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- The Arabidopsis SR45 Splicing Factor, a Negative Regulator of Sugar Signaling, Modulates SNF1-Related Protein Kinase 1 StabilityPublication . Carvalho, Raquel F.; Szakonyi, Dóra; Simpson, Craig G.; Barbosa, Inês C.R.; Brown, John W.S.; Baena-González, Elena; Duque, PaulaThe ability to sense and respond to sugar signals allows plants to cope with environmental and metabolic changes by adjusting growth and development accordingly. We previously reported that the SR45 splicing factor negatively regulates glucose signaling during early seedling development in Arabidopsis thaliana Here, we show that under glucose-fed conditions, the Arabidopsis sr45-1 loss-of-function mutant contains higher amounts of the energy-sensing SNF1-Related Protein Kinase 1 (SnRK1) despite unaffected SnRK1 transcript levels. In agreement, marker genes for SnRK1 activity are upregulated in sr45-1 plants, and the glucose hypersensitivity of sr45-1 is attenuated by disruption of the SnRK1 gene. Using a high-resolution RT-PCR panel, we found that the sr45-1 mutation broadly targets alternative splicing in vivo, including that of the SR45 pre-mRNA itself. Importantly, the enhanced SnRK1 levels in sr45-1 are suppressed by a proteasome inhibitor, indicating that SR45 promotes targeting of the SnRK1 protein for proteasomal destruction. Finally, we demonstrate that SR45 regulates alternative splicing of the Arabidopsis 5PTase13 gene, which encodes an inositol polyphosphate 5-phosphatase previously shown to interact with and regulate the stability of SnRK1 in vitro, thus providing a mechanistic link between SR45 function and the modulation of degradation of the SnRK1 energy sensor in response to sugars.
- 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.
- Pre-mRNA splicing repression triggers abiotic stress signaling in plantsPublication . Ling, Yu; Alshareef, Sahar; Butt, Haroon; Lozano-Juste, Jorge; Li, Lixin; Galal, Aya A.; Moustafa, Ahmed; Momin, Afaque A.; Tashkandi, Manal; Richardson, Dale N.; Fujii, Hiroaki; Arold, Stefan; Rodriguez, Pedro L.; Duque, Paula; Mahfouz, Magdy M.Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress- and ABA-responsive reporters RD29A::LUC and MAPKKK18::uidA in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2C phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling and post-transcriptional regulation in plants.
- 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.
- The Major Facilitator Superfamily Transporter ZIFL2 Modulates Cesium and Potassium Homeostasis in ArabidopsisPublication . Remy, E.; Cabrito, T. R.; Batista, R. A.; Teixeira, M. C.; Sa-Correia, I.; Duque, P.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.
- ZIFL1.1 transporter modulates polar auxin transport by stabilizing membrane abundance of multiple PINs inArabidopsisroot tipPublication . Remy, Estelle; Baster, Pawel; Friml, Jiří; Duque, PaulaCell-to-cell directional flow of the phytohormone auxin is primarily established by polar localization of the PIN auxin transporters, a process tightly regulated at multiple levels by auxin itself. We recently reported that, in the context of strong auxin flows, activity of the vacuolar ZIFL1.1 transporter is required for fine-tuning of polar auxin transport rates in the Arabidopsis root. In particular, ZIFL1.1 function protects plasma-membrane stability of the PIN 2 carrier in epidermal root tip cells under conditions normally triggering PIN 2 degradation. Here, we show that ZIFL1.1 activity at the root tip also promotes PIN 1 plasma-membrane abundance in central cylinder cells, thus supporting the notion that ZIFL1.1 acts as a general positive modulator of polar auxin transport in roots.
- 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.
- 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.
- 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.