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1.
Adrian J. Cutler Mohammed Saleem Hong Wang 《In vitro cellular & developmental biology. Plant》1991,27(3):104-111
Summary Cereal leaf protoplasts are extremely difficult to culture (recalcitrant) in vitro. There have been few reports of division
and the protoplasts typically exhibit excessive enlargement and vacuolization with reduced cell wall deposition. Inasmuch
as leaf base explants are capable of callus formation in vitro, protoplasts derived from this tissue must have lost the ability
to divide as a consequence of changes induced by the wall-digestion process. We review evidence suggesting that the inhibition
of mitosis in these protoplasts is a consequence of a cascade of events initiated at the plasma membrane. The enzyme treatment
necessary for wall removal triggers membrane depolarization and other changes that can lead to the initiation of lipid peroxidation
and oxidative stress. Mitotically inactive cereal leaf protoplasts are unable to mount a protective response to these degradative
processes. Consequently, the resulting membrane perturbations and permeabilization give rise to secondary effects on the cytoskeleton
and the cell wall. These effects include reduced or absent microtubules as well as reduced and uneven wall deposition. Such
abnormalities are observed in cereal leaf protoplasts and are sufficient to account for recalcitrance because the occurrence
of mitosis is strongly dependent on a normal cell wall and cytoskeleton.
This paper is NRCC number 32475. 相似文献
2.
Adrian J. Cutler Mohammed Saleem Maxine A. Coffey Mary K. Loewen 《Plant Cell, Tissue and Organ Culture》1989,18(1):113-127
Cereal leaf protoplasts are often extremely unstable in culture and usually lyse within 24 hours. Using the thiobarbituric acid test and the ferrous thiocyanate test we have shown that corn (Zea mays L. cv. Market Beauty) and wheat (Triticum aestivum L. cv. Benito) leaf protoplasts accumulate peroxides and peroxide degradation products during culture. This increase correlated with an increase in lipoxygenase activity. On the other hand, enzymes involved in detoxification of peroxides such as catalase and peroxidase decreased during culture. The occurrence of lipid peroxidation in leaf protoplasts is likely to be a consequence of a temporary imbalance in the enzymes involved in oxygen metabolism. It has previously been shown that the lipoxygenase inhibitor n-propyl gallate stabilizes the protoplasts in culture and so peroxidation is likely to be the cause of leaf protoplast instability. Protoplasts obtained from suspension cultures are stable in culture and do not undergo lipid peroxidation. This stability is due to a decrease in lipoxygenase activity and increases in catalase and peroxidase activity after protoplast isolation.Abbreviations MDA
malonaldehyde
- 2,4-D
2,4-dichlorophenoxyacetic acid
- PVP
polyvinylpolypyrrolidone
Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday 相似文献
3.
以马铃薯抗青枯病二倍体材料ED13和CE171、炸片颜色好的二倍体材料HS66以及优良性状双单体材料DH401和DH405为供体材料,对马铃薯叶肉原生质体培养进行研究。叶片悬浮黑暗预处理和试管苗黑暗预处理两种预处理方式对原生质体活力无显著影响。以0.5 mol/L甘露醇为渗透调节剂,25℃酶解12 h条件下,适宜CE171和DH401纤维酶浓度略高于ED13、HS66和DH405,分别为0.3%和0.4%。ED13和DH401原生质体在VKM液体培养基中培养3~4周,经愈伤组织生长培养基培养2周,转至芽诱导培养基培养,2~3个月后形成具根茎叶的完整植株。HS66和CE171原生质体培养6~8周也能形3~4 mm愈伤组织,但没有分化出芽;DH405的原生质体不分裂。 相似文献
4.
J. J. Rybczyński 《Plant cell reports》1989,8(7):383-386
Explants from hypcotyls and cotyledons of Browalia speciosa were shown to regenerate plantlets.Protoplasts were isolated from etiolated cotyledon material, and, although callus was readily obtained, plantlet regeneration was not observed using numerous hormone regimes.Abbreviations M
Mannitol
- 2,4-D
Dichlorophenoxyacetic acid
- NAA
Naphthalene-acetic acid
- BAP
Benzylaminopurine
- MS medium
Murashige and Skoog (1962) medium
- UM medium
Uchimiya and Murashige (1974) medium
- COT
cotyledon
- SH
shoot
- R
root 相似文献
5.
6.
7.
Attila Marton Csongor I. Vgsi Orsolya Vincze Veronika Bkony Pter L. Pap Laura Ptra Janka Pnzes Lrinc Brbos Attila Fülp Gergely Osvth Simon Ducatez Mathieu Giraudeau 《Ecology and evolution》2022,12(8)
The mechanistic link between avian oxidative physiology and plumage coloration has attracted considerable attention in past decades. Hence, multiple proximal hypotheses were proposed to explain how oxidative state might covary with the production of melanin and carotenoid pigments. Some hypotheses underscore that these pigments (or their precursors, e.g., glutathione) have antioxidant capacities or function as molecules storing the toxic excess of intracellular compounds, while others highlight that these pigments can act as pro‐oxidants under specific conditions. Most studies addressing these associations are at the intraspecific level, while phylogenetic comparative studies are still scarce, though needed to assess the generality of these associations. Here, we tested whether plumage and bare part coloration were related to oxidative physiology at an interspecific level by measuring five oxidative physiology markers (three nonenzymatic antioxidants and two markers of lipid peroxidative damage) in 1387 individuals of 104 European bird species sampled during the breeding season, and by scoring plumage eumelanin, pheomelanin, and carotenoid content for each sex and species. Only the plasma level of reactive oxygen metabolites was related to melanin coloration, being positively associated with eumelanin score and negatively with pheomelanin score. Thus, our results do not support the role of antioxidant glutathione in driving variation in melanin synthesis across species. Furthermore, the carotenoid scores of feathers and bare parts were unrelated to the measured oxidative physiology parameters, further suggesting that the marked differences in pigmentation across birds does not influence their oxidative state. 相似文献
8.
Summary Degradative changes in tissue during protoplast isolation were a contributing factor to low protoplast yields in the saltsensitive
Grevillea arenaria (R. Brown) and the salt-tolerant Grevillea ilicifolia (R. Brown). Protein and malondialdehyde content decreased significantly during the protoplast isolation procedure. Acid and
neutral proteases were identified, and high acid protease activities were correlated to low protoplast yields. Acid phosphatase,
catalase, polyphenol oxidase and lipoxygenase activities increased in both Grevillea species with cell wall digestion. High activities of catalase and low levels of polyphenol oxidase were correlated with high
protoplast yields. Levels of acid phosphatase and lipoxygenase were not good indicators of final protoplast yields. The addition
of the anti-oxidant, reduced glutathione, and the acid protease inhibitor, pepstatin A, significantly increased protoplast
yields. Strategies were identified to minimize deleterious degradative effects during the isolation of protoplasts, including
strict pH control, testing a number of cell wall digestion enzymes, and the addition of anti-oxidative metabolites and protease
inhibitors. 相似文献
9.
The SH compound glutathione (GSH) is involved in several fundamental functions in the cell, including protection against reactive oxygen species (ROS). Here, we studied the effect on oxidative DNA damage in cultured skin fibroblasts from patients with hereditary GSH synthetase deficiency. Our hypothesis was that GSH-deficient cells are more prone to DNA damage than control cells. Single cell gel electrophoresis (the comet assay) in combination with the formamidopyrimidine DNA glycosylase enzyme, which recognizes oxidative base modifications, was used on cultured fibroblasts from 11 patients with GSH synthetase deficiency and five control subjects. Contrary to this hypothesis, we found no significant difference in background levels of DNA damage between cells from patients and control subjects. To study the induction of oxidative DNA damage without simultaneous DNA repair, the cells were γ-irradiated on ice and DNA single-strand breaks measured. The patient and control cells were equally sensitive to induction of single strand breaks by γ-irradiation. Therefore, factors other than GSH protect DNA from oxidative damage. However, cells with a high background level of oxidative DNA damage were found to be more sensitive to ionizing radiation. This suggests that differences in background levels of oxidative DNA damage may depend on the cells' intrinsic protection against induction of oxidative damage. 相似文献
10.
Hexachlorocyclohexane (HCH) is reported to induce oxidative stress in liver and testis of rat. With an objective to examine its effect on brain tissue acute toxicity of HCH (10 and 20 mg/kg body wt, i.p.) on the antioxidant defense system of cerebral hemisphere of rat was evaluated. Lipid peroxidation (LPX) was elevated after 24 h in the crude homogenate and sub-cellular fractions (nuclear and mitochondrial) except the microsomal fraction in which LPX was induced after 6 h and remained elevated till 24 h. The pesticide elicited decrease in the activities of cytosolic total, CN–-sensitive (not at 24 h) and CN-resistant superoxide dismutases; total, Se-dependent and Se-independent glutathione peroxidases; and catalase throughout the measurement period. In contrast, glutathione reductase activity was elevated till 24 h after a fall at 6 h of pesticide exposure. Cerebral contents of glutathione and ascorbic acid were decreased in response to HCH. The results suggest the possible involvement of reactive oxygen species in the mechanism of HCH-induced neurotoxicity in rat. 相似文献
11.
Bosch M Marí M Gross SP Fernández-Checa JC Pol A 《Traffic (Copenhagen, Denmark)》2011,12(11):1483-1489
Caveolin (CAV) is an essential component of caveolae, cholesterol-enriched invaginations of the plasma membrane of most mammalian cells. However, CAV is not restricted to plasma membrane caveolae, and pools of CAV are present in myriad intracellular membranes. CAV proteins tightly bind cholesterol and contribute to regulation of cholesterol fluxes and distributions within cells. In this context, we recently showed that CAV1 regulates the poorly understood process controlling mitochondrial cholesterol levels. Cholesterol accumulates in mitochondrial membranes in the absence of CAV1, promoting the organelle's dysfunction with important metabolic consequences for cells and animals. In this article, we suggest a working hypothesis that addresses the role of CAV1 within the homeostatic network that regulates the influx/efflux of mitochondrial cholesterol. 相似文献
12.
When segments of rye leaves (Secale cereale L.) grown at 90 μmol m?2 s?1 PAR were incubated at a higher photon flux of 400–500 μ mol m?2 s?1 PAR in the presence of 0.2-0.6 M NaCl, a preferential loss of catalase activity was induced. The extent of this decline increased with the concentration of NaCl. In addition, the accumulation of alternative antioxidative components, such as ascorbate, glutathione, glutathione reductase, or peroxidase, was inhibited. The total content of H2O2 was, however, lower in catalase-depleted than in untreated control leaves. The occurrence of strong oxidative stress in NaCl-treated leaves was indicated by marked declines in the ratios of reduced to oxidized ascorbate and glutathione and by the degradation of chlorophyll in light. The specific elimination of catalase activity by the inhibitor aminotriazole was also accompanied by a rapid decline in the ratio of reduced to oxidized glutathione but other symptoms of oxidative stress were much less severe than in the presence of NaCl. However, all symptoms of photooxidative damage seen in NaCl-treated leaves were closely mimicked by treatment with the translation inhibitor, cycloheximlde, in light. The results suggest that NaCl-induced oxidative damage in light was predominantly mediated by the inhibition of protein synthesis. By this inhibition the resynthesis of catalase, which has a high turnover in light, was blocked and the leaves were thus depleted of catalase activity and, in addition, the intensification of alternative antioxidative systems was also prevented. 相似文献
13.
14.
Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L. 总被引:1,自引:0,他引:1
Theodoropoulos P. A. Roubelakis-Angelakis K. A. 《Plant Cell, Tissue and Organ Culture》1990,20(1):15-23
Axenic shoot cultures of virus-free Vitis vinifera L. cv. Soultanina were a highly efficient source for isolation of viable protoplasts. Optimum results were obtained with leaves of 50–100 mg fresh weight, leaf discs of 0.7 cm in diameter, 100 and 15 U ml-1 Cellulase R-10 and Macerozyme R-10, respectively, and 18 h reaction time in either light or in darkness. Protoplast yield was approx. 25×106 viable protoplasts per g fresh weight and their size ranged from 12 to 44 m. During a 20-day culture period, the maximum survival rate obtained was approx. 40%. A plating density of 10×105 protoplasts per ml resulted in increased survival rates. Various growth regulators and glutamine did not significantly improve survival rates of protoplasts, whereas extract from coconut added to the culture medium caused an increase in the survival rates of protoplasts. Cell elongation at a significant rate and divisions were observed. [14C]glucose uptake was studied as an index of cell membrane integrity and functioning. Uptake rate of glucose by protoplasts was linear for up to 60 min, fully inhibited by NaN3, with an optimum pH of 4.8. Protoplasts 24 h old exhibited significantly lower rates of glucose uptake. 相似文献
15.
Bolandi A.R. Branchard M. Alibert G. Serieys H. Sarrafi A. 《Plant Cell, Tissue and Organ Culture》1999,57(3):189-193
Protoplasts of 6 alloplasmic and 2 euplasmic sunflower inbred lines were isolated from dark grown seedling hypocotyls with
a density of 2×104 protoplasts/ml. The protoplast suspension was mixed with a solution of 0.5% agarose (sigma – type 1), then pipetted in droplets
of about 1000 protoplasts. Droplets were surrounded by two different liquid media. After 30 days droplets from both media
were transferred to solid differentiation medium. Protoplast division, microcolony frequency and the number of calluses produced
were strongly dependent on medium composition and genotype. The number of calluses per 1000 protoplasts plated range from
0.3 to 5.0 according to the genotype and the method used. The alloplasmic line RHA274-PEF1, was the best responding genotype
for calluses produced in both media used. In all cases, the percentage of calluses for alloplasmic lines were significantly
higher when compared with the nucleus donor genotype. H. petiolaris fallax cytoplasm increased both the number of calluses produced and the percentage of microcolonies. The complex interaction among
genotypes tested indicates that protoplast culture responses are affected independently by nuclear-cytoplasm interactions.
Some nucleus-cytoplasm combinations can improve the protoplast culture responses in sunflower.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
16.
To regenerate auxotrophic mutants of Physcomitrella patens, two media of increasing complexity were developed. The survival rate of protoplasts was around 30% higher on full medium when compared to standard minimal medium. Protoplast survival was higher in a medium containing 2.5 mmol/L ammonium tartrate compared to a medium with 5 mmol/L of this compound. Solid medium had a positive effect on protoplast survival compared to either liquid medium or solid medium overlaid with cellophane; the maximum survival rate being 31.6%. However, the number of surviving protoplasts without any cell division during the first ten days increased on solid medium. Density and survival rate of protoplasts were positively correlated, but the formation of long protonema filaments decreased markedly. The effect of different protoplast densities could be explained partly by physiologically active compounds excreted into the medium. 相似文献
17.
《Free radical research》2013,47(3):154-163
AbstractPharmacological ascorbate, via its oxidation, has been proposed as a pro-drug for the delivery of H2O2 to tumors. Pharmacological ascorbate decreases clonogenic survival of pancreatic cancer cells, which can be reversed by treatment with scavengers of H2O2. The goal of this study was to determine if inhibitors of intracellular hydroperoxide detoxification could enhance the cytotoxic effects of ascorbate. Human pancreatic cancer cells were treated with ascorbate alone or in combination with inhibitors of hydroperoxide removal including the glutathione disulfide reductase inhibitor 1,3 bis (2-chloroethyl)-1-nitrosurea (BCNU), siRNA targeted to glutathione disulfide reductase (siGR), and 2-deoxy-D-glucose (2DG), which inhibits glucose metabolism. Changes in the intracellular concentration of H2O2 were determined by analysis of the rate of aminotriazole-mediated inactivation of endogenous catalase activity. Pharmacological ascorbate increased intracellular H2O2 and depleted intracellular glutathione. When inhibitors of H2O2 metabolism were combined with pharmacological ascorbate the increase in intracellular H2O2 was amplified and cytotoxicity was enhanced. We conclude that inclusion of agents that inhibit cellular peroxide removal produced by pharmacological ascorbate leads to changes in the intracellular redox state resulting in enhanced cytotoxicity. 相似文献
18.
Ferrous ion (Fe2+) has been considered to be a cause of neuronal oxidative injury. Since body fluids contain protein and serum is an essential component of tissue culture medium, we have examined the role of serum protein on Fe2+-mediated oxidative stress using PC12 cells and rat cerebral cortices. Fe2+ or the combination of ascorbate and Fe2+ increased concentrations of thiobarbituric acid reactive substances (TBARS) in PC12 cells and cerebrocortical homogenates in medium (RPMI 1640), but did not increase TBARS when the medium was supplemented with 10% fetal bovine serum. Treatment with ascorbate/Fe2+ in serum-free medium reduced endogenous glutathione (GSH) concentration in PC12 cells. However, the medium supplemented with serum did not reduce GSH concentrations. PC12 cell death induced by ascorbate/Fe2+ was alleviated by increasing serum or bovine albumin concentrations in the medium. These observations indicated that oxidative injury caused by the transition metal ion could be lessened by adding fetal bovine serum to culture medium. 相似文献
19.
Effects of dopamine on the glutathione metabolism of cultured astroglial cells: implications for Parkinson's disease 总被引:3,自引:0,他引:3
To investigate the effects of dopamine (DA) on the release of glutathione (GSH) from astrocytes, we used astroglia-rich primary cultures from the brains of newborn rats. In the absence of DA, GSH accumulated in the medium of these cultures with a constant rate. In contrast, during incubation of the cells with 50 micro m DA extracellular GSH was not detectable anymore. This disappearance of extracellular GSH was prevented by superoxide dismutase, indicating that DA does not affect GSH release but rather reacts with the released GSH in a superoxide-dependent reaction. Incubation of astroglial cultures with 0.5 and 1 mm DA established almost constant extracellular concentrations of H2O2 of 5 microm and 15 microm, respectively. Under these conditions astroglial cultures release glutathione disulphide (GSSG). This GSSG export was blocked by catalase and by MK571, an inhibitor of the multidrug resistance protein 1. The effects of DA on the extracellular accumulations of GSH and GSSG were not modulated by inhibitors of DA receptors, DA transport, and monoamine oxidases. The other catecholamines adrenaline and noradrenaline showed similar effects on the accumulation of GSH and GSSG in the medium compared with those obtained for DA. In conclusion, the data presented demonstrate that DA affects astroglial GSH metabolism by two mechanisms: (i) directly by chemical reaction with extracellular GSH, and (ii) indirectly by generation of hydrogen peroxide that leads to the efflux of GSSG from astroglial cells. These observations are discussed in the context of the brain's GSH metabolism in Parkinson's disease. 相似文献
20.
Antioxidant responses to varying degrees of paraquat stress in freshly isolated photosynthesizing pea (Pisum sativum L.) protoplasts from cultivars Progress and Nugget were studied. Leaves of comparable maturity were used for protoplast isolation. Nugget protoplasts were more resistant to paraquat in the micromolar range under our conditions. In Nugget, a non-bleaching paraquat concentration (10 µM) inhibited CO2-dependent O2 evolution ca 50% during the first 40 min, remaining at that rate (“coping behavior”) for up to 100 min. In contrast, Progress protoplasts treated with the same concentration of paraquat did not exhibit coping behavior. Antioxidant enzyme activities were unaltered throughout the time course of the experiment in treated protoplasts from Nugget and in chloroplasts isolated from them. Thus, the coping behavior of Nugget protoplasts cannot be attributed to changes in activities of the three antioxidant enzymes tested. Paraquat treatment did not affect antioxidant enzyme activities in Progress protoplasts nor in chloroplasts isolated from them. When higher doses of paraquat were used (12 h, 0.1 mM paraquat), protoplasts from both cultivars were rapidly bleached and total protein decreased to ca 30% of pre-stress levels. Glutathione reductase (GR, EC 1.6.4.2) activity dropped in protoplasts from both cultivars under the severe stress conditions in concert with declines in protein levels. However, superoxide dismutase (SOD, EC 1.15.1.1) activity remained constant over the first 9 h of the time course, increasing to ca 150& of original levels by the final, 12-h time point. The activity of the plastid Cu,Zn isoform, expressed as a percentage of total SOD activity, declined over the time course of the experiment while that of mitochondrial MnSOD appeared to increase. This change in activity of MnSOD correlated with cell decline, therefore, and was not correlated with protection. These data are in agreement with some earlier reports and are compatible with the hypothesis that SOD activity levels increase in response to reactive oxygen species levels, even under conditions leading to cell death. 相似文献