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See detailCharacterization of FRD3, a iron and zinc homeostasis actor in Arabidopsis relatives
Scheepers, Maxime ULg; Charlier, Jean-Benoit; Spielmann, Julien ULg et al

Poster (2016, June 09)

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See detailCharacterization of FRD3, a zinc and iron homeostasis actor, in Arabidopsis
Scheepers, Maxime ULg; Charlier, Jean-Benoit; Spielmann, Julien ULg et al

Poster (2016, May 13)

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See detailDynamic Distribution and Interaction of the Arabidopsis SRSF1 Subfamily Splicing Factors
Stankovic, Nancy ULg; Schloesser, Marie ULg; Joris, Marine ULg et al

in Plant Physiology (2016), 170

Serine/Arginine-rich (SR) proteins are essential nucleus-localized splicing factors. Our prior studies showed that Arabidopsis RSZ22, a homolog of the human SRSF7 SR factor, exits the nucleus through two ... [more ▼]

Serine/Arginine-rich (SR) proteins are essential nucleus-localized splicing factors. Our prior studies showed that Arabidopsis RSZ22, a homolog of the human SRSF7 SR factor, exits the nucleus through two pathways, either dependent or independent on the XPO1 receptor. Here, we examined the expression profiles and shuttling dynamics of the Arabidopsis SRSF1 subfamily (SR30, SR34, SR34a and SR34b) under control of their endogenous promoter in Arabidopsis and in transient expression assay. Due to its rapid nucleocytoplasmic shuttling and high expression level in transient assay, we analysed the multiple determinants that regulate the localisation and shuttling dynamics of SR34. By site-directed mutagenesis of SR34 RNA-binding sequences and RS domain, we further show that functional RRM1 or RRM2 are dispensable for the exclusive protein nuclear localization and speckle-like distribution. However, mutations of both RRMs induced aggregation of the protein whereas mutation in the RS domain decreased the stability of the protein and suppressed its nuclear accumulation. Furthermore, the RNA-binding motif mutants are defective for their export through the XPO1 (CRM1/Exportin-1) receptor pathway, but retain nucleocytoplasmic mobility. We performed a yeast two hybrid screen with SR34 as bait and discovered SR45 as a new interactor. SR45 is an unusual SR splicing factor bearing two RS domains. These interactions were confirmed in planta by FLIM-FRET and BiFC and the roles of SR34 domains in protein-protein interactions were further studied. Altogether, our report extends our understanding of shuttling dynamics of Arabidopsis SR splicing factors [less ▲]

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See detailMetal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.
Laurent, Clémentine ULg; Lekeux, Gilles ULg; Ukuwela, Ashwinie A et al

in Plant Molecular Biology (2016), 90

PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding ... [more ▼]

PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. <br />Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. <br />The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. <br />Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases. [less ▲]

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See detailFunctional characterization of FRD3,  a citrate transporter, in Arabidopsis relatives
Scheepers, Maxime ULg; Charlier, Jean-Benoit; Spielmann, Julien ULg et al

Poster (2015, December 03)

Transcriptomic studies identified genes which are constitutively over-expressed in A. halleri compared to A. thaliana and which may have a role in metal tolerance or accumulation (1-3). A candidate gene ... [more ▼]

Transcriptomic studies identified genes which are constitutively over-expressed in A. halleri compared to A. thaliana and which may have a role in metal tolerance or accumulation (1-3). A candidate gene encodes FRD3, a member of the MATE family of membrane transporters (56 members in A. thaliana). It is a citrate transporter involved in iron homeostasis (4-6) and playing a role in zinc tolerance in A. thaliana (7). We are aiming to analyse the FRD3 high expression in A. halleri and the FRD3 function in zinc and iron homeostasis in A. thaliana. [less ▲]

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See detailTowards the identification of cis-elements responsible for the high expression of HMA4, an essential gene for Zn hyperaccumulation in Arabidopsis halleri.
Spielmann, Julien ULg; Nouet, Cécile ULg; Scheepers, Maxime ULg et al

Conference (2015, June 26)

A. halleri is a Zn and Cd hypertolerant and hyperaccumulator plant which stores metals in shoot vacuoles. One key step for hyperaccumulation is a highly efficient root-to-shoot translocation of metals ... [more ▼]

A. halleri is a Zn and Cd hypertolerant and hyperaccumulator plant which stores metals in shoot vacuoles. One key step for hyperaccumulation is a highly efficient root-to-shoot translocation of metals. The Zn/Cd plasmamembrane pump HMA4 (heavy metal ATPase 4) plays a central role in this process and is involved in xylem loading/unloading. Moreover, HMA4 is overexpressed in A. halleri compare to its non-tolerant and non-hyperaccumulator sister, Arabidopsis thaliana. The HMA4 overexpression is determined by a combination of gene triplication and cis-regulatory changes increasing transcription levels of each three HMA4 copies. In this study, we are focusing on the two most active HMA4 promoters and we aim to identify the cis-elements underlying high expression of HMA4 in A. halleri using promoter-reporter constructs. Through deletion analysis, two important regions for a high expression have been identified. Ongoing analysis of more detailed deletions will be presented. [less ▲]

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See detailTowards the identification of mechanisms underlying the high expression of HMA4, an essential gene for zinc hyperaccumulation in Arabidopsis halleri.
Spielmann, Julien ULg; Nouet, Cécile ULg; Scheepers, Maxime ULg et al

Poster (2015, June 08)

To maintain metal homeostasis within physiological limits independently of metal concentrations present in soil, plants developed a complex network including metal uptake, chelation, trafficking, and ... [more ▼]

To maintain metal homeostasis within physiological limits independently of metal concentrations present in soil, plants developed a complex network including metal uptake, chelation, trafficking, and storage processes. In this network, transporters have a central role. To study the molecular basis of zinc homeostasis in plants, we are using A. halleri, a zinc- and cadmium-tolerant and zinc-hyperaccumulating species representing the extreme end of natural variation in terms of metal homeostasis. Indeed, A. halleri stores high amount of metals in vacuoles of above-ground tissues (>1% Zn or 0.01% Cd in shoot dry weight). Cross-species transcriptomic studies identified about thirty genes which are constitutively over-expressed in A. halleri compared to its non-tolerant and non-hyperaccumulator sister species, Arabidopsis thaliana (1-3). HMA4 is one of these genes. It encodes a Zn/Cd plasmamembrane pump involved in xylem loading/unloading which is a key step in the hyperaccumulation process (4-5). The HMA4 overexpression is determined by a combination of gene triplication and cis-regulatory changes increasing transcription levels of each three HMA4 copies (5). We aim to identify the cis-elements underlying high expression of HMA4 in A. halleri using promoter-reporter constructs. [less ▲]

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See detailExpression study of FRD3 in Arabidopsis relatives
Scheepers, Maxime ULg; Charlier, Jean-Benoit ULg; Spielmann, Julien ULg et al

Poster (2015, June 08)

Transcriptomic studies identified genes which are constitutively over-expressed in A. halleri compared to A. thaliana and which may have a role in metal tolerance or accumulation (1-3). A candidate gene ... [more ▼]

Transcriptomic studies identified genes which are constitutively over-expressed in A. halleri compared to A. thaliana and which may have a role in metal tolerance or accumulation (1-3). A candidate gene encodes FRD3, a member of the MATE family of membrane transporters (56 members in A. thaliana). It is a citrate transporter involved in iron homeostasis (4-6) and playing a role in zinc tolerance in A. thaliana (7). We are aiming to analyse the FRD3 high expression in A. halleri and the FRD3 function in zinc and iron homeostasis in A. thaliana. [less ▲]

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See detailFunctional analysis of FRD3 in Arabidopsis
Scheepers, Maxime ULg; Charlier, Jean-Benoit ULg; Spielmann, Julien ULg et al

Poster (2015, June)

Zinc and iron are two essential micronutrients for plants. The homeostasis networks of the two metals are intertwined. The FRD3 (FERRIC REDUCTASE DEFECTIVE 3) protein, a member of the MATE family of ... [more ▼]

Zinc and iron are two essential micronutrients for plants. The homeostasis networks of the two metals are intertwined. The FRD3 (FERRIC REDUCTASE DEFECTIVE 3) protein, a member of the MATE family of membrane transporters, is a citrate transporter involved in iron homeostasis and playing a role in zinc tolerance in Arabidopsis. The FRD3 gene displays a complex regulation. Alternative transcript initiation for FRD3 determines two transcripts, which differ in their 5'UTRs and have differential translation efficiency. The two transcripts are selectively regulated under stress conditions: iron and zinc depletion, zinc excess or cadmium presence. We are aiming to determine the FRD3 function in zinc and iron homeostasis in Arabidopsis. We will present data (i) on the functional characterization of the alternative transcripts and their role in metal homeostasis in Arabidopsis and (ii) on the zinc phenotypes of the frd3 mutant. [less ▲]

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See detailTowards the identification of cis-elements responsible for the high expression of HMA4, an essential gene for zinc hyperaccumulation in Arabidopsis halleri.
Spielmann, Julien ULg; Nouet, Cécile ULg; Scheepers, Maxime ULg et al

Poster (2015, May 13)

A. halleri is a Zn and Cd hypertolerant and hyperaccumulator plant which stores metals in shoot vacuoles. One key step for hyperaccumulation is a highly efficient root-to-shoot translocation of metals ... [more ▼]

A. halleri is a Zn and Cd hypertolerant and hyperaccumulator plant which stores metals in shoot vacuoles. One key step for hyperaccumulation is a highly efficient root-to-shoot translocation of metals. The Zn/Cd plasmamembrane pump HMA4 (heavy metal ATPase 4) plays a central role in this process and is involved in xylem loading/unloading. Moreover, HMA4 is overexpressed in A. halleri compare to its non-tolerant and non-hyperaccumulator sister, Arabidopsis thaliana. The HMA4 overexpression is determined by a combination of gene triplication and cis-regulatory changes increasing transcription levels of each three HMA4 copies. In this study, we are focusing on the two most active HMA4 promoters and we aim to identify the cis-elements underlying high expression of HMA4 in A. halleri using promoter-reporter constructs. Through deletion analysis, two important regions for a high expression have been identified. Ongoing analysis of more detailed deletions will be presented. [less ▲]

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See detailZinc triggers a complex transcriptional and post-transcriptional regulation of the metal homeostasis gene FRD3 in Arabidopsis relatives
Charlier, Jean_Benoit; Polese, Catherine; Nouet, Cécile ULg et al

in Journal of Experimental Botany (2015), 66

In Arabidopsis thaliana, FRD3 (Ferric Chelate Reductase Defective 3) plays a central role in metal homeostasis. FRD3 is among a set of metal homeostasis genes that are constitutively highly expressed in ... [more ▼]

In Arabidopsis thaliana, FRD3 (Ferric Chelate Reductase Defective 3) plays a central role in metal homeostasis. FRD3 is among a set of metal homeostasis genes that are constitutively highly expressed in roots and shoots of Arabidopsis halleri, a zinc hyperaccumulating and hypertolerant species. Here, we examined the regulation of FRD3 by zinc in both species to shed light on the evolutionary processes underlying the evolution of hyperaccumulation in A. halleri. We combined gene expression studies with the use of GUS and GFP reporter constructs to compare the expression profile, transcriptional and post-transcriptional regulation of FRD3 in both species. The AtFRD3 and AhFRD3 genes display a conserved expression profile. In A. thaliana, alternative transcription initiation sites from two promoters determine transcript variants which are differentially regulated by zinc supply in roots and shoots to favour the most highly translated variant under zinc excess conditions. In A. halleri, a single transcript variant with higher transcript stability and enhanced translation has been maintained. The FRD3 gene thus undergoes complex transcriptional and post-transcriptional regulation in Arabidopsis relatives. Our study reveals that a diverse set of mechanisms underlie increased gene dosage in the A. halleri lineage and illustrates how an environmental challenge can alter gene regulation. [less ▲]

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See detailFunctional analysis of the three HMA4 copies of the metal hyperaccumulator Arabidopsis halleri
Nouet, Cécile ULg; Charlier, Jean-Benoit; Carnol, Monique ULg et al

in Journal of Experimental Botany (2015), 66

In Arabidopsis halleri, the AhHMA4 gene has an essential function in Zn/Cd hypertolerance and hyperaccumulation by mediating root to shoot translocation of metals. Constitutive high expression of AhHMA4 ... [more ▼]

In Arabidopsis halleri, the AhHMA4 gene has an essential function in Zn/Cd hypertolerance and hyperaccumulation by mediating root to shoot translocation of metals. Constitutive high expression of AhHMA4 results from a tandem triplication and cis-activation of the promoter of all three copies. The three AhHMA4 copies possess divergent promoter sequences, but highly conserved coding sequences, and display identical expression profiles in the root and shoot vascular system. Here, we expressed an AhHMA4::GFP fusion under the control of each three A. halleri HMA4 promoters in a hma2hma4 double mutant of Arabidopsis thaliana to individually examine the function of each A. halleri AhHMA4 copy. The protein localized non-polarly at the plasma membrane of the root pericycle cells of both A. thaliana and A. halleri. The expression of each AhHMA4::GFP copy complemented the severe Zn deficiency phenotype of the hma2hma4 mutant by restoring root-to-shoot translocation of zinc. However, each copy had different impact on metal homeostasis in the A. thaliana genetic background: AhHMA4 copies 2 and 3 were more highly expressed and provided higher Zn tolerance in roots and accumulation in shoots than copy 1, whereas AhHMA4 copy 3 also increased Cd tolerance in roots. Our data suggest a certain extent of functional differentiation among the three A. halleri HMA4 copies, stemming from differences in expression levels rather than in expression profile. HMA4 is a key node of the Zn homeostasis network and small changes in expression level can have major impact on Zn allocation to root or shoot tissues. [less ▲]

Detailed reference viewed: 142 (7 ULg)