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The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulosic fermentations. Based on the screening of EUROSCARF haploid mutant collection, 242 and 216 determinants of tolerance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate were identified, respectively. Genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics are among the newly found determinants of WSH resistance. Moreover, PRS3, VMA8, ERG2, RAV1 and RPB4 were confirmed as key genes on yeast tolerance and fermentation of industrial WSH. [ABSTRACT FROM AUTHOR]

Background: The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC) Superfamily and Major Facilitator Superfamily (MFS) in S. cerevisiae were scrutinized for a role in ethanol stress resistance. Results: A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased 3 H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. Conclusions: PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic fermentation performance for sustainable bio-ethanol production. [ABSTRACT FROM AUTHOR]

The success of food and beverage production processes carried out by Saccharomyces cerevisiae and the thriving of food spoilage fungi are dependent on the ability of a cell to cope with the many environmental insults imposed during food production and preservation processes. Post-genomic approaches, especially transcriptomics, proteomics and chemogenomics, applied to S. cerevisiae made possible the unveiling of general and specific genome-wide adaptive response programs against stress induced by weak acids, ethanol, sulfite, heat and cold shock, osmotic pressure and nutrient limitation. These programs and the underlying signaling pathways are overviewed herein, highlighting the recent identification of genes and pathways found to be involved in stress response and tolerance. These are good candidate targets for genetic engineering aiming at the development of improved strains. The extension of the data gathered in S. cerevisiae to food spoilage fungi is considered. The relevance of the different genome-wide approaches in this context is also discussed. [Copyright &y& Elsevier]

The understanding of the molecular mechanisms underlying acquired herbicide resistance is crucial in dealing with the emergence of resistant weeds. Saccharomyces cerevisiae has been used as a model system to gain insights into the mechanisms underlying resistance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The TPO1 gene, encoding a multidrug resistance (MDR) plasma membrane transporter of the major facilitator superfamily (MFS), was previously found to confer resistance to 2,4-D in yeast and to be transcriptionally activated in response to the herbicide. In this work, we demonstrate that Tpo1p is required to reduce the intracellular concentration of 2,4-D. ScTpo1p homologs encoding putative plasma membrane MFS transporters from the plant model Arabidopsis thaliana were analyzed for a possible role in 2,4-D resistance. At5g13750 was chosen for further analysis, as its transcript levels were found to increase in 2,4-D stressed plants. The functional heterologous expression of this plant open reading frame in yeast was found to confer increased resistance to the herbicide in Δtpo1 and wild-type cells, through the reduction of the intracellular concentration of 2,4-D. Heterologous expression of At5g13750 in yeast also leads to increased resistance to indole-3-acetic acid (IAA), Al3+ and Tl3+. At5g13750 is the first plant putative MFS transporter to be suggested as possibly involved in MDR. [ABSTRACT FROM AUTHOR]

The understanding of the molecular basis of yeast resistance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. In this study, the yeast disruptome was screened for mutants with differential susceptibility to stress induced by high ethanol concentrations in minimal growth medium. Over 250 determinants of resistance to ethanol were identified. The most significant gene ontology terms enriched in this data set are those associated with intracellular organization, biogenesis, and transport, in particular, regarding the vacuole, the peroxisome, the endosome, and the cytoskeleton, and those associated with the transcriptional machinery. Clustering the proteins encoded by the identified determinants of ethanol resistance by their known physical and genetic interactions highlighted the importance of the vacuolar protein sorting machinery, the vacuolar H+-ATPase complex, and the peroxisome protein import machinery. Evidence showing that vacuolar acidification and increased resistance to the cell wall lytic enzyme β-glucanase occur in response to ethanol-induced stress was obtained. Based on the genome-wide results, the particular role of the FPS1 gene, encoding a plasma membrane aquaglyceroporin which mediates controlled glycerol efflux, in ethanol stress resistance was further investigated. FPSI expression contributes to decreased [3H]ethanol accumulation in yeast cells, suggesting that Fps1p may also play a role in maintaining the intracellular ethanol level during active fermentation. The increased expression of FPS1 confirmed the important role of this gene in alcoholic fermentation, leading to increased final ethanol concentration under conditions that lead to high ethanol production. [ABSTRACT FROM AUTHOR]

Abstract: FLR1 gene, encoding a multidrug resistance (MDR) transporter of the major facilitator superfamily (MFS) was found to confer resistance to the fungicide mancozeb in Saccharomyces cerevisiae. This agrochemical has been linked to the development of Parkinson disease and cancer. Yeast response to mancozeb was proved to involve the strong activation of FLR1 transcription (20-fold) during the fungicide-induced growth latency. This activation of FLR1 transcription is fully dependent on Yap1p and is reduced (by 50%) in the absence of Rpn4p, Yrr1p or Pdr3p. A model for the coordinate action over FLR1 transcription activation, in response to mancozeb, of these transcription factors that mediate oxidative stress response (Yap1p), proteasome gene expression (Rpn4p), and pleiotropic drug resistance (Pdr3p and Yrr1p), is proposed. [Copyright &y& Elsevier]

Genomic information and tools are beginning to be used to increase our understanding of how organisms of all types interact with their environment. The study of the expression of all genes, at the genome, transcriptome, proteome and metabolome level, in response to exposure to a toxicant, is known as toxicogenomics. Here, we show how this new field of environmental genomics has enhanced the development of fundamental knowledge on the mechanisms behind the toxicity of and resistance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Although 2,4-D is one of the most successfully and widely used herbicides, its intensive use has led to the emergence of resistant weeds and might give rise to several toxicological problems when present in concentrations above those recommended. This review summarizes recent mechanistic insights into 2,4-D toxicity and the corresponding adaptive responses based on studies carried out using Saccharomyces cerevisiae and Arabidopsis thaliana as model organisms. [Copyright &y& Elsevier]

The global gene transcription pattern of the eukaryotic experimental model Saccharomyces cerevisiae in response to sudden aggression with the widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was analysed. Under acute stress, 14% of the yeast transcripts suffered a greater than twofold change. Theyeastract database was used to predict the transcription factors mediating the response registered in this microarray analysis. Most of the up-regulated genes in response to 2,4-D are known targets of Msn2p, Msn4p, Yap1p, Pdr1p, Pdr3p, Stp1p, Stp2p and Rpn4p. The major regulator of ribosomal protein genes, Sfp1p, is known to control 60% of the down-regulated genes, in particular many involved in the transcriptional and translational machinery and in cell division. The yeast response to the herbicide includes the increased expression of genes involved in the oxidative stress response, the recovery or degradation of damaged proteins, cell wall remodelling and multiple drug resistance. Although the protective role of TPO1 and PDR5 genes was confirmed, the majority of the responsive genes encoding multidrug resistance do not confer resistance to 2,4-D. The increased expression of genes involved in alternative carbon and nitrogen source metabolism, fatty acid β-oxidation and autophagy was also registered, suggesting that acute herbicide stress leads to nutrient limitation. [ABSTRACT FROM AUTHOR]

The pro-oxidant action of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is demonstrated in this study using Saccharomyces cerevisiae as a eukaryotic experimental model. Evidence is presented for the generation of hydroxyl-radicals, in yeast cells suddenly exposed to 2,4-D, detected by in vivo electron paramagnetic resonance (EPR) spectroscopy using 5,5′-dimethyl-1-pyrroline N-oxide and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide as spin-traps. The intensity of the EPR spectra was dependent on the concentration of herbicide tested and was consistently higher in a mutant (Δsod1) devoid of the cytosolic CuZn-superoxide dismutase. A time-course-dependent variation of the level of free-radical adducts was registered upon sudden exposure of an yeast cell population to concentrations of 2,4-D that lead to an initial period of viability loss, before resumption of inhibited growth by the viable adapted population. The variation pattern of the level of hydroxyl-radical adducts correlated with the one determined for the activity of Sod1p, cytosolic catalase Ctt1p, and the dithiol glutaredoxins Grx1p and Grx2p. [Copyright &y& Elsevier]

The transcription regulator Pdr1p is a determinant of Saccharomyces cerevisiae resistance to 2-methyl-4-chlorophenoxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The Pdr1p-regulated genes, TPO1 and PDR5, encoding putative multidrug transporters belonging to the major facilitator superfamily (MFS) and to the ATP-binding cassette (ABC) superfamily, respectively, are required for yeast resistance to sudden exposure to these herbicides. A rapid and transient activation of TPO1 (sixfold) and PDR5 (twofold) transcription takes place during the adaptation period preceding cell division under MCPA or 2,4-D moderate stress. These activations are mediated by both Pdr1p and Pdr3p and, as soon as adapted cells start duplication under herbicide stress, mRNA levels are drastically reduced to basal values. The longer duration of the adaptation period, observed for the Δpdr1 population, may involve the abolishment of the Pdr1p-mediated transcriptional activation of TPO1 and PDR5 genes, whose expression is critical to surpass the viability loss during the initial period of adaptation to the herbicides. [Copyright &y& Elsevier]

A metabolomic analysis using high resolution 1H NMR spectroscopy coupled with multivariate statistical analysis was used to characterize the alterations in the endo- and exo-metabolome of S. cerevisiae BY4741 during the exponential phase of growth in minimal medium supplemented with different ethanol concentrations (0, 2, 4 and 6% v/v). This study provides evidence that supports the notion that ethanol stress induces reductive stress in yeast cells, which, in turn, appears to be counteracted by the increase in the rate of NAD+ regenerating bioreactions. Metabolomics data also shows increased intra- and extra-cellular accumulation of most amino acids and TCA cycle intermediates in yeast cells growing under ethanol stress suggesting a state of overflow metabolism in turn of the pyruvate branch-point. Given its previous implication in ethanol stress resistance in yeast, this study also focused on the effect of the expression of the aquaglyceroporin encoded by FPS1 in the yeast metabolome, in the absence or presence of ethanol stress. The metabolomics data collected herein shows that the deletion of the FPS1 gene in the absence of ethanol stress partially mimics the effect of ethanol stress in the parental strain. Moreover, the results obtained suggest that the reported action of Fps1 in mediating the passive diffusion of glycerol is a key factor in the maintenance of redox balance, an important feature for ethanol stress resistance, and may interfere with the ability of the yeast cell to accumulate trehalose. Overall, the obtained results corroborate the idea that metabolomic approaches may be crucial tools to understand the function and/or the effect of membrane transporters/porins, such as Fps1, and may be an important tool for the clear-cut design of improved process conditions and more robust yeast strains aiming to optimize industrial fermentation performance. [ABSTRACT FROM AUTHOR]

The emergence of widespread multidrug resistance (MDR) is a serious challenge for therapeutics, food-preservation and crop protection. Frequently, MDR is a result of the action of drug-efflux pumps, which are able to catalyze the extrusion of unrelated chemical compounds. This review summarizes the current knowledge on the Saccharomyces cerevisiae drug:H+ antiporters of the major facilitator superfamily (MFS), a group of MDR transporters that is still characterized poorly in eukaryotes. Particular focus is given here to the physiological role and expression regulation of these transporters, while we provide a unified view of new data emerging from functional genomics approaches. Although traditionally described as drug pumps, evidence reviewed here corroborates the hypothesis that several MFS-MDR transporters might have a natural substrate and that drug transport might occur only fortuitously or opportunistically. Their role in MDR might even result from the transport of endogenous metabolites that affect the partition of cytotoxic compounds indirectly. Finally, the extrapolation of the gathered knowledge on the MDR phenomenon in yeast to pathogenic fungi and higher eukaryotes is discussed. [Copyright &y& Elsevier]

Abstract: Yeast cells adapted to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) exhibit a plasma membrane less susceptible to 2,4-D-induced disruption and are more tolerant than unadapted cells to lethal concentrations of the herbicide. These cells, adapted to grow in the presence of increasing concentrations of 2,4-D, were found to exhibit a dose-dependent increase of the saturation degree of membrane fatty acids, associated to the higher percentage of stearic (C18:0) and palmitic (C16:0) acids, and to the decreased percentage of palmitoleic (Δ9-cisC16:1) and oleic (Δ9-cisC18:1) acids. The decreased transcription of the OLE1 gene (encoding the Δ9 fatty acid desaturase that catalyses the conversion of palmitic and stearic acids to palmitoleic and oleic acids, respectively) registered in 2,4-D adapted cells suggests that yeast adaptation to the herbicide involves the enhancement of the ratio of saturated (C16:0 and C18:0) to monounsaturated (C16:1 and C18:1) membrane fatty acids through a reduced OLE1 expression. [Copyright &y& Elsevier]

Abstract: Saccharomyces cerevisiae ORF YBR043c, predicted to code for a transporter of the major facilitator superfamily required for multiple drug resistance, encodes a plasma membrane protein that confers resistance to quinidine and barban, as observed before for its close homologues QDR1 and QDR2. This ORF was, thus, named the QDR3 gene. The increased expression of QDR3, or QDR2, also leads to increased resistance to the anticancer agents cisplatin and bleomycin. However, no evidence for increased QDR3 expression in yeast cells exposed to all these inhibitory compounds was found. Transport assays support the concept that Qdr3 is involved, even if opportunistically, in the active export of quinidine out of yeast cell. A correlation was established between the efficiency of quinidine active export mediated by Qdr3p, Qdr2p or Qdr1p, and the efficacy of the expression of the encoding genes in alleviating the deleterious action of quinidine, as well as of the other compounds (QDR2>QDR3>>>QDR1). [Copyright &y& Elsevier]

Responding to the recent interest of the yeast research community in non- Saccharomyces cerevisiae species of biotechnological relevance, the N.C.Yeastract (http://yeastract-plus.org/ncyeastract/) was associated to YEASTRACT + (http://yeastract-plus.org/). The YEASTRACT + portal is a curated repository of known regulatory associations between transcription factors (TFs) and target genes in yeasts. N.C.Yeastract gathers all published regulatory associations and TF-binding sites for Komagataella phaffii (formerly Pichia pastoris), the oleaginous yeast Yarrowia lipolytica , the lactose fermenting species Kluyveromyces lactis and Kluyveromyces marxianus , and the remarkably weak acid-tolerant food spoilage yeast Zygosaccharomyces bailii. The objective of this review paper is to advertise the update of the existing information since the release of N.C.Yeastract in 2019, and to raise awareness in the community about its potential to help the day-to-day work on these species, exploring all the information available in the global YEASTRACT + portal. Using simple and widely used examples, a guided exploitation is offered for several tools: (i) inference of orthologous genes; (ii) search for putative TF binding sites and (iii) inter-species comparison of transcription regulatory networks and prediction of TF-regulated networks based on documented regulatory associations available in YEASTRACT + for well-studied species. The usage potentialities of the new CommunityYeastract platform by the yeast community are also discussed. [ABSTRACT FROM AUTHOR]

Although Saccharomyces cerevisiae and S. cerevisiae var. boulardii share more than 95% genome sequence homology, only S. cerevisiae var. boulardii displays probiotic activity. In this study, the transcriptomic differences exhibited by S. cerevisiae and S. cerevisiae var. boulardii in intestinal like medium were evaluated. S. cerevisiae was found to display stress response overexpression, consistent with higher ability of S. cerevisiae var. boulardii to survive within the human host, while S. cerevisiae var. boulardii exhibited transcriptional patterns associated with probiotic activity, suggesting increased acetate biosynthesis. Resorting to the creation of a S. cerevisiae var. boulardii genomic database within Yeastract+, a possible correlation between loss or gain of transcription factor binding sites in S. cerevisiae var. boulardii promoters and the transcriptomic pattern is discussed. This study suggests that S. cerevisiae var. boulardii probiotic activity, when compared to S. cerevisiae , relies, at least partially, on differential expression regulation, based on promoter variability. • A comparative transcriptome analysis on S. boulardii versus S. cerevisiae is conducted for the first time. • A genomic database for S. boulardii strains, focusing on predicted transcription regulation is presented. • Transcriptomic data suggests that S. boulardii is better adapted to the human GI tract environment and exhibits probiotic properties. • Global promoter analysis points out transcription factor binding site divergence. [ABSTRACT FROM AUTHOR]

The capacity of living cells to adapt to different environmental, sometimes adverse, conditions is achieved through differential gene expression, which in turn is controlled by a highly complex transcriptional network. We recovered the full network of transcriptional regulatory associations currently known for Saccharomyces cerevisiae, as gathered in the latest release of the YEASTRACT database. We assessed topological features of this network filtered by the kind of supporting evidence and of previously published networks. It appears that in-degree distribution, as well as motif enrichment evolve as the yeast transcriptional network is being completed. Overall, our analyses challenged some results previously published and confirmed others. These analyses further pointed towards the paucity of experimental evidence to support theories and, more generally, towards the partial knowledge of the complete network. [ABSTRACT FROM AUTHOR]

Saccharomyces boulardii is a probiotic yeast often used for the treatment of GI tract disorders such as diarrhea symptoms. It is genetically close to the model yeast Saccharomyces cerevisiae and its classification as a distinct species or a S. cerevisiae variant has long been discussed. Here, we review the main genetic divergencies between S. boulardii and S. cerevisiae as a strategy to uncover the ability to adapt to the host physiological conditions by the probiotic. S. boulardii does possess discernible phenotypic traits and physiological properties that underlie its success as probiotic, such as optimal growth temperature, resistance to the gastric environment and viability at low pH. Its probiotic activity has been elucidated as a conjunction of multiple pathways, ranging from improvement of gut barrier function, pathogen competitive exclusion, production of antimicrobial peptides, immune modulation, and trophic effects. This review summarizes the participation of S. boulardii in these mechanisms and the multifactorial nature by which this yeast modulates the host microbiome and intestinal function. [ABSTRACT FROM AUTHOR]

The action of benzoic acid in the food and beverage industries is compromised by the ability of spoilage yeasts to cope with this food preservative. Benzoic acid occurs naturally in many plants and is an intermediate compound in the biosynthesis of many secondary metabolites. The understanding of the mechanisms underlying the response and resistance to benzoic acid stress in the eukaryotic model yeast is thus crucial to design more suitable strategies to deal with this toxic lipophilic weak acid. In this study, the Saccharomyces cerevisiae multidrug transporter Tpo1 was demonstrated to confer resistance to benzoic acid. TPO1 transcript levels were shown to be up-regulated in yeast cells suddenly exposed to this stress agent. This up-regulation is under the control of the Gcn4 and Stp1 transcription factors, involved in the response to amino acid availability, but not under the regulation of the multidrug resistance transcription factors Pdr1 and Pdr3 that have binding sites in TPO1 promoter region. Benzoic acid stress was further shown to affect the intracellular pool of amino acids and polyamines. The observed decrease in the concentration of these nitrogenous compounds, registered upon benzoic acid stress exposure, was not found to be dependent on Tpo1, although the limitation of yeast cells on nitrogenous compounds was found to activate Tpo1 expression. Altogether, the results described in this study suggest that Tpo1 is one of the key players standing in the crossroad between benzoic acid stress response and tolerance and the control of the intracellular concentration of nitrogenous compounds. Also, results can be useful to guide the design of more efficient preservation strategies and the biotechnological synthesis of benzoic acid or benzoic acid-derived compounds. [ABSTRACT FROM AUTHOR]

5-Flucytosine is currently used as an antifungal drug in combination therapy, but fungal pathogens are rapidly able to develop resistance against this drug, compromising its therapeutic action. The understanding of the underlying resistance mechanisms is crucial to deal with this problem. In this work, the S. cerevisiae deletion mutant collection was screened for increased resistance to flucytosine. Through this chemogenomics analysis, 183 genes were found to confer resistance to this antifungal agent. Consistent with its known effect in DNA, RNA and protein synthesis, the most significant Gene Ontology terms over-represented in the list of 5-flucytosine resistance determinants are related to DNA repair, RNA and protein metabolism. Additional functional classes include carbohydrate and nitrogen—particularly arginine—metabolism, lipid metabolism and cell wall remodeling. Based on the results obtained for S. cerevisiae as a model system, further studies were conducted in the pathogenic yeast Candida glabrata. Arginine supplementation was found to relieve the inhibitory effect exerted by 5-flucytosine in C. glabrata. Lyticase susceptibility was found to increase within the first 30min of 5-flucytosine exposure, suggesting this antifungal drug to act as a cell wall damaging agent. Upon exponential growth resumption in the presence of 5-flucytosine, the cell wall exhibited higher resistance to lyticase, suggesting that cell wall remodeling occurs in response to 5-flucytosine. Additionally, the aquaglyceroporin encoding genes CgFPS1 and CgFPS2, from C. glabrata, were identified as determinants of 5-flucytosine resistance. CgFPS1 and CgFPS2 were found to mediate 5-flucytosine resistance, by decreasing 5-flucytosine accumulation in C. glabrata cells. [ABSTRACT FROM AUTHOR]

Potassium (K+) is an essential mineral nutrient for plant growth and development, with numerous membrane transporters and channels having been implicated in the maintenance and regulation of its homeostasis. The cation cesium (Cs+) is toxic for plants but shares similar chemical properties to the K+ ion and hence competes with its transport. Here, we report that K+ and Cs+ homeostasis in Arabidopsis thaliana also requires the action of ZIFL2 (Zinc-Induced Facilitator-Like 2), a member of the Major Facilitator Superfamily (MFS) of membrane transporters. We show that the Arabidopsis ZIFL2 is a functional transporter able to mediate K+ and Cs+ influx when heterologously expressed in yeast. Promoter–reporter, reverse transcription–PCR and fluorescent protein fusion experiments indicate that the predominant ZIFL2.1 isoform is targeted to the plasma membrane of endodermal and pericyle root cells. ZIFL2 loss of function and overexpression exacerbate and alleviate plant sensitivity, respectively, upon Cs+ and excess K+ supply, also influencing Cs+ whole-plant partitioning. We propose that the activity of this Arabidopsis MFS carrier promotes cellular K+ efflux in the root, thereby restricting Cs+/K+ xylem loading and subsequent root to shoot translocation under conditions of Cs+ or high K+ external supply. [ABSTRACT FROM AUTHOR]

We report results on the functional analysis of Saccharomyces cerevisiae ORF YNL065w, predicted to code for a protein belonging to the poorly characterized major facilitator superfamily (MFS) of transporters that are involved in multidrug resistance (MDR). YNL065w is important for a moderate increase of yeast tolerance to ketoconazole and to the cationic dye crystal violet; it protects the cell against short-chain monocarboxylic acids (C2–C6), but not against highly liposoluble acids such as octanoic acid or the phenoxyacetic-acid herbicides 2,4-D and MCPA; it is also a determinant of resistance to the antiarrhytmic and antimalarial drug quinidine. The encoding ORF was, thus, denominated the AQR1 gene. Results obtained using an AQR1-lacZ fusion indicate that gene expression is very low and it is not stimulated under weak acid stress. The encoded putative transporter was localized in the plasma membrane by fluorescence microscopy observation of the overproduced Aqr1-GFP fusion protein distribution. [Copyright &y& Elsevier]

The cryoprotection of cell activity is a key determinant in frozen-dough technology. Although several factors that contribute to freezing tolerance have been reported, the mechanism underlying the manner in which yeast cells respond to freezing and thawing (FT) stress is not well established. Therefore, the present study demonstrated the relationship between DaMDHAR encoding monodehydroascorbate reductase from Antarctic hairgrass Deschampsia antarctica and stress tolerance to repeated FT cycles (FT2) in transgenic yeast Saccharomyces cerevisiae. DaMDHAR-expressing yeast (DM) cells identified by immunoblotting analysis showed high tolerance to FT stress conditions, thereby causing lower damage for yeast cells than wild-type (WT) cells with empty vector alone. To detect FT2 tolerance-associated genes, 3′-quant RNA sequencing was employed using mRNA isolated from DM and WT cells exposed to FT (FT2) conditions. Approximately 332 genes showed ≥2-fold changes in DM cells and were classified into various groups according to their gene expression. The expressions of the changed genes were further confirmed using western blot analysis and biochemical assay. The upregulated expression of 197 genes was associated with pentose phosphate pathway, NADP metabolic process, metal ion homeostasis, sulfate assimilation, β-alanine metabolism, glycerol synthesis, and integral component of mitochondrial and plasma membrane (PM) in DM cells under FT2 stress, whereas the expression of the remaining 135 genes was partially related to protein processing, selenocompound metabolism, cell cycle arrest, oxidative phosphorylation, and α-glucoside transport under the same condition. With regard to transcription factors in DM cells, MSN4 and CIN5 were activated, but MSN2 and MGA1 were not. Regarding antioxidant systems and protein kinases in DM cells under FT stress, CTT1, GTO, GEX1, and YOL024W were upregulated, whereas AIF1, COX2, and TRX3 were not. Gene activation represented by transcription factors and enzymatic antioxidants appears to be associated with FT2-stress tolerance in transgenic yeast cells. RCK1, MET14, and SIP18, but not YPK2, have been known to be involved in the protein kinase-mediated signalling pathway and glycogen synthesis. Moreover, SPI18 and HSP12 encoding hydrophilin in the PM were detected. Therefore, it was concluded that the genetic network via the change of gene expression levels of multiple genes contributing to the stabilization and functionality of the mitochondria and PM, not of a single gene, might be the crucial determinant for FT tolerance in DaMDAHR-expressing transgenic yeast. These findings provide a foundation for elucidating the DaMDHAR-dependent molecular mechanism of the complex functional resistance in the cellular response to FT stress. [ABSTRACT FROM AUTHOR]