Glucose repression and autolysis ofSaccharomyces cerevisiae cells: alterations in the cytochemical localization of acid phosphatase

Summary Allerations in the localization of acid phosphatase inSaccharomyces cerevisiae during glucose repression and during autolysis have been studied. Cell morphology becomes distinctly changed after only 2 h in the presence of high glucose concentration while after 3 h of glucose repression the majority of the mitochondirial structures resemble promitochondria. Yeast cells repressed for 6 h contain almost completely degraded mitochondrial structures and numerous lipid droplets in the central vacuole and cytoplasm. Destruction of mitochondria is accompanied by the accumulation of acid phosphatase in these organelles and in the cytoplasm whereas its activity in the central vacuole is lowered, most probably because of the leakage of the enzyme into the cytoplasm.No preferential breakdown of mitochondria is observed during autolysis. On the contrary, mitochondria are apparently the last to be degraded. Digestion of cytoplasmic regions and membranous elements occurs intravacuolarly after sequestration by protrusions of the central vacuole which are formed at the initial stages of autolysis. Acid phosphatase is not released from the central vacuole, suggesting indirectly that vacuole enzymes do not migrate into the cytoplasm during autolysis.     

Direct determination of the chromosomal location of bunching onion and bulb onion markers using bunching onion–shallot monosomic additions and allotriploid-bunching onion single alien deletions

To determine the chromosomal location of bunching onion (Allium fistulosum L.) simple sequence repeats (SSRs) and bulb onion (A. cepa L.) expressed sequence tags (ESTs), we used a complete set of bunching onion–shallot monosomic addition lines and allotriploid bunching onion single alien deletion lines as testers. Of a total of 2,159 markers (1,198 bunching onion SSRs, 324 bulb onion EST–SSRs and 637 bulb onion EST-derived non-SSRs), chromosomal locations were identified for 406 markers in A. fistulosum and/or A. cepa. Most of the bunching onion SSRs with identified chromosomal locations showed polymorphism in bunching onion (89.5%) as well as bulb onion lines (66.1%). Using these markers, we constructed a bunching onion linkage map (1,261 cM), which consisted of 16 linkage groups with 228 markers, 106 of which were newly located. All linkage groups of this map were assigned to the eight basal Allium chromosomes. In this study, we assigned 513 markers to the eight chromosomes of A. fistulosum and A. cepa. Together with 254 markers previously located on a separate bunching onion map, we have identified chromosomal locations for 766 markers in total. These chromosome-specific markers will be useful for the intensive mapping of desirable genes or QTLs for agricultural traits, and to obtain DNA markers linked to these.     

Production of Pectic Enzymes by Fusarium oxysporum f. sp. cepae and its Involvement in Onion Bulb Rot

Fusarium oxysporum f. sp. cepae produced an exo-polygalacturonase (exo-PG) and endopectin-trans-eliminase (endo-PTE) in a mineral medium supplemented with a restricted supply of either D-galacturonic acid or onion cell walls. These enzymes were also extracted from infected onion tissue, but only endo-PTE caused tissue maceration and cell death. The patterns of host tissue colonization and pectic enzyme production were followed during bulb rot development. Stem plates were invaded within two weeks of inoculation. The pathogen then remained confined to the stem plates for several weeks or months, before spreading to the outer fleshy scales to initiate a basal rot. In most cases the inner leaf sheaths containing the lateral bud remained healthy. Exo-PG activity m stem plate tissue was greatest at two weeks after inoculation, then it declined. Endo-PTE was not detected in newly invaded stem plate tissue, but was recovered from infected stem plates before decay and from the bases of bulb scales and leaf sheaths at the onset of bulb rot. There was no pectic enzyme activity in uninvaded onion tissue. Spread of the fungus and pectic enzyme production in two Caledon Globe genotypes susceptible or tolerant to F. oxysporum f. sp. cepae were similar, but the onset of bulb rot in tolerant genotypes was considerably delayed.     

Quercetin-4′-glucoside: a physiological inhibitor of the activities of dominant glutathione <Emphasis Type="Italic">S</Emphasis>-transferases in onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.) bulb

The onion (Allium cepa L.) bulb has a high level of glutathione S-transferase (GST) activity, and it is a rich source of sulfur compounds as well as flavonoids. To investigate interactions between onion bulb GSTs and metabolites, we separated onion bulb GSTs (GSTa and GSTb as minor GSTs and GSTc, GSTd and GSTe as dominant GSTs) by DEAE-cellulose chromatography. In Western blot analysis with anti-CmGSTF1 antiserum, GSTc and GSTd fractions showed a thick band. A cDNA (AcGSTF1) corresponding to GSTc was immunoscreened with the same antiserum from an onion bulb cDNA library and its bacterial expression product was also subjected to investigation. Among the sulfur compounds, nonphysiological compounds, S-hexyl glutathione (GSH) and S-butyl GSH, showed strong inhibitory effects on 1-chloro-2,4-dinitrobenezene (CDNB)-conjugating activities of GSTa, GSTb and GSTe. However, physiological sulfur compounds, S-methyl GSH, S-propyl GSH, S-lactoyl GSH and S-ethyl-l-cysteine sulfoxide, had small or almost no inhibitory effects. Therefore, onion sulfur compounds might have the least possibility to be substantial inhibitors of onion GSTs. On the other hand, the activities of GSTc, GSTd and AcGSTF1 were strongly inhibited by flavonoids, quercetin, luteolin, apigenin and kaempferol. Ethylacetate (EtOAc) extract of onion bulb contained quercetin-4′-glucoside as a major inhibitory substance. The strong inhibitory effects of quercetin-4′-glucoside on GSTc and GSTd as well as on AcGSTF1 (50% inhibitory concentration (IC₅₀): 9.5, 7.5 and 11.2 μM, respectively) along with its high concentration (226 μM) in the onion bulb indicates that quercetin-4′-glucoside is a physiological inhibitor of dominant GSTs in the onion bulb.     

A new fluorescent test for cell vitality using calcofluor white M2R

The fluorescent fabric-brightener dye, Calcofluor white M2R (CFW), can be used to distinguish between living and dead cells from a variety of animal and plant sources. CFW does not stain living mouse fibroblasts or trout red blood cells and stains only the cell walls in living cells from the epidermis of onion bulb scale, staminal hairs of Tradescantia, and longitudinal sections of broad bean stems and roots. Heat-killed plant or animal cells are recognized by their lightly stained cytoplasm and brightly stained nuclei. The optimum staining concentrations were very low (0.01% to 0.03%) and nontoxic. Using onion scale epidermis in which some cells had been killed by heating as a test system, and the plasmolysis-deplasmolysis rection as the ultimate test for cell vitality, results from CFW staining correctly predicted cell vitality for about 98% of the cells tested. This success rate was comparable to those for Evans blue, uranin or neutral red in this test system.     

A New Fluorescent Test for Cell Vitality Using Calcofluor White M2R

The fluorescent fabric-brightener dye, Calcofluor white M2R (CFW), can be used to distinguish between living and dead cells from a variety of animal and plant sources. CFW does not stain living mouse fibroblasts or trout red blood cells and stains only the cell walls in living cells from the epidermis of onion bulb scale, staminal hairs of Tradescantia, and longitudinal sections of broad bean stems and roots. Heat-killed plant or animal cells are recognized by their lightly stained cytoplasm and brightly stained nuclei. The optimum staining concentrations were very low (0.01% to 0.03%) and nontoxic. Using onion scale epidermis in which some cells had been killed by heating as a test system, and the plasmolysisdeplasmolysis rection as the ultimate test for cell vitality, results from CFW staining correctly predicted cell vitality for about 98% of the cells tested. This success rate was comparable to those for Evans blue, uranin or neutral red in this test system.     

Sequence analysis of a chloroplast intergenic spacer for phylogenetic estimates in Allium section Cepa and a PCR-based polymorphism detecting mixtures of male-fertile and male-sterile cytoplasmic onion

Restriction-enzyme analysis of the chloroplast (cp) DNA yielded maternal phylogenies supporting a close phylogenetic relationship among normal (N) male-fertile and male-sterile (S) cytoplasmic bulb onion (Allium cepa), Allium altaicum, Allium fistulosum, Allium galanthum, Allium roylei, and Allium vavilovii. The S cytoplasm of onion is most likely an alien cytoplasm introduced in antiquity into onion populations. We previously showed that size differences in an intergenic spacer in the cp DNA distinguish N and S cytoplasms of onion. We cloned and sequenced this intergenic spacer from the N and S cytoplasms of onion, A. altaicum, A. fistulosum, A. galanthum, Allium pskemense, Allium oschaninii, A. roylei, and Allium ampeloprasm (outgroup) to identify the nature of previously described RFLPs and to develop a PCR-based marker revealing N-cytoplasmic contamination of S-cytoplasmic hybrid seed lots. Phylogenies based on restriction-enzyme analysis of the entire cp DNA were similar, but not identical, to those based on sequence divergence in this intergenic region. Received: 29 November 1999 / Accepted: 28 April 2000     

快速诱导细胞凋亡及检测方法研究

目的：探讨Ca2＋和Na＋诱导细胞凋亡的最佳浓度及时间,并用甲基绿一派诺宁染色法检测凋亡细胞的形态变化。方法：分别用不同浓度梯度及时间梯度的Ca2＋和Na＋胁迫处理洋葱鳞茎内表皮细胞,得诱导的最佳浓度及时间;用甲基绿一派诺宁染色法检测诱导凋亡的洋葱鳞茎内表皮细胞、大蒜根尖细胞和鸡血红细胞的形态特征变化。结果：诱导处理的最佳Ca2＋和Na＋浓度为0．4mol／L,最适时间约为8h,且CaCl2的诱导效果较NaCl好;经甲基绿一派诺宁染色,洋葱鳞茎内表皮细胞、大蒜根尖细胞、鸡血红细胞凋亡细胞的细胞核均呈蓝紫色,细胞质呈红色。结论：找出了诱导细胞凋亡的最适Ca2＋和Na＋浓度和时间,并检测到细胞凋亡。     

Observations of the pattern of secondary wall development in the hypodermis of onion (Allium cepa) roots

Summary This brief communication reports the appearance, under certain circumstances, of root hypodermal cells with transfer cell labyrinths. These cells lie at regular intervals around the hypodermis at the bases of onion bulb roots. They are narrower (smaller tangential dimension) than unmodified cells but have the same radial dimension. These narrow cells contain small vacuoles, their main volume being composed of a cytoplasm rich in organelles, especially mitochondria. When treated with a low concentration of lanthanum nitrate solution, the tracer accumulates in the outer tangential wall and in small vacuoles and vesicles.     

Sulfur and nitrogen fertility affects flavour of field-grown onions

Although glasshouse studies have conclusively demonstrated that S nutrition can affect onion (Allium cepaL.) pungency this has been rarely observed in field-based studies due to difficulties in controlling S nutrition and lack of efficient methods for measurement of flavour bioactives. We have developed a rapid automated method for determination of onion lachrymatory factor ((Z, E)-thiopropanal-S-oxide; LF) based on juice extraction into dichloromethane and gas chromatography (GC) analysis with flame photometric detection. We evaluated this in a field trial of a mild (cv. ‘Encore’) and a pungent (cv. ‘Kojak’) onion cultivar grown on a low S soil with and without S addition, under high or low N treatments. No treatments significantly affected bulb fresh weight but S fertilisation significantly increased bulb total S, sulfate, pungency, LF and flavour precursor levels in both varieties. Analysis of bulb flavour precursors by HPLC confirmed that juice LF levels paralleled levels of the flavour precursor S-1-propenyl cysteine sulfoxide. The pungent cultivar also exhibited significant N main effects on bulb LF, total S and sulfate. We also assayed the key S-assimilatory enzyme, APS reductase (APR) in leaves before and during bulbing. Specific activities in the range of 1 to 11 nmol mg⁻¹·min⁻¹ were observed in youngest leaves, but only the milder cultivar exhibited significant stage × N × S effects. These findings suggest that sulfur metabolism of mild and pungent onions respond differently to N fertility, and that GC of LF is practical for field-based studies of onion flavour.     

Electron Microscope Studies of the Human Epidermis The Clear Cell of Masson (Dendritic Cell or Melanocyte)

The human epidermis has been studied by electron microscopy following osmium tetroxide and potassium permanganate fixation. An anatomically distinct cell in the human epidermis has been demonstrated with features similar to the melanocyte of the hair bulb described by Barnicot, Birbeck and Cuckow (3). It is dendritic in form and does not contain tonofilaments. "Intercellular bridges" are not formed. The mitochondria are larger and more numerous than those of other epidermal cells and the endoplasmic reticulum is more complex. Some of these cells contain melanin but others are melanin-free. The cell has been interpreted as being identical with the dopa-positive, clear cell of Masson (dendritic cell of Bloch or melanocyte). We have found that many membranous structures in the human epidermis are better preserved by permanganate fixation than by osmium tetroxide fixation.     

Electron microscope studies of the human epidermis: the clear cell of Masson (dendritic cell or melanocyte)

The human epidermis has been studied by electron microscopy following osmium tetroxide and potassium permanganate fixation. An anatomically distinct cell in the human epidermis has been demonstrated with features similar to the melanocyte of the hair bulb described by Barnicot, Birbeck and Cuckow (3). It is dendritic in form and does not contain tonofilaments. "Intercellular bridges" are not formed. The mitochondria are larger and more numerous than those of other epidermal cells and the endoplasmic reticulum is more complex. Some of these cells contain melanin but others are melanin-free. The cell has been interpreted as being identical with the dopa-positive, clear cell of Masson (dendritic cell of Bloch or melanocyte). We have found that many membranous structures in the human epidermis are better preserved by permanganate fixation than by osmium tetroxide fixation.     

Electronic nose evaluation of onion headspace volatiles and bulb quality as affected by nitrogen, sulphur and soil type

Edaphic factors affect the quality of onions (Allium cepa). Two experiments were carried out in the field and glasshouse to investigate the effects of N (field: 0,120 kg ha⁻¹; glasshouse: 0,108 kg ha⁻¹), S (field: 0, 20 kg ha⁻¹; glasshouse: 0, 4.35 kg ha⁻¹) and soil type (clay, sandy loam) on onion quality. A conducting polymer sensor electronic nose (E-nose) was used to classify onion headspace volatiles. Relative changes in the E-nose sensor resistance ratio (%dR/R) were reduced following N and S fertilisation. A 2D Principal Component Analysis (PCA) of the E-nose data sets accounted for c. 100% of the variations in onion headspace volatiles in both experiments. For the field experiment, E-nose data set clusters for headspace volatiles for no N-added onions overlapped (D²= 1.0) irrespective of S treatment. Headspace volatiles of N-fertilised onions for the glasshouse sandy loam also overlapped (D²=1.1) irrespective of S treatment as compared with distinct separations among clusters for the clay soil. N fertilisation significantly (P < 0.01) reduced onion bulb pyruvic acid concentration (flavour) in both experiments. S fertilisation increased pyruvic acid concentration significantly (P < 0.01) in the glasshouse experiment, especially for the clay soil, but had no effect on pyruvic acid concentration in the field. N and S fertilisation significantly (P < 0.01) increased lachrymatory potency (pungency), but reduced total soluble solids (TSS) content in the field experiment. In the glasshouse experiment, N and S had no effect on TSS. TSS content was increased on the clay by 1.2-fold as compared with the sandy loam. Onion tissue N: water-soluble SO₄²⁻ ratios of between five and eight were associated with greater %dR/R and pyruvic acid concentration values. N did not affect inner bulb tissue microbial load. In contrast, S fertilisation reduced inner bulb tissue microbial load by 80% in the field experiment and between 27% (sandy loam) and 92% (clay) in the glasshouse experiment. Overall, onion bulb quality discriminated by the E-nose responded to N, S and soil type treatments, and reflected their interactions. However, the conventional analytical and sensory measures of onion quality did not correlate with %dR/R.     

Determination of Onion yellow dwarf virus concentration levels on onion bulb and leaf by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA)

With the aim of understanding virus movement and fluctuations in the virus concentration in bulb and leaves of onion (Allium cepa L.) plants after infection, Onion yellow dwarf virus (OYDV) was analysed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). OYDV concentrations were higher in onion leaves of plants grown from tested bulbs compared with bulbs, although the virus was successfully detected in bulb of onion.     

THE USE OF BISMUTH AS AN ELECTRON STAIN FOR NUCLEIC ACIDS

Evidence is presented to show that bismuth combines in vitro with the phosphate of nucleic acids in a manner similar to its reaction with inorganic phosphate. When tested under similar conditions, protein exhibited no attraction for bismuth. The results of the in vitro experiments, which are of interest within themselves, may be indirectly applicable to in vivo staining. Dividing cells of onion root tips were fixed in OsO₄, stained with bismuth, and examined in the electron microscope. The electron opacity of cell structures known to contain nucleic acids was enhanced by bismuth, while organelles known to lack appreciable quantities of DNA or RNA showed little, if any, change. Bismuth is particularly effective as a stain for the chromatin material during interphase and for the chromosomes during division.     

Evidence for nuclear-cytoplasmic incompatibility between Allium fistulosum and A. cepa

An F₂ population (Allium fistulosum x A. cepa) of 20plants, 10 BC₁,[(A. fistulosum x A. cepa) x A. cepa], and 50 BC₂ plants, [(A. fistulosum x A. cepa) x A. cepa] x A. cepa were studied cytogenetically and characterized for four isozyme alleles plus various morphological characteristics. All of the progenies were in A. fistulosum (the bunching onion) cytoplasm. In the F₂ population we observed non-random chromosomal and allelic segregation, suppression of bulb onion allelic expression, and abnormalities in mitosis and meiosis. Most BC₂ plants resembled A. cepa (the bulbing onion) morphologically, but anthers, filaments, pistils, and petals were abnormal. Only 3 plants, and these were most nearly like the F₁ hybrid morphologically, produced any seeds.The data and observations support the hypothesis of nuclear-cytoplasmic incompatibility interactions between the bunching and bulb onion species.Use of trade names does not imply endorsement of the products named nor criticism of similar ones not named. This research was supported by the New Mexico Agricultural Experiment Station.     

An electron-microscopical analysis of capture and initial stages of penetration of nematodes byArthrobotrys oligospora

A detailed analysis was made of the capture and subsequent penetration of nematodes by the nematophagous fungusArthrobotrys oligospora using different electron-microscopical techniques. Capture of nematodes by this fungus occurred on complex hyphal structures (traps) and was effectuated by an adhesive coating, present on these trap cells. The adhesive layer was largely fibrillar in nature and was absent on cells of normal hyphae. Following capture, penetration hyphae were formed at those sites where the trap cell wall was anchored to the nematode cuticle by the adhesive. New walls of these hyphae were formed underneath the original trap cell walls, which were partly hydrolysed to allow growth and development of the penetration tubes through the adhesive coating towards the cuticle. Our observations indicated that the cuticle of the nematode was subsequently penetrated by the penetration tubes by mechanical means. After penetration a large infection bulb was formed from which trophic hyphae arose. Cytochemical experiments indicated that the sites of penetration of the cuticle were intensely stained for acid phosphatase activity. At later stages of infection activity of this enzyme was present throughout the nematode contents; the enzyme was most probably secreted by complex membranous structures associated with the cytoplasmic membrane of the infection bulb and the trophic hyphae.     

Phosphorylation mediates the nuclear targeting of the maize Rab17 protein

The maize abscisic acid-responsive Rab17 protein localizes to the nucleus and cytoplasm in maize cells. In-frame fusion of Rab17 to the reporter protein β-glucuronidase (GUS) directed GUS to the nucleus and cytoplasm in transgenic Arabidopsis thaliana and in transiently transformed onion cells. Analysis of chimeric constructs identified one region between amino acid positions 66–96, which was necessary for targeting GUS to the nucleus. This region contains a serine cluster followed by a putative consensus site for protein kinase CK2 phosphorylation, and a stretch of basic amino acids resembling the simian virus 40 large T antigen-type nuclear localization signal (NLS). Mutation of two basic amino acids in the putative NLS had a weak effect on nuclear targeting in the onion cell system and did not modify the percentage of nuclear fusion protein in the Arabidopsis cells. The mutation of three amino acids in the consensus site for CK2 recognition resulted in the absence of in vitro phosphorylated forms of Rab17 and in a strong decrease of GUS enzymatic activity in isolated nuclei of transgenic Arabidopsis. These results suggest that phosphorylation of Rab17 by protein kinase CK2 is the relevant step for its nuclear location, either by facilitating binding to specific proteins or as a direct part of the nuclear targeting apparatus.