Heterologous expression of the yeast Tpo1p or Pdr5p membrane transporters in Arabidopsis confers plant xenobiotic tolerance.

Soil contamination is a major hindrance for plant growth and development. The lack of effective strategies to remove chemicals released into the environment has raised the need to increase plant resilience to soil pollutants. Here, we investigated the ability of two Saccharomyces cerevisiae plasma-membrane transporters, the Major Facilitator Superfamily (MFS) member Tpo1p and the ATP-Binding Cassette (ABC) protein Pdr5p, to confer Multiple Drug Resistance (MDR) in Arabidopsis thaliana. Transgenic plants expressing either of the yeast transporters were undistinguishable from the wild type under control conditions, but displayed tolerance when challenged with the herbicides 2,4-D and barban. Plants expressing ScTPO1 were also more resistant to the herbicides alachlor and metolachlor as well as to the fungicide mancozeb and the Co2+, Cu2+, Ni2+, Al3+ and Cd2+ cations, while ScPDR5-expressing plants exhibited tolerance to cycloheximide. Yeast mutants lacking Tpo1p or Pdr5p showed increased sensitivity to most of the agents tested in plants. Our results demonstrate that the S. cerevisiae Tpo1p and Pdr5p transporters are able to mediate resistance to a broad range of compounds of agricultural interest in yeast as well as in Arabidopsis, underscoring their potential in future biotechnological applications.

Yeast response and tolerance to benzoic acid involves the Gcn4- and Stp1-regulated multidrug/multixenobiotic resistance transporter Tpo1.

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.

The major facilitator superfamily transporter ZIFL2 modulates cesium and potassium homeostasis in Arabidopsis.

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.

Intron retention in the 5'UTR of the novel ZIF2 transporter enhances translation to promote zinc tolerance in arabidopsis.

Root vacuolar sequestration is one of the best-conserved plant strategies to cope with heavy metal toxicity. Here we report that zinc (Zn) tolerance in Arabidopsis requires the action of a novel Major Facilitator Superfamily (MFS) transporter. We show that ZIF2 (Zinc-Induced Facilitator 2) localises primarily at the tonoplast of root cortical cells and is a functional transporter able to mediate Zn efflux when heterologously expressed in yeast. By affecting plant tissue partitioning of the metal ion, loss of ZIF2 function exacerbates plant sensitivity to excess Zn, while its overexpression enhances Zn tolerance. The ZIF2 gene is Zn-induced and an intron retention event in its 5'UTR generates two splice variants (ZIF2.1 and ZIF2.2) encoding the same protein. Importantly, high Zn favours production of the longer ZIF2.2 transcript, which compared to ZIF2.1 confers greater Zn tolerance to transgenic plants by promoting higher root Zn immobilization. We show that the retained intron in the ZIF2 5'UTR enhances translation in a Zn-responsive manner, markedly promoting ZIF2 protein expression under excess Zn. Moreover, Zn regulation of translation driven by the ZIF2.2 5'UTR depends largely on a predicted stable stem loop immediately upstream of the start codon that is lost in the ZIF2.1 5'UTR. Collectively, our findings indicate that alternative splicing controls the levels of a Zn-responsive mRNA variant of the ZIF2 transporter to enhance plant tolerance to the metal ion.

Candida glabrata drug:H+ antiporter CgQdr2 confers imidazole drug resistance, being activated by transcription factor CgPdr1.

The widespread emergence of antifungal drug resistance poses a severe clinical problem. Though predicted to play a role in this phenomenon, the drug:H(+) antiporters (DHA) of the major facilitator superfamily have largely escaped characterization in pathogenic yeasts. This work describes the first DHA from the pathogenic yeast Candida glabrata reported to be involved in antifungal drug resistance, the C. glabrata QDR2 (CgQDR2) gene (ORF CAGL0G08624g). The expression of CgQDR2 in C. glabrata was found to confer resistance to the antifungal drugs miconazole, tioconazole, clotrimazole, and ketoconazole. By use of a green fluorescent protein (GFP) fusion, the CgQdr2 protein was found to be targeted to the plasma membrane in C. glabrata. In agreement with these observations, CgQDR2 expression was found to decrease the intracellular accumulation of radiolabeled clotrimazole in C. glabrata and to play a role in the extrusion of this antifungal from preloaded cells. Interestingly, the functional heterologous expression of CgQDR2 in the model yeast Saccharomyces cerevisiae further confirmed the role of this gene as a multidrug resistance determinant: its expression was able to complement the susceptibility phenotype exhibited by its S. cerevisiae homologue, QDR2, in the presence of imidazoles and of the antimalarial and antiarrhythmic drug quinidine. In contrast to the findings reported for Qdr2, CgQdr2 expression does not contribute to the ability of yeast to grow under K(+)-limiting conditions. Interestingly, CgQDR2 transcript levels were seen to be upregulated in C. glabrata cells challenged with clotrimazole or quinidine. This upregulation was found to depend directly on the transcription factor CgPdr1, the major regulator of multidrug resistance in this pathogenic yeast, which has also been found to be a determinant of quinidine and clotrimazole resistance in C. glabrata.

A major facilitator superfamily transporter plays a dual role in polar auxin transport and drought stress tolerance in Arabidopsis.

Many key aspects of plant development are regulated by the polarized transport of the phytohormone auxin. Cellular auxin efflux, the rate-limiting step in this process, has been shown to rely on the coordinated action of PIN-formed (PIN) and B-type ATP binding cassette (ABCB) carriers. Here, we report that polar auxin transport in the Arabidopsis thaliana root also requires the action of a Major Facilitator Superfamily (MFS) transporter, Zinc-Induced Facilitator-Like 1 (ZIFL1). Sequencing, promoter-reporter, and fluorescent protein fusion experiments indicate that the full-length ZIFL1.1 protein and a truncated splice isoform, ZIFL1.3, localize to the tonoplast of root cells and the plasma membrane of leaf stomatal guard cells, respectively. Using reverse genetics, we show that the ZIFL1.1 transporter regulates various root auxin-related processes, while the ZIFL1.3 isoform mediates drought tolerance by regulating stomatal closure. Auxin transport and immunolocalization assays demonstrate that ZIFL1.1 indirectly modulates cellular auxin efflux during shootward auxin transport at the root tip, likely by regulating plasma membrane PIN2 abundance. Finally, heterologous expression in yeast revealed that ZIFL1.1 and ZIFL1.3 share H(+)-coupled K(+) transport activity. Thus, by determining the subcellular and tissue distribution of two isoforms, alternative splicing dictates a dual function for the ZIFL1 transporter. We propose that this MFS carrier regulates stomatal movements and polar auxin transport by modulating potassium and proton fluxes in Arabidopsis cells.

Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation.

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 ³H-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.

Yeast toxicogenomics: genome-wide responses to chemical stresses with impact in environmental health, pharmacology, and biotechnology.

The emerging transdisciplinary field of Toxicogenomics aims to study the cell response to a given toxicant at the genome, transcriptome, proteome, and metabolome levels. This approach is expected to provide earlier and more sensitive biomarkers of toxicological responses and help in the delineation of regulatory risk assessment. The use of model organisms to gather such genomic information, through the exploitation of Omics and Bioinformatics approaches and tools, together with more focused molecular and cellular biology studies are rapidly increasing our understanding and providing an integrative view on how cells interact with their environment. The use of the model eukaryote Saccharomyces cerevisiae in the field of Toxicogenomics is discussed in this review. Despite the limitations intrinsic to the use of such a simple single cell experimental model, S. cerevisiae appears to be very useful as a first screening tool, limiting the use of animal models. Moreover, it is also one of the most interesting systems to obtain a truly global understanding of the toxicological response and resistance mechanisms, being in the frontline of systems biology research and developments. The impact of the knowledge gathered in the yeast model, through the use of Toxicogenomics approaches, is highlighted here by its use in prediction of toxicological outcomes of exposure to pesticides and pharmaceutical drugs, but also by its impact in biotechnology, namely in the development of more robust crops and in the improvement of yeast strains as cell factories.

The yeast ABC transporter Pdr18 (ORF YNR070w) controls plasma membrane sterol composition, playing a role in multidrug resistance.

The action of multidrug efflux pumps in MDR (multidrug resistance) acquisition has been proposed to partially depend on the transport of physiological substrates which may indirectly affect drug partition and transport across cell membranes. In the present study, the PDR18 gene [ORF (open reading frame) YNR070w], encoding a putative PDR (pleiotropic drug resistance) transporter of the ATP-binding cassette superfamily, was found to mediate plasma membrane sterol incorporation in yeast. The physiological role of Pdr18 is demonstrated to affect plasma membrane potential and is proposed to underlie its action as a MDR determinant, conferring resistance to the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid). The action of Pdr18 in yeast tolerance to 2,4-D, which was found to contribute to reduce [(14)C]2,4-D intracellular accumulation, may be indirect, given the observation that 2,4-D exposure deeply affects the sterol plasma membrane composition, this effect being much stronger in a Δpdr18 background. PDR18 activation under 2,4-D stress is regulated by the transcription factors Nrg1, controlling carbon source availability and the stress response, and, less significantly, Yap1, involved in oxidative stress and MDR, and Pdr3, a key regulator of the yeast PDR network, consistent with a broad role in stress defence. Taken together, the results of the present study suggest that Pdr18 plays a role in plasma membrane sterol incorporation, this physiological trait contributing to an MDR phenotype.

Yeast response and tolerance to polyamine toxicity involving the drug : H+ antiporter Qdr3 and the transcription factors Yap1 and Gcn4.

The yeast QDR3 gene encodes a plasma membrane drug : H(+) antiporter of the DHA1 family that was described as conferring resistance against the drugs quinidine, cisplatin and bleomycin and the herbicide barban, similar to its close homologue QDR2. In this work, a new physiological role for Qdr3 in polyamine homeostasis is proposed. QDR3 is shown to confer resistance to the polyamines spermine and spermidine, but, unlike Qdr2, also a determinant of resistance to polyamines, Qdr3 has no apparent role in K(+) homeostasis. QDR3 transcription is upregulated in yeast cells exposed to spermine or spermidine dependent on the transcription factors Gcn4, which controls amino acid homeostasis, and Yap1, the main regulator of oxidative stress response. Yap1 was found to be a major determinant of polyamine stress resistance in yeast and is accumulated in the nucleus of yeast cells exposed to spermidine-induced stress. QDR3 transcript levels were also found to increase under nitrogen or amino acid limitation; this regulation is also dependent on Gcn4. Consistent with the concept that Qdr3 plays a role in polyamine homeostasis, QDR3 expression was found to decrease the intracellular accumulation of [(3)H]spermidine, playing a role in the maintenance of the plasma membrane potential in spermidine-stressed cells.

Heterologous expression of a Tpo1 homolog from Arabidopsis thaliana confers resistance to the herbicide 2,4-D and other chemical stresses in yeast.

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 Deltatpo1 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), Al(3+) and Tl(3+). At5g13750 is the first plant putative MFS transporter to be suggested as possibly involved in MDR.

Drug:H+ antiporters in chemical stress response in yeast.

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.