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

This deposit is composed by the main article plus the supplementary materials of the publication.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 Co(2+), Cu(2+), Ni(2+), Al(3+) and Cd(2+) 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.Fundação para a Ciência e a Tecnologia grants: (EXPL/AGR-PRO/1013/2013, PTDC/BIA-PLA/1084/2014, SFRH/BPD/44640/2008, SFRH/BPD/81221/2011, PD/BD/105735/2014, PD/00133/2012, SFRH/BD/92552/2013, UID/BIO/04565/2013, UID/Multi/04551/2013). Programa Operacional Regional de Lisboa 2020 grant: (Project N. 007317).info:eu-repo/semantics/publishedVersio

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.FCT PostDoctoral Fellowships: SFRH/BPD/44640/2008, SFRH/BPD/81221/2011

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 [14C]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

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

<p>Abstract</p> <p>Background</p> <p>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 <it>S. cerevisiae</it> were scrutinized for a role in ethanol stress resistance.</p> <p>Results</p> <p>A yeast multidrug resistance ABC transporter encoded by the <it>PDR18</it> 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. <it>PDR18</it> 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 <it>PDR18</it>, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of <it>PDR18</it> was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the <it>PDR18</it> promoter was replaced in the genome by the stronger <it>PDR5</it> promoter, leading to increased <it>PDR18</it> 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 <it>PDR18</it> was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation.</p> <p>Conclusions</p> <p><it>PDR18</it> gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. <it>PDR18</it> overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic fermentation performance for sustainable bio-ethanol production.</p

The <i>Arabidopsis ZIF2</i> gene encodes multiple transcripts.

<p>(A) Exon/intron organization of the <i>ZIF2</i> gene and T-DNA insertion site in the <i>zif2-1</i> mutant. Boxes and lines between boxes denote exons and introns, respectively. The triangle depicts the site of the T-DNA insertion. F1, F1′, F2, F3, R1, R2, and R3 indicate the location of the primers used to detect <i>ZIF2</i> expression in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004375#pgen-1004375-g003" target="_blank">Figures 3A, 3C</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004375#pgen-1004375-g007" target="_blank">7A, S</a>1 and S4. Scale bar, 200 bp. (B) Structure of the alternative <i>ZIF2</i> transcripts. The coding sequence (CDS) is shown in black and the UTRs in white. Grey boxes indicate portions of the 5′UTR overlapping with the retained intron. RACE1 to RACE6 indicate the location of the gene-specific primers used in the 5′RLM-RACE experiment. Occurrence of each <i>ZIF2</i> transcript was determined according to its detection (number of corresponding 5′RLM-RACE clones) or not (-) by RLM-5′RACE in the indicated tissue. Scale bar, 200 nt.</p

The <i>ZIF2</i> promoter is active in most <i>Arabidopsis</i> tissues.

<p>(A–H) Differential interference contrast microscopy images of GUS-stained transgenic plants carrying the <i>ProZIF2:GFP-GUS</i> reporter construct. <i>ZIF2</i> promoter activity in floral buds (A), a mature flower (B), a young leaf (C), the primary root (D), the primary root tip (E), a lateral root primordium at stage V (F) and stage VII (G) and a mature lateral root (H). Scale bars, 1 mm (A), 200 µm (B, C), or 50 µm (D–H). (I–L) Confocal laser scanning microscopy images of transgenic root tissues carrying the <i>ProZIF2:GFP-GUS</i> reporter construct. <i>ZIF2</i> promoter activity in the primary root (I, K) and a mature lateral root (J, L). ep, epidermis; c, cortex; en, endodermis; cc, central cylinder. Cell walls were stained with propidium iodide. The GFP and propidium iodide signals are visualized by green and red coloration, respectively. Scale bars, 25 µm (I, J), or 10 µm (K, L).</p

The ZIF2 transporter affects root-shoot partitioning in <i>Arabidopsis</i>.

<p>Zn concentration, expressed on a dry weight (DW) basis, in the shoot (upper panel) and root (middle panel), as well as the corresponding shoot/root ratio (lower panel), of 21-d old seedlings of the wild type (Col-0), the <i>zif2-1</i> mutant and <i>ZIF2</i>-overexpressing lines (<i>ZIF2.1</i>OX2 and <i>ZIF2.2</i>OX1) grown on control medium (30 µM Zn²⁺) or under excess Zn supply (125 and 250 µM Zn²⁺). Bars represent means ± SD (<i>n</i> = 4). Different letters indicate statistically significant differences between genotypes under each condition (<i>P</i><0.05; Student's <i>t</i>-test).</p