Adaptation of Yeast Cell Membranes to Ethanol

A highly ethanol-tolerant Saccharomyces wine strain is able, after growth in the presence of ethanol, to efficiently improve the ethanol tolerance of its membrane. A less-tolerant Saccharomyces laboratory strain, however, is unable to adapt its membrane to ethanol. Furthermore, after growth in the presence of ethanol, the membrane of the latter strain becomes increasingly sensitive, although this is a reversible process. Reversion to a higher tolerance occurs only after the addition of an energy source and does not take place in the presence of cycloheximide.     

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress.     

玉米籽粒胚乳细胞增殖及其与淀粉充实的关系

用纤维素酶解离胚乳、滤膜法统计玉米胚乳细胞的数目,进一步借助Logistic方程模拟胚乳细胞增殖动态的结果表明,整个灌浆期间胚乳细胞增殖呈现“慢-快-慢”的变化趋势。授粉15d后,不同类型胚乳的细胞数目依序为普通玉米〉糯玉米〉甜玉米〉爆裂玉米;胚乳细胞数目主要取决于细胞的增殖速率,并与淀粉充实和粒重关系密切。胚乳发育前期以胚乳细胞增殖为主,后期以淀粉积累为主。     

Fermentation Rates and Ethanol Accumulation in Relation to Flooding Tolerance in Rhizomes of Monocotyledonous Species

Under anoxia an accumulation of ethanol took place in all species.Lactic fermentation was found to be of less importance in everycase. The amount of ethanol accumulated depended on the abilityof the rhizome to eliminate it from its tissues. Despite greatvariations in fermentative capacity as seen in the ADH measurementsthe wetland species did not accumulate ethanol to concentrationsgreater than 30 µmol g^–1 fresh weight. This representeda plateau above which the ethanol concentration did not riseeven with continued anaerobic incubation. No such plateau ofethanol accumulation was found in the dryland species Iris germanicawhich accumulated ethanol steadily reaching concentrations of70 µmol g^–1 fresh weight. If ethanol is toxic to higher plant tissues then the steadystate condition of low ethanol accumulation found in wetlandspecies will minimize this danger. Key words: Ethanol, Flooding, Rhizomes     

絮凝特性对自絮凝颗粒酵母耐酒精能力的影响及作用机制

首次报道絮凝特性提高酵母菌耐酒精能力的现象及其机制。融合株SPSC与其两亲本粟酒裂殖酵母变异株和酿酒酵母变异株于 30℃经 18% (V/V)酒精冲击 7h的存活率分别为 52%、37%和 9%。细胞膜磷脂脂肪酸组成分析表明 ,两絮凝酵母 (融合株SPSC和粟酒裂殖酵母变异株 )的棕榈酸含量均约为非絮凝酵母 (酿酒酵母变异株 )的两倍 ,而棕榈油酸和油酸的含量明显低于后者。研究表明 ,当两絮凝酵母在培养中由于柠檬酸钠的作用 (抑制絮凝体的形成 )而以游离细胞生长存在时 ,其细胞膜磷脂棕榈酸含量显著下降 ,而棕榈油酸和油酸的含量明显增加 ,结果细胞膜磷脂脂肪酸组成特点与酿酒酵母变异株相似 ;而且实验表明 ,絮凝特性的消失伴随菌体耐酒精能力的急剧下降 ,变得与酿酒酵母变异株的水平相当。这些结果提示两絮凝酵母具有较强的耐酒精能力与其细胞膜磷脂脂肪酸组成中含有更高比例的棕榈酸有关。     

Ethanol-Induced Leakage in Saccharomyces cerevisiae: Kinetics and Relationship to Yeast Ethanol Tolerance and Alcohol Fermentation Productivity

Ethanol stimulated the leakage of amino acids and 260-nm-light-absorbing compounds from cells of Saccharomyces cerevisiae. The efflux followed first-order kinetics over an initial period. In the presence of lethal concentrations of ethanol, the efflux rates at 30 and 36°C were an exponential function of ethanol concentration: k_e^X = k_e^Xme^{E (X-X_m)}, where k_e^X and k_e^Xm are the efflux rate constants, respectively, in the presence of a concentration X of ethanol or the minimal concentration of ethanol, X_m, above which the equation was applicable, coincident with the minimal lethal concentration of ethanol. E is the enhancement constant. At 36°C, as compared with the corresponding values at 30°C, the efflux rates were higher and the minimal concentration of ethanol (X_m) was lower. The exponential constants for the enhancement of the rate of leakage (E) had similar values at 30 or 36°C and were of the same order of magnitude as the corresponding exponential constants for ethanol-induced death. Under isothermic conditions (30°C) and up to 22% (vol/vol) ethanol, the resistance to ethanol-induced leakage of 260-nm-light-absorbing compounds was found to be closely related with the ethanol tolerance of three strains of yeasts, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Saccharomyces bayanus. The resistance to ethanol-induced leakage indicates the possible adoption of the present method for the rapid screening of ethanol-tolerant strains. The addition to a fermentation medium of the intracellular material obtained by ethanol permeabilization of yeast cells led to improvements in alcohol fermentation by S. cerevisiae and S. bayanus. The action of the intracellular material, by improving yeast ethanol tolerance, and the advantages of partially recycling the fermented medium after distillation were discussed.     

Comparative Evolutionary Analysis of Cell Cycle Proteins Networks in Fission and Budding Yeast

Fission yeast and budding yeast are the two distantly related species with common ancestors. Various studies have shown significant differences in metabolic networks and regulatory networks. Cell cycle regulatory proteins in both species have differences in structural as well as in functional organization. Orthologous proteins in cell cycle regulatory protein networks seem to play contemporary role in both species during the evolution but little is known about non-orthologous proteins. Here, we used system biology approach to compare topological parameters of orthologous and non-orthologous proteins to find their contributions during the evolution to make an efficient cell cycle regulation. Observed results have shown a significant role of non-orthologous proteins in fission yeast in maintaining the efficiency of cell cycle regulation with less number of proteins as compared to budding yeast.     

Polyamine Accumulation in Transgenic Tomato Enhances the Tolerance to High Temperature Stress

Polyamines play an important role in plant response to abiotic stress. S-adenosyl-I-methionine decarboxylase (SAMDC) is one of the key regulatory enzymes in the biosynthesis of polyamines. In order to better understand the effect of regulation of polyamine biosynthesis on the tolerance of high-temperature stress in tomato, SAMDC Cdna isolated from Saccharomyces cerevisiae was introduced into tomato genome by means of Agrobacterium tumefaciens through leaf disc transformation. Transgene and expression was confirmed by Southern and Northern blot analyses, respectively. Transgenic plants expressing yeast SAMDC produced 1.7- to 2.4-fold higher levels of spermidine and spermine than wild-type plants under high temperature stress, and enhanced antioxidant enzyme activity and the protection of membrane lipid peroxidation was also observed. This subsequently improved the efficiency of CO2 assimilation and protected the plants from high temperature stress, which indicated that the transgenic tomato presented an enhanced tolerance to high temperature stress (38℃) compared with wild-type plants, Our results demonstrated clearly that increasing polyamine biosynthesis in plants may be a means of creating high temperature-tolerant germplasm.     

两种生态型香根草对镉的耐受和积累特性比较

以Monto和Veriveria nermorlis两种生态型香根草［Chrysopogon zizanioides (L.) Roberty］为材料,采用室内水培试验,在不同浓度(0、200、800、2 000 μmol·L^-1)硝酸镉溶液处理7 d后,比较、分析两类型香根草的叶片生长状况、叶绿素荧光参数、镉的积累、亚细胞分布和化学形态分布等指标的差异,明确两类香根草对镉的耐受能力和积累特性。结果表明,(1)在中度(800 μmol·L^-1)和重度(2 000 μmol·L^-1)镉胁迫条件下,Monto的叶片生长状况优于Veriveria nermorlis,其F_m、F_v/F_m和F_v/F_o等叶绿素荧光参数显著高于Veriveria nermorlis,而其叶片、根部镉含量、富集系数和镉转移系数则显著低于Veriveria nermorlis。(2)镉主要分布在香根草根和叶细胞壁组分和可溶性组分中,两者镉含量占比之和达95%以上;对于两种生态型香根草的叶片和Monto的根部而言,细胞壁组分和可溶组分镉占比随着镉胁迫浓度增加分别呈增加和下降趋势,但两参数在Veriveria nermorlis根中的变化趋势则相反;Monto叶中细胞壁组分的镉占比要高于Veriveria nermorlis。(3)镉的化学形态分布分析显示,两种生态型香根草叶片中以氯化钠提取态镉为主,而根中以乙醇提取态和氯化钠提取态镉为主;Monto根中乙醇提取态的镉含量占比远大于Veriveria nermorlis。研究发现,Monto香根草的镉耐受能力较强,主要因为其根、叶中的镉积累量、镉富集系数和转移系数均低于Veriveria nermorlis,且Monto叶片中细胞壁组分的镉占比和根中乙醇提取态镉的占比更高。     

Effect of Ethanol on Cell Growth of Budding Yeast: Genes That Are Important for Cell Growth in the Presence of Ethanol

The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds of alcoholic beverages. But the effect of ethanol on the cell growth of this yeast is poorly understood. This study shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion of F-actin cytoskeleton, resulting in an increase in cell size. We found that the tyrosine kinase Swe1, the negative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol. Indeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and the amount of Swe1 protein increased transiently in the presence of ethanol. These results indicated that Swe1 is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes a transient up-regulation of Swe1. Further we investigated comprehensively the ethanol-sensitive strains in the complete set of 4847 non-essential gene deletions and identified at least 256 genes that are important for cell growth in the presence of ethanol.     

Differential Regulation of Mitochondrial Pyruvate Carrier Genes Modulates Respiratory Capacity and Stress Tolerance in Yeast

Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.     

钙离子通过降低细胞膜透性提高自絮凝颗粒酵母耐酒精能力

生长阶段和冲击阶段均添加 1 6 4mmol LCa2 能显著提高自絮凝颗粒酵母于 30℃在 2 0 % (V V)酒精冲击下的存活率 ,经过 9h冲击 ,对照组的存活率为 0 ,而添加Ca2 试验组的存活率为 5 0 0 % ,表明添加适当浓度的Ca2 能显著提高菌体的耐酒精能力。通过考察Ca2 对菌体于 30℃在 15 % (V V)酒精冲击下细胞膜透性的影响发现 ,生长阶段和冲击阶段均添加 1 6 4mmol LCa2 的试验组的菌体胞外核苷酸平衡浓度和细胞膜透性系数 (P′)分别仅为对照组水平的 5 0 0 %和 2 9 3% ,表明添加适当浓度的Ca2 能显著降低受冲击菌体的细胞膜透性 ;而且 ,添加Ca2 提高存活率与添加Ca2 降低胞外核苷酸浓度和P′存在直接的对应关系。因此 ,Ca2 提高自絮凝颗粒酵母耐酒精能力是与其降低受冲击菌体的细胞膜透性密切相关的。     

重组酵母菌的高密度发酵表达

酵母作为一类外源基因的表达系统具有很多优点 ,有许多外源基因在工程酵母菌表达生产重要的外源蛋白[1] 。从生理学角度看 ,作为外源基因的受体细胞 ,酵母的生理和遗传特性影响着外源基因的表达。同时 ,外源基因的表达 ,特别是高表达的外源蛋白对宿主细胞有抑制作用甚至会导致细胞中毒或死亡。再加上工程菌的培养过程中的不稳定性问题的存在 ,因此 ,菌体细胞的高浓度和目的基因的高表达是一对矛盾 ,它们互相制约 ,构成生物工程研究的重要工作之一。1 .工程酵母菌的高密度发酵酵母的高密度发酵是一个相对概念 ,一般指发酵液中酵母浓度在 30 …     

Overexpression of Constitutively Active OsRab11 in Plants Enhances Tolerance to High Salinity Levels

Rab proteins are key regulators of intracellular trafficking between specific compartments in a cell. Among them, Rab11, a widely conserved sub-group, mainly regulates plasma membrane (PM) trafficking. Previously, we reported that Oryza sativa Rab11 (OsRab11) plays an important role in the intracellular trafficking from the trans-Golginetwork (TGN) to the plasma membrane (PM) and prevacuolar compartments (PVCs), and in the plant’s response to high salt stress. In this study, when the constitutively active mutant of OsRab11, (CA OsRab11(Q73L)) was co-transformed with Arabidopsis Ca²⁺-ATPase8-GFP (ACA8-GFP) or sporamin-GFP (Spo-GFP) into Arabidopsis protoplasts, the PM or vacuolar trafficking proportion of the reporter proteins was highly increased. Transgenic Arabidopsis plants overexpressing (OE) CA OsRab11(Q73L) exhibited enhanced tolerance to high salt stress and exogenous abscisic acid (ABA) compared to Col plants. Moreover, certain stress-responsive genes were expressed under high salt stress and ABA treatment in OEOsRab11(Q73L) plants. Thus, these results suggest that the active conformation of OsRab11 may be required to modulate plant responses to salt and ABA via the regulation of the expression of stress-responsive genes.