Plant Molecular Biology
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The Plant Molecular Biology lab uses Arabidopsis thaliana as a model system to investigate how plants perceive and respond to environmental signals and endogenous developmental cues at the molecular level. In particular, we are focusing on the role of pre-mRNA splicing in the regulation of gene expression. The versatility of this mode of regulation suggests that it is likely to play an important contribution in ensuring the developmental plasticity and stress tolerance that are essential for plant survival. Another major ongoing project in the lab is examining the role of mutiple drug resistance (MDR) transporters in plant resistance to pesticides and other chemical stresses.
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Alternative Splicing in Plant Development and Stress Response
Herbicide resistance in Arabidopsis thaliana: Role of plant multidrug resistance transporters
Alternative Splicing of an Arabidopsis RING E3 ubiquitin ligase involved in plant stress and development
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- Abscisic Acid (ABA) Regulation of Arabidopsis SR Protein Gene ExpressionPublication . Cruz, Tiago; Carvalho, Raquel; Richardson, Dale; Duque, PaulaSerine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses.
- Alternative Splicing as a Regulator of Early Plant DevelopmentPublication . Szakonyi, Dóra; Duque, PaulaMost plant genes are interrupted by introns and the corresponding transcripts need to undergo pre-mRNA splicing to remove these intervening sequences. Alternative splicing (AS) is an important posttranscriptional process that creates multiple mRNA variants from a single pre-mRNA molecule, thereby enhancing the coding and regulatory potential of genomes. In plants, this mechanism has been implicated in the response to environmental cues, including abiotic and biotic stresses, in the regulation of key developmental processes such as flowering, and in circadian timekeeping. The early plant development steps - from embryo formation and seed germination to skoto- and photomorphogenesis - are critical to both execute the correct body plan and initiate a new reproductive cycle. We review here the available evidence for the involvement of AS and various splicing factors in the initial stages of plant development, while highlighting recent findings as well as potential future challenges.
- Alternative Splicing Control of Abiotic Stress ResponsesPublication . Laloum, Tom; Martín, Guiomar; Duque, PaulaAlternative splicing, which generates multiple transcripts from the same gene, is an important modulator of gene expression that can increase proteome diversity and regulate mRNA levels. In plants, this post-transcriptional mechanism is markedly induced in response to environmental stress, and recent studies have identified alternative splicing events that allow rapid adjustment of the abundance and function of key stress-response components. In agreement, plant mutants defective in splicing factors are severely impaired in their response to abiotic stress. Notably, mounting evidence indicates that alternative splicing regulates stress responses largely by targeting the abscisic acid (ABA) pathway. We review here current understanding of post-transcriptional control of plant stress tolerance via alternative splicing and discuss research challenges for the near future.
- Assessing Tolerance to Heavy-Metal Stress in Arabidopsis thaliana SeedlingsPublication . Remy, Estelle; Duque, PaulaHeavy-metal soil contamination is one of the major abiotic stress factors that, by negatively affecting plant growth and development, severely limit agricultural productivity worldwide. Plants have evolved various tolerance and detoxification strategies in order to cope with heavy-metal toxicity while ensuring adequate supply of essential micronutrients at the whole-plant as well as cellular levels. Genetic studies in the model plant Arabidopsis thaliana have been instrumental in elucidating such mechanisms. The root assay constitutes a very powerful and simple method to assess heavy-metal stress tolerance in Arabidopsis seedlings. It allows the simultaneous determination of all the standard growth parameters affected by heavy-metal stress (primary root elongation, lateral root development, shoot biomass, and chlorophyll content) in a single experiment. Additionally, this protocol emphasizes the tips and tricks that become particularly useful when quantifying subtle alterations in tolerance to a given heavy-metal stress, when simultaneously pursuing a large number of plant lines, or when testing sensitivity to a wide range of heavy metals for a single line.
- Beyond cellular detoxification: a plethora of physiological roles for MDR transporter homologs in plantsPublication . Remy, Estelle; Duque, PaulaHigher plants possess a multitude of Multiple Drug Resistance (MDR) transporter homologs that group into three distinct and ubiquitous families-the ATP-Binding Cassette (ABC) superfamily, the Major Facilitator Superfamily (MFS), and the Multidrug And Toxic compound Extrusion (MATE) family. As in other organisms, such as fungi, mammals, and bacteria, MDR transporters make a primary contribution to cellular detoxification processes in plants, mainly through the extrusion of toxic compounds from the cell or their sequestration in the central vacuole. This review aims at summarizing the currently available information on the in vivo roles of MDR transporters in plant systems. Taken together, these data clearly indicate that the biological functions of ABC, MFS, and MATE carriers are not restricted to xenobiotic and metal detoxification. Importantly, the activity of plant MDR transporters also mediates biotic stress resistance and is instrumental in numerous physiological processes essential for optimal plant growth and development, including the regulation of ion homeostasis and polar transport of the phytohormone auxin.
- Heterologous expression of the yeast Tpo1p or Pdr5p membrane transporters in Arabidopsis confers plant xenobiotic tolerancePublication . Remy, Estelle; Niño-González, María; Godinho, Cláudia P.; Cabrito, Tânia R.; Teixeira, Miguel C.; Sá-Correia, Isabel; Duque, PaulaSoil 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.
- Intron retention in the 5'UTR of the novel ZIF2 transporter enhances translation to promote zinc tolerance in arabidopsisPublication . Remy, Estelle; Cabrito, Tânia R; Batista, Rita A; Hussein, Mohamed A M; Teixeira, Miguel C; Athanasiadis, Alekos; Sá-Correia, Isabel; Duque, PaulaRoot 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.
- LEAFDATA: a literature-curated database for Arabidopsis leaf developmentPublication . Szakonyi, DóraIn the post-genomic era, biological databases provide an easy access to a wide variety of scientific data. The vast quantity of literature calls for curated databases where existing knowledge is carefully organized in order to aid novel discoveries. Leaves, the main photosynthetic organs are not only vital for plant growth but also essential for maintaining the global ecosystem by producing oxygen and food. Therefore, studying and understanding leaf formation and growth are key objectives in biology. Arabidopsis thaliana to this date remains the prime experimental model organism in plant science.
- A Major Facilitator Superfamily Transporter Plays a Dual Role in Polar Auxin Transport and Drought Stress Tolerance in ArabidopsisPublication . Remy, E.; Cabrito, T. R.; Baster, P.; Batista, R. A.; Teixeira, M. C.; Friml, J.; Sa-Correia, I.; Duque, P.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.
- On the physiological significance of alternative splicing events in higher plantsPublication . Carvalho, Raquel F.; Feijão, Carolina V.; Duque, PaulaAlternative splicing, which generates multiple transcripts from the same gene and potentially different protein isoforms, is a key posttranscriptional regulatory mechanism for expanding proteomic diversity and functional complexity in higher eukaryotes. The most recent estimates, based on whole transcriptome sequencing, indicate that about 95 % of human and 60 % of Arabidopsis multi-exon genes undergo alternative splicing, suggesting important roles for this mechanism in biological processes. However, while the misregulation of alternative splicing has been associated with many human diseases, its biological relevance in plant systems is just beginning to unfold. We review here the few plant genes for which the production of multiple splice isoforms has been reported to have a clear in vivo functional impact. These case studies implicate alternative splicing in the control of a wide range of physiological and developmental processes, including photosynthetic and starch metabolism, hormone signaling, seed germination, root growth and flowering, as well as in biotic and abiotic stress responses. Future functional characterization of alternative splicing events and identification of the transcripts targeted by major regulators of this versatile means of modulating gene expression should uncover the breadth of its physiological significance in higher plants.