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1.
Summary The utility of numerous dyes for determining the viability of barley ( Hordeum vulgare L. cv. Himalaya) aleurone protoplasts was studied. Protoplasts isolated from the barley aleurone layer synthesize and secrete -amylase isozymes in response to treatment with gibberellic acid (GA) and Ca 2+. These cells also undergo dramatic morphological changes which eventually result in cell death. To monitor the viability of protoplasts during incubation in GA and Ca 2+, several types of fluorescent and nonfluorescent dyes were tested. Evans blue and methylene blue were selected as nonfluorescent dyes. Living cells exclude Evans blue, but dead cells and cell debris stain blue. Both living and dead cells take up methylene blue, but living cells reduce the dye to its colorless form whereas dead cells and cell debris stain blue. The relatively low extinction coefficient of these dyes sometimes makes it difficult to distinguish blue-stained cells against a background of blue dye. Several types of fluorescent dyes were tested for their ability to differentially stain dead or living cells. Tinopal CBS-X, for example, stains only dead cells, and its high extinction coefficient allows its ultraviolet fluorescence to be recorded even when preparations are simultaneously illuminated with visible light. To double-stain protoplasts, the most effective stain was a combination of fluorescein diacetate (FDA) and propidium iodide (PI). By employing a double-exposure method to record the fluorescence from cells stained with both FDA and PI, dead and living cells could be distinguished on the basis of fluorochromasia. 相似文献
2.
A technique has been developed which permits mechanistic studies of phloem unloading in developing seeds of soybean ( Glycine max cv Clark) and other legumes. An opening is cut in the pod wall and the embryo surgically removed from the seedcoat without diminishing the capacity of that tissue for assimilate import, phloem unloading, or efflux. The sites of phloem unloading were accessible via the seedcoat apoplast and were challenged with inhibitors, solutes, buffers, etc., to characterize the unloading process. Unloading is stimulated by divalent metal chelators and diethylstilbestrol, and inhibited by metabolic uncouplers and sulfhydryl group modifiers. Solutes released from the seed coat had a carbon/nitrogen ratio of 31 milligrams carbon per milligram nitrogen; sucrose represented 90% of the carbon present and various nitrogenous solutes contributed the remaining 10%. Unloading could be maintained for up 8 hours at rates of 0.5 to 1.0 micromoles per hour, providing a valid, convenient in vivo technique for studies of phloem unloading and seed growth mechanisms. 相似文献
3.
The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed. 相似文献
4.
The rates of CO 2 exchange and 14CO 2 incorporation in the light and dark and the activities of several photosynthetic, photorespiratory, and respiratory enzymes of soybean ( Glycine max [L.] Merr. cv. Wye) reproductive structures were determined at weekly intervals from anthesis to pod maturity. At all stages of pod development soybean reproductive structures were found to be incapable of net photosynthesis under the experimental conditions employed, but capable of gross photosynthesis and light-induced 14CO 2 uptake. Consistent with the lack of net photosynthesis throughout the development of the reproductive structure, the maximum in vitro activity of ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) in pod tissue was only 3% of that in leaf extracts when expressed on a fresh weight basis. We concluded that the major role of the reproductive structure of the soybean with respect to photosynthetic carbon metabolism is the reassimilation of its respiratory CO 2. 相似文献
5.
The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed. 相似文献
6.
Multiple skin sections from three nonhuman primates ( Macaca mulatta) and three hairless guinea pigs ( Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference. 相似文献
7.
In plant cells, Ca 2+ is required for both structural and biophysical roles. In addition, changes in cytosolic Ca 2+ concentration ([Ca 2+] cyt) orchestrate responses to developmental and environmental signals. In many instances, [Ca 2+] cyt is increased by Ca 2+ influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca 2+-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca 2+-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca 2+-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca 2+ channels, the genetic counterparts of specific Ca 2+-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca 2+ channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca 2+ channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La 3+ and nifedipine, and their increased activity as [Ca 2+] cyt is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca 2+-permeable) K + channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels ( CNGC) and ionotropic glutamate receptor ( GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels. 相似文献
8.
Soybean looper, Chrysodeixis includens (Walker), is one of the most destructive pests of soybean in the southern U.S. Soybean looper defoliation exceeding 20% from R3 (pod initiation) to R5 (pod fill) can result in significant yield loss. In addition, soybean looper is highly resistant to many insecticides. An alternative to insecticide control is induced host plant resistance. In this study, a total of four experiments over 2 years were conducted in which three different elicitors of SAR (systemic acquired resistance), Actigard 50WG (acibenzolar-S-methyl), Regalia (extract of Reynoutria sachalinensis), and methyl jasmonate (MeJA), were applied to soybean at different plant stages to determine if these chemicals could induce plant resistance and lower soybean looper fitness. None of the elicitors of SAR significantly affected soybean looper mortality. However, Actigard 50WG, MeJA, and Regalia had adverse effects on developmental time, defoliation, and pupal weight of soybean looper. Induced effects by Regalia on soybean looper were very limited compared to Actigard 50WG and MeJA. A single application of MeJA reduced pupal weight by 6.8% and delayed larval development by 14.3%. Soybean seed production was not affected by application of elicitors. In conclusion, the results suggest that exogenous elicitors applied in the field can trigger plant resistance against herbivores and this low level of host plant resistance may effectively lessen pest pressure by favoring natural enemy population regulation without reducing seed production. 相似文献
9.
An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca2+ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KCl to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca2+. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca2+ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate. 相似文献
10.
A new naturally occurring chalcone glycoside, chalcone, and other chalcones, aurones, flavonols, and anthocyanins are reported from Megalodonta ( Bidens) beckii. The plan is an amphibious aquatic with heterophyllous leaves, but the presence of certain flavonoids is correlated with vegetative versus floral tissues rather than vegetative leaf form. The implications of flavonoid chemistry with respect to the relationship of M. beckii to several sections of Bidens are discussed. 相似文献
11.
An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca 2+ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KC1 to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca 2+. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca 2+ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate. 相似文献
12.
The environmental sensitivity of the processes associated with the import of photosynthate by developing soybean seeds was investigated within intact fruit and with excised, immature embryos. Intact pods of field-grown ( Glycine max [L.] Merr.) Amsoy 71 soybeans were subjected to localized regimes of 0, 21, or 100% O 2 and 15, 25, or 35°C during pulsechase translocation experiments and, 2.5 hours later, the uptake and distribution of 14C-photosynthate among dissected fruit tissues determined. In other experiments, excised embryos were incubated in [ 14C]sucrose solutions under various experimental conditions to separate the effects of these treatments on accumulation by the embryos from those which may operate on phloem unloading in the maternal seedcoat. 相似文献
13.
Mitochondria isolated from etiolated shoots of corn ( Zea mays), wheat ( Triticum aestivum), barley ( Hordeum vulgare), soybean ( Glycine max L. Merr.), and mung bean ( Phaseolus aureus) exhibited a proline-dependent O 2 uptake subject to respiratory control. ADP/O ratios with proline as substrate were intermediate between ratios obtained with exogenous NADH and malate + pyruvate as substrates. Isotope studies showed proline metabolism to be dependent on O 2, but not NAD. The major ninhydrin-positive product formed via Δ 1-pyrroline-5-carboxylic acid was glutamate. Mitochondria were capable of further metabolism of glutamate, as radioactive CO 2, organic acids, and aspartate were recovered after [ 14C]proline feeding experiments. These results demonstrate the mitochondrial association and O 2 dependence of plant proline metabolism. 相似文献
14.
The new ultrasensitive photochemically derived silver stain described for polypeptides in polyacrylamide gels (Merril et al., Science211, 1437–1438 (1981)) also stains nucleic acid in polyacrylamide gels. Reovirus genome double-stranded (ds) RNA segments were clearly detected in gels at about 0.03 ng/mm 2 with the silver staining technique when either purified virions or isolated, purified dsRNA was analyzed. The silver stain was about 10 to 30 times more sensitive than ethidium bromide for detecting reovirus dsRNA. 相似文献
15.
Fura-2 and its lipid analogue, FFP-18, were used to measure changes in cytosolic free Ca 2+concentration within human neutrophils. Whereas fura-2 was employed to monitor cytosolic Ca 2+increases throughout the cytosol, FFP-18 was used to monitor Ca 2+changes only near the membrane. This latter probe was incorporated into the plasma membrane as its acetoxymethyl ester (FFP-18-AM) but as de-esterification was catalysed by cytosolic esterases, the Ca 2+-sensing probe (FFP-18 acid) accumulated on the inner face of membrane. The fluorescence of esterified probe on the extracellularly facing membrane leaflet was quenched by the membrane-impermeant ion Ni 2+. Under these conditions, near membrane Ca 2+changes which resulted from the release of Ca 2+from intracellular stores was possible by conventional ratio fluorescence measurement of FFP-18. From the timing of arrival of Ca 2+at the plasma membrane, it was proposed that there were two Ca 2+storage sites, liberated by different stimuli, one close to the plasma membrane and the other more distant. In order to discover whether organelles within the neutrophil had distributions which correlate with the Ca 2+release sites, fluorescent dyes for structures within the cytosol were employed. We have previously shown that the location of the intracellular membrane stain, DiOC 6(3) corresponds to the distant Ca 2+release site. Here a second stain, BODIPY-C 5ceramide, has also been used and is shown to stain a peripheral region of the neutrophil, in a similar pattern to the near membrane Ca 2+storage site. These data therefore raise the question of whether these stains mark the organelles in neutrophils which are the two Ca 2+storage and release sites. 相似文献
16.
Many abiotic stresses induce the generation of nitric oxide (NO) in plant tissues, where it functions as a signal molecule in stress responses. Plants modulate NO by oxidizing it to NO 3− with plant hemoglobin (GLB), because excess NO is toxic to cells. At least eight genes encoding GLB have been identified in soybean, in three clades: GLB1, GLB2, and GLB3. However, it is still unclear which GLB genes are responsible for NO regulation under abiotic stress in soybean. We exposed soybean roots to flooding, salt, and two NO donors—sodium pentacyanonitrosylferrate (III) dihydrate (SNP) and S-nitroso- N-acetyl- d,l-penicillamine (SNAP)—and analyzed expression of GLB genes. GmGLB1, one of two GLB1 genes of soybean, significantly responded to both SNP and SNAP, and its induction was almost completely repressed by a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. GmGLB1 responded to flooding but not to salt, suggesting that it is responsible for NO regulation under NO-inducing abiotic stresses such as flooding. GmGLB3, one of two GLB3 genes of soybean, did not respond to NO donors at all but did respond to flooding, at a lower level than GmGLB1. These results suggest that flooding induces not only NO but also unknown factor(s) that induce GmGLB3 gene in soybean. 相似文献
17.
Locust neural lamella and Calpodes connective tissue fixed in glutaraldehyde have a fibrous component which stains after reaction with DAB and osmication and after staining sections with PTA. The fibers also stain when fixed in glutaraldehyde with tannic acid followed by osmication and section staining with lead citrate. 相似文献
18.
Radioactive photosynthetic assimilates, translocated to a soybean ( Glycine max [L.] Merr. `Fiskeby V') pod can be measured directly by excising the stylar tip of the pod under 20 m m ethylenediaminetetraacetate solution (pH 7.0) and allowing the material to leak into the solution. Pods at the source node received approximately 50% of the 14C exported from the source leaf to the pod and leaked approximately 1 to 3% of this into the solution. More than 90% of the 14C that leaked from the pods was found in the neutral fraction and, of this, about 93% was in sucrose. Fifteen amino acids were identified in the leakage including: alanine, arginine, asparagine, γ-aminobutyric acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, serine, threonine, tyrosine, and valine. The majority of the 14C in the basic fraction was found in serine (30%) and asparagine (23%). The inorganic ions K, Ca, P, Mg, Zn, and Fe were found in the leakage component. Nitrate was not detectable in the collected leakage solution. The absence of NO 3− and the large proportion of the label in sucrose suggest a possible phloem origin for most of the material. The technique provides an uncomplicated, reproducible means of analyzing the material translocated into and through the soybean pod, as well as following the time course of label arrival at the pod. 相似文献
19.
A bench study was carried out to characterize the kinetics of two plant growth promoting rhizobacteria (PGPR) Azotobacter chroococcum and Bacillus megaterium to adsorb heavy metals from solution. Adsorption of Pb 2+ and Cd 2+ by bacterial cells was processed quickly with an equilibration achieved within 5 min. The adsorptions were fitted well with Freundlich and Langmuir isotherm models. The comparison of isotherm parameters indicated that A. chroococcum had a stronger capacity to bind metal ions than B. megaterium, with an average increase of 59.8% for Pb 2+ and 75.6% for Cd 2+, respectively. Both bacteria had a stronger affinity to Pb 2+ than Cd 2+ since Pb 2+ was more easily bound with the phosphoryl groups on the cell surface than Cd 2+. This demonstrated that the presence of bacteria in the rhizosphere may result in the reduction of mobile ions in soil solution. 相似文献
20.
Field experiments were conducted in 1981 and 1982 to study the effects of low-irradiance supplemental light on soybean ( Glycine max [L.] Merr. cv Evans) flower and pod abscission. Cool-white and red fluorescent lights illuminated the lower part of the soybean canopy during daylight hours for 3 weeks late in flowering. At the same time, flowers and young pods on half the plants were shaded with aluminum foil. Flowers were tagged at anthesis and monitored through abscission or pod maturity. Responses to red and white lights were similar. Supplemental light tended to reduce abscission and increase seed weight per node compared to natural light. Shading flowers and pods increased abscission and reduced seed weight per node. Number of flowers produced per node, individual seed weight, and seeds per pod were not affected by light or shade treatments. Further studies examined the effects of shading reproductive structures on their capacity to accumulate 14C-photoassimilates. Individual leaves were pulse labeled with 14CO2 1, 2, and 4 weeks post anthesis. Flowers and pods in the axil of the labeled leaf were covered with aluminum foil 0, 24, 72, and 120 hours before pulsing. Shading flowers and pods resulted in a 30% reduction in the relative amount of radiolabel accumulated from the source leaf. The reduction in 14C accumulation due to shading was evident regardless of the length of the shading period and was most pronounced when the shades were applied early in reproductive development. We conclude that light perceived by soybean flowers and young pods has a role in regulating both their abscission and their capacity to accumulate photoassimilates. 相似文献
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