Effects of gas pressurization with ethylene on the ultrastructure of the yeast Saccharomyces cerevisiae

We investigated ultrastructural changes in the yeast Saccharomyces cerevisiae when exposed to compressed ethylene gas. Transmission electron microscopy (TEM) revealed that intracellular organelles in yeast cells treated with compressed ethylene at up to 0.640 MPa (6.4 atm), especially the nuclear and plasma membranes, were seriously damaged.     

Some Physical and Chemical Properties of Nuclease P1

The inhibitory action of compressed hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated quantitatively by microcalorimetry. Both the 50% inhibitory pressure (IP₅₀) and the minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of hydrocarbon gases, were determined from growth thermograms. Based on these values, the inhibitory potency of the hydrocarbon gases increased in the order methane << ethane < propane < i-butane < n-butane. The toxicity of these hydrocarbon gases correlated to their hydrophobicity, suggesting that hydrocarbon gases interact with some hydrophobic regions of the cell membrane. In support of this, we found that UV absorbing materials at 260 nm were released from yeast cells exposed to compressed hydrocarbon gases. Additionally, scanning electron microscopy indicated that morphological changes occurred in these cells.     

Evolution of ethylene by Saccharomyces cerevisiae as influenced by the carbon source for growth and the presence of air.

Effects of the carbon source and oxygen on ethylene production by the yeast Saccharomyces cerevisiae have been studied. The amounts of ethylene evolved by the yeast culture were less than those detected in the blank (an equal volume of uninoculated medium), suggesting a net absorption of ethylene by the yeast cells. Addition of glucose to the lactate-grown yeast culture induced ethylene production. This glucose-induced stimulation of ethylene production was inhibited to a great extent by cycloheximide. Results suggested that the yeast cells in the presence of glucose synthesized an ethylene precursor and passed it into the medium. The conversion of this precursor to ethylene might be stimulated by oxygen. The fact that ethylene was produced by the yeast growing anaerobically and also by respiration-deficient mutants isolated from the wild-type yeast suggested that mitochondrial ATP synthesis was not an absolute requirement for ethylene biogenesis.     

面包酵母细胞出芽率与产品质量关系研究

速用压榨酵母(Compressed yeast,CY)和活性干酵母(Active dry yeast,ADY)是用于面点制作的主要两类酵母产品。发酵力则是其共同质量指标。目前,国产压榨酵母的发酵力波动很大,活性干酵母不仅发酵力低,而且保质期短。面包酵母的发酵力定义为由一定数量的酵母样品,面粉和水制成的面团在恒温下产生CO_2的能力。本文采用Burrows法测量面包酵母发酵力,面团由0.15克干酵母,20克面粉和15mol脱离子水混合而成,并以该面团在30℃、180min内产生的CO₂总量作为发酵力的表征,文献^[2]表明,面包酵母的发酵力与终细胞群体中的带芽细胞分率(Fraction of budding cells,FBC)有密切的关系,传统的概念是,为提高酵母产品质量,FBC越低愈好。经研究发现,压榨酵母的耐贮存力及活性干酵发酵力与FBC成反比,但对速用压榨酵母而言,其发酵力FBC的正相关关系甚为明显,这与传统有着根本的差异。     

The effect of yeast dehydration on the activity of a number of enzymes of cell energetic metabolism

Summary It has been shown that dehydration markedly affects the activity of a number of enzymes connected with energy metabolism in the yeastSaccharomyces cerevisiae. Independently of the drying method used, there was found to be an inverse relationship between the activity of mitochondrial enzymes — NADH-dehydrogenase (EC 1.6.2.1), succinate dehydrogenase (EC 1.3.99.1) and cytochrome C oxidase (EC 1.9.3.1) - and the viability of yeast cells at the stationary growth phase. Dehydration led to an increase in activity only in exogenous NADH-dehydrogenase compared with activity in the initial compressed yeast. On the basis of alcohol dehydrogenase (EC 1.1.1.1) and catalase (EC 1.11.1.6) as examples, an ambivalent effect of the dehydration process on the activity of cytoplasmic enzymes has been demonstrated. The results obtained lead to the conclusion that the activity of individual electron-transport enzymes in yeastSaccharomyces cerevisiae is a sufficiently sensitive to be used as an indicator of the physiological state and to monitor a microbial biomass dehydration procedure.     

Studies on fermentative production of squalene

Fermentative production of squalene under anaerobic conditions using commercially available compressed baker's yeast (Saccharomyces cerevisiae), and a strain of Torulaspora delbrueckii isolated from molasses was studied. Yield of squalene from S. cerevisiae and T. delbrueckii were found to be 41.16 and 237.25 g g^–1 respectively, dry weight of yeast cells. Isolation and purification of squalene from the lipid extracts obtained by cell lysis of either strain were achieved chromatographically. The purified squalene was characterized spectroscopically against an authentic standard.     

Association of cytochrome b5 with ETR1 ethylene receptor signaling through RTE1 in Arabidopsis

Ethylene plays important roles in plant growth, development and stress responses, and is perceived by a family of receptors that repress ethylene responses when ethylene is absent. Repression by the ethylene receptor ETR1 depends on an integral membrane protein, REVERSION TO ETHYLENE SENSITIVITY1 (RTE1), which acts upstream of ETR1 in the endoplasmic reticulum (ER) membrane and Golgi apparatus. To investigate RTE1 function, we screened for RTE1‐interacting proteins using the yeast split‐ubiquitin assay, which yielded the ER‐localized cytochrome b₅ (Cb5) isoform D. Cb5s are small hemoproteins that perform electron transfer reactions in all eukaryotes, but their roles in plants are relatively uncharacterized. Using bimolecular fluorescence complementation (BiFC), we found that all four ER‐localized Arabidopsis Cb5 isoforms (AtCb5–B, ‐C, ‐D and ‐E) interact with RTE1 in plant cells. In support of this interaction, atcb5 mutants exhibited phenotypic parallels with rte1 mutants in Arabidopsis. Phenotypes included partial suppression of etr1–2 ethylene insensitivity, and no suppression of RTE1‐independent ethylene receptor isoforms. The single loss‐of‐function mutants atcb5–b, ‐c and ‐d appeared similar to the wild‐type, but double mutant combinations displayed slight ethylene hypersensitivity. Over‐expression of AtCb5–D conferred reduced ethylene sensitivity similar to that conferred by RTE1 over‐expression, and genetic analyses suggested that AtCb5–D acts upstream of RTE1 in the ethylene response. These findings suggest an unexpected role for Cb5, in which Cb5 and RTE1 are functional partners in promoting ETR1‐mediated repression of ethylene signaling.     

Effects of compressed hydrocarbon gases on the growth activity of Saccharomyces cerevisiae

The inhibitory action of compressed hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated quantitatively by microcalorimetry. Both the 50% inhibitory pressure (IP(50)) and the minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of hydrocarbon gases, were determined from growth thermograms. Based on these values, the inhibitory potency of the hydrocarbon gases increased in the order methane < ethane < propane < i-butane < n-butane. The toxicity of these hydrocarbon gases correlated to their hydrophobicity, suggesting that hydrocarbon gases interact with some hydrophobic regions of the cell membrane. In support of this, we found that UV absorbing materials at 260 nm were released from yeast cells exposed to compressed hydrocarbon gases. Additionally, scanning electron microscopy indicated that morphological changes occurred in these cells.     

Ethylene perception by the ERS1 protein in Arabidopsis

Ethylene perception in Arabidopsis is controlled by a family of five genes, including ETR1, ERS1 (ethylene response sensor 1), ERS2, ETR2, and EIN4. ERS1, the most highly conserved gene with ETR1, encodes a protein with 67% identity to ETR1. To clarify the role of ERS1 in ethylene sensing, we biochemically characterized the ERS1 protein by heterologous expression in yeast. ERS1, like ETR1, forms a membrane-associated, disulfide-linked dimer. In addition, yeast expressing the ERS1 protein contains ethylene-binding sites, indicating ERS1 is also an ethylene-binding protein. This finding supports previous genetic evidence that isoforms of ETR1 also function in plants as ethylene receptors. Further, we used the ethylene antagonist 1-methylcyclopropene (1-MCP) to characterize the ethylene-binding sites of ERS1 and ETR1. We found 1-MCP to be both a potent inhibitor of the ethylene-induced seedling triple response, as well as ethylene binding by yeast expressing ETR1 and ERS1. Yeast expressing ETR1 and ERS1 showed nearly identical sensitivity to 1-MCP, suggesting that the ethylene-binding sites of ETR1 and ERS1 have similar affinities for ethylene.     

Cytosolic Neutral Proteinases of Paracoccidioides brasiliensis

Cytosolic proteinases were assayed in both morphological phases of Paracoccidioides brasiliensis. Preparations from the mycelial phase were more active in vitro than those from the yeast cells. Optimal proteinase activities for both phases occurred at pH's between 6.0 and 9.0, and at 45°C. Gelatin-SDS-PAGE electrophoresis separated several bands (58–112 kDa) in mycelial preparations; a single band (70 kDa) was seen in yeast preparations. Enzymatic activities were inhibited by antipain, phenyl methyl sulfonyl fluoride (PMSF), and chymostatin, suggestive of serine proteinases. Partial inhibition of the mycelial enzymes by ethylene diamine tetraacetic acid (EDTA), 1,10-phenanthroline, and iodoacetamide, also suggested the presence of cysteine- and metallo-proteinases. The enzymatic activity increased in preparations extracted from yeast cells transforming to mycelia, and decreased in preparations obtained from the reverse process. Received: 29 September 1997 / Accepted: 19 February 1998     

Studies on the possible role of protein phosphorylation in the transduction of the ethylene signal

Previous work in our laboratory has demonstrated the existence of high affinity binding sites for the plant growth regulator ethylene. The ethylene binding protein (EBP), from Phaseolus cotyledons, shows many of the characteristics of a functional receptor for ethylene, has been purified on SDS-PAGE and polyclonal antibodies raised in rabbits. Current work involves the investigation of the ethylene transduction signal in a number of ethylene-responsive tissues.In peas, it has been shown that ethylene promotes the phosphorylation of specific proteins of similar molecular weight to the EBP from Phaseolus. Such ethylene-induced phosphorylation can be inhibited by the ethylene antagonist, 2,5-NBD. The antibodies raised to the EBP from Phaseolus have been shown to immunoprecipitate ³²P-labelled proteins from membrane protein preparations obtained from pea tissue. Studies on ethylene binding in pea have also shown that the binding of ethylene may be regulated by phosphorylation. Thus, under conditions which promote phosphorylation, binding is inhibited, whereas the reverse is true under conditions which enhance dephosphorylation.Further work is described which examines the effect of protein kinase, protein phosphatase and calcium channel inhibitors on ethylene-induced phosphorylation in peas, together with wild-type (WT) and ethylene insensitive (eti) mutants of Arabidopsis thaliana. The effects of these treatments can be monitored in vivo using the ethylene-induced triple response as a screen. Furthermore, the protein profiles of such treated seedlings can then be compared by labelling protein extracts with ³²P and subjecting the samples to SDS-PAGE followed by autoradiography.     

The Relationship between Ethylene Binding and Dominant Insensitivity Conferred by Mutant Forms of the ETR1 Ethylene Receptor

Ethylene responses in Arabidopsis are mediated by a small family of receptors, including the ETR1 gene product. Specific mutations in the N-terminal ethylene-binding domain of any family member lead to dominant ethylene insensitivity. To investigate the mechanism of ethylene insensitivity, we examined the effects of mutations on the ethylene-binding activity of the ETR1 protein expressed in yeast. The etr1-1 and etr1-4 mutations completely eliminated ethylene binding, while the etr1-3 mutation severely reduced binding. Additional site-directed mutations that disrupted ethylene binding in yeast also conferred dominant ethylene insensitivity when the mutated genes were transferred into wild-type Arabidopsis plants. By contrast, the etr1-2 mutation did not disrupt ethylene binding in yeast. These results indicate that dominant ethylene insensitivity may be conferred by mutations that disrupt ethylene binding or that uncouple ethylene binding from signal output by the receptor. Increased dosage of wild-type alleles in triploid lines led to the partial recovery of ethylene sensitivity, indicating that dominant ethylene insensitivity may involve either interactions between wild-type and mutant receptors or competition between mutant and wild-type receptors for downstream effectors.     

The Role of Ethylene Diisothiocyanate (EDI) in the Antifungal Action of Disodium Ethylene Bisdithiocarbamate (Nabam). I. The Mode of Action of EDI on the Metabolism of Yeast

With glucose as a substrate, the oxygen consumption in yeast in inhibited by 2· 10^-5M ethylene diisothiocyanate. The degree of inhibition was only to a small extent dependant on pH. Radiorespirometric experiments with uniformely labelled glucose showed that the CO₂-production from glucose increased, probably due to increased glycolytic activity. Conversion of C-1 to CO₂ was unaffected by the inhibitor, while the evolution of CO₂ from C-6 was strongly inhibited. The same was the case with CO₂ from C-1 in acetate. Respiration of ethanol was more strongly inhibited than that of glucose or acetate. Experiments with dual wavelength spectrophotometry showed the inhibition to be located on the Krebs cycle side of the respiratory flavoproteins. It is concluded that the action of ethylene diisothiocyanate on respiration must be located at the mitochondria.     

Two androecious mutations reveal the crucial role of ethylene receptors in the initiation of female flower development in Cucurbita pepo

Ethylene is the key regulator of sex determination in monoecious species of the family Cucurbitaceae. This hormone determines which individual floral meristems develop as female or male flowers and the female flowering transition. The sex determination genes discovered so far code for ethylene biosynthesis enzymes, but little is known about the importance of ethylene signaling components. In this paper we characterize two novel ethylene‐insensitive mutations (etr1a‐1 and etr1b) which block the female flowering transition of Cucurbita pepo; this makes plants produce male flowers indefinitely (androecy). Two missense mutations in the ethylene‐binding domain of the ethylene receptors CpETR1A or CpETR1B were identified as the causal mutations of these phenotypes by using whole‐genome resequencing. The distinctive phenotypes of single and double mutants for four etr mutations have demonstrated that the final level of ethylene insensitivity depends upon the strength and dosage of mutant alleles for at least three cooperating ETR genes, and that the level of ethylene insensitivity determines the final sex phenotype of the plant. The sex phenotype ranges from monoecy in ethylene‐sensitive wild‐type plants to androecy in the strongest ethylene‐insensitive ones, via andromonoecy in partially ethylene‐insensitive plants. The induction of female flowering transition was found to be associated with upregulation of CpACS11, CpACO2 and CpACS27, three ethylene biosynthesis genes required for female flower development. A model is proposed herein, integrating both ethylene biosynthesis and receptor genes into the genetic network which regulates sex determination in C. pepo.     

Effects of ethylene on the metabolism of Saccharomyces cerevisiae.

Supply of exogenous ethylene to lactate-grown yeast initially accelerated the rate of ethanol production from glucose, but later reduced the rate, with the overall effect being to reduce the total ethanol production. The rate of ethanol production by ethylene-treated yeast was not changed by removal of metabolic carbon dioxide. However, if CO2 was allowed to build up in the absence of applied ethylene, the ethanol production decreased. Ethylene increased the activities of a number of pentose phosphate and glycolytic pathway enzymes. The largest increase in activity was observed for phosphofructokinase (EC 2.7.1.11), regulatory enzyme of the glycolytic pathway. After an initial stimulation, glucose (and also 3-O-methyl glucose) uptake was reduced by ethylene. Ethylene appears to inhibit non-competitively the glucose transport system.     

An Efficient Method for Separating Ascospores from Sporulating Cultures in Saccharomyces cerevisiae by Hydrostatic Pressure

A new oxidative reaction of ethylene glycol was found with two alcohol oxidases from methanol yeast, Candida sp. and Pichia pastoris. Both alcohol oxidases oxidized ethylene glycol to glyoxal via glycolaldehyde. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Candida alcohol oxidase were around 8.5 and 5.5, respectively, and their apparent K_ms were 2.96 m and 28.6 mm, respectively. The optimum temperature was 40°C at pH 7.0. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Pichia alcohol oxidase were around 8.0 and 6.0, respectively, and their optimum temperatures were 50 and 45°C, respectively, at pH 7.0. The apparent K_m for glycolaldehyde was found to be 83.3 mm. For the accumulation of glyoxal, addition of catalase was effective, and a higher amount of glyoxal was obtained at a much lower temperature than the optimum for the alcohol oxidase. When 0.1 m ethylene glycol and glycolaldehyde were incubated with 80 units of the Pichia enzyme at 10°C, both substrates were almost completely converted to glyoxal after 10 and 3h of incubation, respectively.