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1.
2.
Changes in the limiting porosity of cell walls, i.e. the size limit for permeation of neutral molecules through the wall,
were studied in several higher-plant cell-suspension cultures. For this purpose, samples of biomass fixed at different cultivation
times were investigated using a method based on size-exclusion chromatography of polydisperse dextrans before and after equilibration
with the extracted cell clusters. In suspension cultures of Chenopodium album L., Dioscorea deltoidea Wall. and Medicago sativa L., the mean size limit (MSL; critical Stokes' radius for exclusion of neutral polymers from half of the intracellular space)
was found to vary between 2.4 and 3.8 nm. It decreased significantly during transition from the growth phase to the stationary
phase. In the case of the C. album culture this change was found to be irrespective of whether sucrose in the medium was completely depleted at the end of the
growth phase or not. The MSL was kept constant for long periods of the stationary phase if cell viability was maintained by
repeated sucrose supplement. In a suspension strain of Triticum aestivum L., the MSL of cell wall permeation was comparatively small (1.75 nm) and remained constant during all cultivation phases.
Relations between limiting porosity and cell wall growth, loss of pectic compounds to the medium, cross-linking activities
and cell wall stiffening are discussed.
Received: 19 December 1996 / Accepted: 23 April 1997 相似文献
3.
Suspension-cultured carrot cells and intact leaves respond to crude and purified protein elicitors from the non-host fungus
Pythium aphanidermatum by activating the general phenylpropanoid pathway and incorporating de-novo-synthesized 4-hydroxybenzoic acid (4-HBA) into
the cell wall. The cultured cells undergo a very rapid elicitor-induced cell death. Both reactions are directly correlated
in their time course and their dose dependency. Cell death in elicitor-treated protoplasts resulted in early membrane damage
and the digestion of DNA into oligonucleosomal fragments. The same pattern of DNA degradation could be induced in protoplasts
by the G-protein activators Mas-7 or mastoparan. In cell cultures, both activators induced a rapid loss of viability without
the activation of the general phenylpropanoid pathway. The elicitor-induced reactions, the loss of viability and the induction
of 4-HBA biosynthesis were blocked by the calcium-channel blocker nifedipine. Neomycin and U73122, two inhibitors of phospholipase
C, blocked the induction of 4-HBA biosynthesis but did not affect the loss in viability. The injection of the elicitor into
the leaves of intact carrot plants confirmed the results obtained with cell cultures with regard to the induction of the hypersensitive
response. The purification of the active compound revealed a 25-kDa protein which triggers both cell death and 4-HBA synthesis.
The signalling pathways to both reactions could be independently blocked or induced.
Received: 27 February 1998 / Accepted: 25 May 1998 相似文献
4.
The intracellular localization of prenyltransferases involved in the biosynthesis of the phytoalexins glyceollin in soybean
(Glycine max L.) and phaseollin in French bean (Phaseolus vulgaris L.) has been investigated. By sucrose- and Percoll-gradient centrifugation of microsomes of an elicitor-challenged soybean
cell culture, the membranes containing prenyltransferase were separated from the endoplasmic reticulum and shown to be lighter
in density. In a continuous Percoll gradient the peak of prenyltransferase activity coincided with the peak of galactolipid
synthesis, as determined by incorporation of uridine 5′-diphospho-[14C]galactose (UDP-[14C]galactose). Intact chloroplasts isolated from cupricchloride-treated bean leaves contained both prenyltransferase and UDP-galactose
transferase activity. Both activities increased during chloroplast isolation. Fractionation of swollen chloroplasts on a discontinuous
sucrose gradient showed prenyltransferase and UDP-galactose transferase activity in the envelope membrane subfraction. It
is concluded that in both plants prenyltransferase is located in the envelope membrane of plastids.
Dedicated to Professor Hans Mohr on the occasion of his 60th birthday 相似文献
5.
Maize (Zea mays L.) cell cultures incorporated radioactivity from [14C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the
CoA pool had lost its 14C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a
pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age.
In young (1–3 d) cultures, polysaccharide-bound [14C]feruloyl- and [14C]diferuloyl residues were both detectable within 1 min of [14C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least
the first 2.3 h after [14C]cinnamate feeding, polysaccharide-bound [14C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [14C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter,
and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H2O2 (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In
older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [14C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [14C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H2O2 induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H2O2-limited. In both 2- and 4-d-old cultures, polysaccharide-bound 14C-trimers and larger coupling products exceeded [14C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually
quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides
in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely
to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response
of cell walls to an oxidative burst are discussed.
Received: 19 January 2000 / Accepted: 13 April 2000 相似文献
6.
Phosphorus deficiency increases the argon-induced decline of nodule nitrogenase activity in soybean and alfalfa 总被引:1,自引:0,他引:1
Open-flow assays of H2 evolution in Ar:O2 (80:20, v/v) by nodulated roots were performed in situ with soybean [Glycine max (L.) Merr.] and alfalfa [Medicago sativa L.) grown in sand with orthophosphate (Pi) nutrition either limiting (low-P) or non-limiting (control) for plant growth.
Nodule growth was more limited than shoot growth by P deficiency. Phosphorus concentration was less affected in nodules than
in other parts of the low-P plants. During assays, nitrogenase activity declined a few minutes after exposure of the nodulated
roots to Ar. The magnitude of this argon-induced decline (Ar-ID) was less in alfalfa than in soybean. In both symbioses the
magnitude of the Ar-ID was larger in low-P than control plants. Moreover, the minimum H2 evolution after the Ar-ID, was reached earlier in low-P plants. The Ar-ID was partly reversed by raising the external partial
pressure of O2 in the rhizosphere. The magnitude of the Ar-ID in soybean was correlated negatively to nodule and shoot mass per plant, individual
nodule mass, H2 evolution in air prior to the assay, and nodule N and P concentrations. Possible reasons, including nodule size and nodule
O2 permeability, for the increase in Ar-ID in P-deficient plants are discussed and an interpretation of the P effect on nodule
respiration and energetic metabolism is proposed.
Received: 17 May 1996 / Accepted: 16 September 1996 相似文献
7.
Alfin1 transcription factor overexpression enhances plant root growth under normal and saline conditions and improves salt tolerance in alfalfa 总被引:11,自引:0,他引:11
Plant root development is an essential determinant of plant growth and crop yield that could be enhanced by induced changes in the expression of root-specific regulatory factors. We reported previously that Alfin1 binds DNA in a sequence-specific manner and that Alfin1 overexpression in transgenic alfalfa (Medicago sativa L.) enhances expression of the salt-inducible MsPRP2 gene in roots, suggesting that Alfin1 functions to regulate gene expression in roots. Here we show that Alfin1 is an essential gene for root growth and that its overexpression in transgenic plants confers a many-fold increase in root growth under normal and saline conditions. Alfin1-binding sites occur in promoters of genes expressed in roots of a wide variety of plant species and we propose that it is a general root growth regulator. Even though Alfin1 overexpression was under the control of the CaMV 35S promoter, plant shoot growth was not adversely affected. We show further that introduction of the Alfin1 transgene in plants confers a dominant characteristic that significantly increases plant growth and salt tolerance. 相似文献
8.
The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA
copy DNA
- CHS
chalcone synthase
- PAL
phenylalanine ammonia-lyase 相似文献
9.
The use of plants for production of recombinant proteins is becoming widely accepted. More recently, plant cell cultures have been proposed as valuable systems for producing a wide range of biologically active proteins. Such systems provide certain advantages over whole plants, but yields are still considered a limitation. In this study we established a Medicago truncatula cell suspension line expressing phytase from Aspergillus niger. Phytase is an N-glycosylated enzyme that breaks down indigestible phytate, resulting in an increased availability of phosphorus and other minerals in monogastric animals and reduced levels of phosphorus output in their manure. Various production systems have previously been used to express heterologous phytase, including several plant species. In this work, remarkable amounts of enzymatically active recombinant phytase were produced and secreted into the culture medium. Recombinant phytase accumulated to at least 25 mg/L and remained stable along the growth curve, and an enriched fraction with high enzymatic activity was easily obtained. We therefore propose M. truncatula cell suspension cultures as a potential system for the production of recombinant proteins. Most importantly, we have shown that, contrary to general belief, it is possible to achieve high levels of a functional recombinant protein in plant cell culture systems. 相似文献
10.
Wolfgang Pfeiffer 《Trees - Structure and Function》1998,13(2):112-116
Vacuolar ATPase (EC 3.6.1.3) and PPase (EC 3.6.1.1) were studied in suspension cells and seedlings from spruce [Picea abies (L.) Karst. Proton transport activity and uncoupler (1 μM nigericin) stimulated substrate hydrolysis were measured in tonoplast
enriched membrane vesicles. In suspension cells the vacuolar PPase exhibited 1.8-fold activity of the ATPase. In roots and
needles from 12-week-old spruce seedlings the vacuolar PPase was inactive, whereas the ATPase was active. Therefore, we investigated
whether the preparation of spruce tonoplast vesicles from roots and needles inactivates the vacuolar PPase but not the ATPase.
For this purpose, maize (Zea mays L.) tonoplast membranes exhibiting vacuolar PPase as well as ATPase activity were used as a probe and added to the homogenization
medium prior to the preparation of spruce vesicles. The preparation of spruce vesicles was more inhibitory to the vacuolar
ATPase than to the PPase. The comparison of vacuolar PPases from spruce suspension cells and maize roots revealed similar
enzymatic properties. After isopycnic centrifugation on continuous sucrose gradients the vacuolar PPase from spruce suspension
cells co-purified with the vacuolar ATPase. Together, these data show: (1) vacuolar PPases from spruce suspension cells and
maize roots are similar, (2) the preparation of tonoplast vesicles from spruce roots and needles does not inactivate the vacuolar
PPase, (3) tonoplasts of suspension cultured cells and seedlings from spruce are differentially energized by the vacuolar
pyrophosphatase that may indicate a difference in pyrophosphate metabolism between embryogenic and differentiated spruce cells,
and (4) tonoplast vesicles from spruce seedlings may allow investigations of the effect of pyrophosphate on the vacuolar ATPase
in the absence of vacuolar PPase activity.
Received: 2 July 1998 / Accepted: 14 September 1998 相似文献
11.
12.
Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures
Summary. In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H2O2 is induced by excess concentrations of copper (up to 100 μM). This specific and early response towards copper stress was
shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H2O2. Superoxide dismutase (5 U/ml) induced an increase in H2O2 production by 22.2%. This indicates that at least part of the H2O2 is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene
iodonium (2 and 10 μM) and quinacrine (1 and 5 mM) prevented the generation of H2O2 under copper stress for 90%. The influence of the pH on the H2O2 production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species
after copper stress.
Received May 20, 2002; accepted July 26, 2002; published online May 21, 2003
RID="*"
ID="*" Correspondence and reprints: Plant Physiology, Department of Biology, University of Antwerp (RUCA), Groenenborgerlaan
171, 2020 Antwerp, Belgium. 相似文献
13.
Kylie J. Nunan Ian M. Sims Antony Bacic Simon P. Robinson Geoffrey B. Fincher 《Planta》1997,203(1):93-100
Cell walls have been isolated from the mesocarp of mature grape (Vitis vinifera L.) berries. Tissue homogenates were suspended in 80% (v/v) ethanol to minimise the loss of water-soluble wall components
and wet-sieved on nylon mesh to remove cytoplasmic material. The cell wall fragments retained on the sieve were subsequently
treated with buffered phenol at pH 7.0, to inactivate any wall-bound enzymes and to dislodge small amounts of cytoplasmic
proteins that adhered to the walls. Finally, the wall preparation was washed with chloroform/methanol (1:1, v/v) to remove
lipids and dried by solvent exchange. Scanning electron microscopy showed that the wall preparation was essentially free of
vascular tissue and adventitious protein of cytoplasmic origin. Compositional analysis showed that the walls consisted of
approximately 90% by weight of polysaccharide and less than 10% protein. The protein component of the walls was shown to be
rich in arginine and hydroxyproline residues. Cellulose and polygalacturonans were the major constituents, and each accounted
for 30–40% by weight of the polysaccharide component of the walls. Substantial varietal differences were observed in the relative
abundance of these two polysaccharides. Xyloglucans constituted approximately 10% of the polysaccharide fraction and the remainder
was made up of smaller amounts of mannans, heteroxylans, arabinans and galactans.
Received: 26 November 1996 / Accepted: 30 January 1997 相似文献
14.
A microsomal preparation from suspension-cultured potato stem cells (Solanum tuberosum L. cv. AZY) was incubated with [14C]acetyl-CoA resulting in a precipitable radiolabeled product. Analysis of the product revealed that it consisted mostly of
acetylated proteins and cell wall polysaccharides, including xyloglucan, homogalacturonan and rhamnogalacturonan I. Thus,
acetyl-CoA is a donor-substrate for the O-acetylation of wall polysaccharides. A rhamnogalacturonan acetylesterase was used to develop an assay to measure and characterize
rhamnogalacturonan O-acetyl transferase activity in the microsomal preparation. Using this assay, it was shown that the transferase activity was
highest during the linear growth phase of the cells, had a pH-optimum at pH 7.0, a temperature optimum at 30 °C, an apparent
K
m of 35 μM and an apparent V
max of 0.9 pkat per mg protein. Further analysis of the radiolabeled acetylated product revealed that it had a molecular mass
>500 kDa.
Received: 3 July 1999; Accepted: 27 September 1999 相似文献
15.
Previous mechanical studies using algae have concentrated on cell extension and growth using creep-type experiments, but
there appears to be no published study of their failure properties. The mechanical strength of single large internode cell
walls (up to 2 mm diameter and 100 mm in length) of the charophyte (giant alga) Chara corallina was determined by dissecting cells to give sheets of cell wall, which were then notched and fractured under tension. Tensile
tests, using a range of notch sizes, were conducted on cell walls of varying age and maturity to establish their notch sensitivity
and to investigate the propagation of cracks in plant cell walls. The thickness and stiffness of the walls increased with
age whereas their strength was little affected. The strength of unnotched walls was estimated as 47 ± 13 MPa, comparable to
that of some grasses but an order of magnitude higher than that published for model bacterial cellulose composite walls. The
strength was notch-sensitive and the critical stress intensity factor K
1c was estimated to be 0.63 ± 0.19 MNm−3/2, comparable to published values for grasses.
Received: 4 April 2000 / Accepted: 21 July 2000 相似文献
16.
Somatic embryoids differentiated in suspension cultures of G. klotzschianum after 3–4 weeks of culture in a liquid medium containing glutamine (optimally, 10–15 mM). Embryogenesis occurred after a preculture of callus on a medium containing 10 mg/l of the cytokinin, 2iP. The embryoids had meristematic regions, a well formed epidermis, and formed roots and vestigial leaves. Asparagine was much less effective than glutamine in promoting embryoid differentiation. The presence of 2,4-D in the medium resulted in increased vigor of the suspension cultures and subsequently in the formation of many embryoids, but does not seem to be necessary for somatic embryogenesis in cotton.Technical Article 14646 from the Texas Agricultural Experiment Station 相似文献
17.
Summary Cell suspension cultures of alfalfa (Medicago sativa L.) accumulated phenolic secondary metabolites in a pattern similar to that seen in alfalfa roots. Upon treatment with a crude elicitor preparation from the bean pathogen Colletotrichum lindemuthianum, the pterocarpan phytoalexin medicarpin accumulated in cells and culture medium. The extractable activities of six enzymes involved in medicarpin biosynthesis (including three cytochrome P450 activities) were induced by treatment with elicitor, and their induction kinetics correlated with the rate of medicarpin accumulation. However, protoplasts prepared from these cultures accumulated neither medicarpin nor other secondary products after treatment with elicitor. The cytochrome P450 activities were induced during the preparation of the protoplasts, but could be further induced by treatment with fungal elicitor. The results are discussed in relation to the use of alfalfa protoplasts as a system for functional analysis of cloned defense genes.Abbreviations AUFS
absorption unit full scale
- CHI
chalcone isomerase (EC 5.5.1.6)
- CHS
chalcone synthase (EC 2.3.1.74)
- C40H
cinnamic acid 4-hydroxylase (EC 1.14.13.11)
- CLE
elicitor from Colletotrichum lindemuthianum
- IFOH
isoflavone 2-hydroxylase
- IFS
isoflavone synthase
- PAL
L-phenylalanine ammonia-lyase (EC 4.3.1.5) 相似文献
18.
Role of extensin peroxidase in tomato (Lycopersicon esculentum Mill.) seedling growth 总被引:3,自引:0,他引:3
It is proposed that inhibition of extensin peroxidase activity leads to a less rigid cell wall and thus promotes cell expansion
and plant growth. A low-molecular-weight inhibitor derived from the cell walls of suspension-cultured tomato cells was found
to completely inhibit extensin peroxidase-mediated extensin cross-linking in vitro at a concentration of 260 μg/ml. The inhibitor
had no effect upon guaiacol oxidation catalyzed by extensin peroxidase or horseradish peroxidase. We have demonstrated that
the light-irradiated inhibition of plant growth may be partially offset by inhibition of endogenous extensin peroxidase activity.
Overall plant growth was enhanced by up to 15% in the presence of inhibitor relative to control plants. Inhibitor-treated
and illuminated tomato hypocotyls grew up to 15% taller than untreated controls. The inhibitor had no effect upon etiolated
plants over a 15-d period, suggesting that only low levels of peroxidase-mediated cross-linking can be found in the cell walls
of etiolated plants. SDS-PAGE/Western blots of ionically bound protein from both etiolated and illuminated hypocotyls identified
a doublet at 57/58.5 kDa which is immuno-reactive with antibodies raised to tomato extensin peroxidase. Levels of the 58.5-kDa
protein, determined by SDS-PAGE, were at least threefold higher in illuminated tomato hypocotyls than in etiolated hypocotyls.
Three fold higher levels of extensin peroxidase, elevated in-vitro extensin cross-linking activity and 15% higher levels of
cross-linked, non-extractable extensin were observed in illuminated tomato hypocotyls compared with etiolated tomato hypocotyls.
This suggests that white-light inhibition of tomato hypocotyl growth appears to be mediated, at least partially, by deposition
of cell wall extensin, a process regulated by Mr-58,500 extensin peroxidase. Our results indicate that the contribution of peroxidase-mediated extensin deposition to plant
cell wall architecture may have an important role in plant growth.
Received: 22 July 1999 / Accepted: 11 October 1999 相似文献
19.
Mapping of simple sequence repeat (SSR) DNA markers in diploid and tetraploid alfalfa 总被引:8,自引:3,他引:8
N. Diwan J. H. Bouton G. Kochert P. B. Cregan 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,101(1-2):165-172
Cultivated alfalfa (Medicago sativa) is an autotetraploid. However, all three existing alfalfa genetic maps resulted from crosses of diploid alfalfa. The current
study was undertaken to evaluate the use of Simple Sequence Repeat (SSR) DNA markers for mapping in diploid and tetraploid
alfalfa. Ten SSR markers were incorporated into an existing F2 diploid alfalfa RFLP map and also mapped in an F2 tetraploid population. The tetraploid population had two to four alleles in each of the loci examined. The segregation of
these alleles in the tetraploid mapping population generally was clear and easy to interpret. Because of the complexity of
tetrasomic linkage analysis and a lack of computer software to accommodate it, linkage relationships at the tetraploid level
were determined using a single-dose allele (SDA) analysis, where the presence or absence of each allele was scored independently
of the other alleles at the same locus. The SDA diploid map was also constructed to compare mapping using SDA to the standard
co-dominant method. Linkage groups were generally conserved among the tetraploid and the two diploid linkage maps, except
for segments where severe segregation distortion was present. Segregation distortion, which was present in both tetraploid
and diploid populations, probably resulted from inbreeding depression. The ease of analysis together with the abundance of
SSR loci in the alfalfa genome indicated that SSR markers should be a useful tool for mapping tetraploid alfalfa.
Received: 10 September 1999 / Accepted: 11 November 1999 相似文献
20.
The pathogenesis-related accumulation of superoxide radical anions (O·−
2) and hydrogen peroxide (H2O2) was comparatively analyzed in a barley line (Hordeum vulgare L. cv Sultan-5) carrying the powdery mildew (Blumeria graminis f.sp. hordei, Speer, Bgh) resistance gene Mla12, and in susceptible mutants defective in Mla12 or in genes “required for Mla12-specified disease resistance” (Rar1 and Rar2). In-situ localization of reactive oxygen intermediates was performed both by microscopic detection of azide-insensitive
nitroblue tetrazolium (NBT) reduction or diaminobenzidine (DAB) polymerization, and by an NBT-DAB double-staining procedure.
The Mla12-mediated hypersensitive cell death occurred either in attacked epidermal cells or adjacent mesophyll cells of wild-type plants.
Whole-cell H2O2 accumulation was detected in dying cells, while O·−
2 emerged in adjacent cells. Importantly, all susceptible mutants lacked these reactions. An oxalate oxidase, which is known
to generate H2O2 and has been implicated in barley resistance against the powdery mildew fungus, was not differentially expressed between
the wild type and all mutants. The results demonstrate that the Rar1 and Rar2 gene products, which are control elements of R-gene-mediated programmed cell death, also control accumulation of reactive oxygen intermediates but not the pathogenesis-related
expression of oxalate oxidase.
Received: 7 January 2000 / Accepted: 2 June 2000 相似文献