheat plasmalemma vesicles should be used to avoid serious problems with non-linearity of response of 9-aminoacridine fluorescence, ii) mannitol can be used instead of sucrose as the osmoticum, iii) the vesicles were ruptured in the absence of osmoticum allowing us to monitor both sides of the membranes, iv) plasmalemma vesicles from oat roots are more negative than vesicles from wheat roots, and v) oat and wheat root plasmalemma vesicles are isolated with about the same amounts of bound Ca2+ and Mg2+. These bound divalent cations may not, however, reflect the in-vivo conditions since the tissues were homogenised in the presence of ethylenediaminetetraacetic acid.Abbreviations EDTA
ethylenediaminetetraacetic acid
- EGTA
ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid
-
c1/2 value
concentration at which half of the maximum effect is observed
- Mops
3-(N-morpholino)propanesulfonic acid 相似文献
20.
Generation of active oxygen species during enzymic isolation of protoplasts from oat leaves 总被引:2,自引:0,他引:2
Shigetaka Ishii 《In vitro cellular & developmental biology. Plant》1987,23(9):653-658
Summary Protoplasts were isolated from oat (Avena sativa L.) leaves by the combination of highly purified preparations of pectin lyase, xylanase, and cellulase C1. During the enzymic isolation, superoxide radical (O
−
2
) was generated from the tissues. Both the protoplasts themselves and the cell walls, exposed to enzyme treatment, produced
O
−
2
. Hydrogen peroxide (H2O2) apparently accumulated in the reaction mixture due to the spontaneous dismutation reaction of O
−
2
, while a part of H2O2 may have been produced directly from cell walls by the action of enzymes. Singlet molecular oxygen (1O2) generated in the reaction mixture was detected by cholesterol oxidation in small unilamellar liposomes. It seems likely
that1O2 may be generated by the peroxidase-H2O2-halide system during enzymic treatment of the leaves.
The work was partially supported by the Research Project “Research and development of the improvement of bacterial and plant
cells by cell fusion” of the Food and Agriculture Research and Development Association (Japan). 相似文献