Divergent roles of IREG/Ferroportin transporters from the nickel hyperaccumulator Leucocroton havanensis.

In response to our ever-increasing demand for metals, phytotechnologies are being developed to limit the environmental impact of conventional metal mining. However, the development of these technologies, which rely on plant species able to tolerate and accumulate metals, is partly limited by our lack of knowledge of the underlying molecular mechanisms. In this work, we aimed to better understand the role of metal transporters of the IRON REGULATED 1/FERROPORTIN (IREG/FPN) family from the nickel hyperaccumulator Leucocroton havanensis from the Euphorbiaceae family. Using transcriptomic data, we identified two homologous genes, LhavIREG1 and LhavIREG2, encoding divalent metal transporters of the IREG/FPN family. Both genes are expressed at similar levels in shoots, but LhavIREG1 shows higher expression in roots. The heterologous expression of these transporters in A. thaliana revealed that LhavIREG1 is localized to the plasma membrane, whereas LhavIREG2 is located on the vacuole. In addition, the expression of each gene induced a significant increase in nickel tolerance. Taken together, our data suggest that LhavIREG2 is involved in nickel sequestration in vacuoles of leaf cells, whereas LhavIREG1 is mainly involved in nickel translocation from roots to shoots, but could also be involved in metal sequestration in cell walls. Our results suggest that paralogous IREG/FPN transporters may play complementary roles in nickel hyperaccumulation in plants.

The X-ray fluorescence screening of multiple elements in herbarium specimens from the Neotropical region reveals new records of metal accumulation in plants.

Plants have developed a diversity of strategies to take up and store essential metals in order to colonize various types of soils including mineralized soils. Yet, our knowledge of the capacity of plant species to accumulate metals is still fragmentary across the plant kingdom. In this study, we have used the X-ray fluorescence technology to analyze metal concentration in a wide diversity of species of the Neotropical flora that was not extensively investigated so far. In total, we screened more than 11 000 specimens representing about 5000 species from herbaria in Paris and Cuba. Our study provides a large overview of the accumulation of metals such as manganese, zinc, and nickel in the Neotropical flora. We report 30 new nickel hyperaccumulating species from Cuba, including the first records in the families Connaraceae, Melastomataceae, Polygonaceae, Santalaceae, and Urticaceae. We also identified the first species from this region of the world that can be considered as manganese hyperaccumulators in the genera Lomatia (Proteaceae), Calycogonium (Melastomataceae), Ilex (Aquifoliaceae), Morella (Myricaceae), and Pimenta (Myrtaceae). Finally, we report the first zinc hyperaccumulator, Rinorea multivenosa (Violaceae), from the Amazonas region. The identification of species able to accumulate high amounts of metals will become instrumental to support the development of phytotechnologies in order to limit the impact of soil metal pollution in this region of the world.

Wide cross-species RNA-Seq comparison reveals convergent molecular mechanisms involved in nickel hyperaccumulation across dicotyledons.

The Anthropocene epoch is associated with the spreading of metals in the environment increasing oxidative and genotoxic stress on organisms. Interestingly, c. 520 plant species growing on metalliferous soils acquired the capacity to accumulate and tolerate a tremendous amount of nickel in their shoots. The wide phylogenetic distribution of these species suggests that nickel hyperaccumulation evolved multiple times independently. However, the exact nature of these mechanisms and whether they have been recruited convergently in distant species is not known. To address these questions, we have developed a cross-species RNA-Seq approach combining differential gene expression analysis and cluster of orthologous group annotation to identify genes linked to nickel hyperaccumulation in distant plant families. Our analysis reveals candidate orthologous genes encoding convergent function involved in nickel hyperaccumulation, including the biosynthesis of specialized metabolites and cell wall organization. Our data also point out that the high expression of IREG/Ferroportin transporters recurrently emerged as a mechanism involved in nickel hyperaccumulation in plants. We further provide genetic evidence in the hyperaccumulator Noccaea caerulescens for the role of the NcIREG2 transporter in nickel sequestration in vacuoles. Our results provide molecular tools to better understand the mechanisms of nickel hyperaccumulation and study their evolution in plants.

Vacuolar Iron Stores Gated by NRAMP3 and NRAMP4 Are the Primary Source of Iron in Germinating Seeds.

During seed germination, iron (Fe) stored in vacuoles is exported by the redundant NRAMP3 and NRAMP4 transporter proteins. A double nramp3 nramp4 mutant is unable to mobilize Fe stores and does not develop in the absence of external Fe. We used RNA sequencing to compare gene expression in nramp3 nramp4 and wild type during germination and early seedling development. Even though sufficient Fe was supplied, the Fe-responsive transcription factors bHLH38, 39, 100, and 101 and their downstream targets FRO2 and IRT1 mediating Fe uptake were strongly upregulated in the nramp3 nramp4 mutant. Activation of the Fe deficiency response was confirmed by increased ferric chelate reductase activity in the mutant. At early stages, genes important for chloroplast redox control (FSD1 and SAPX), Fe homeostasis (FER1 and SUFB), and chlorophyll metabolism (HEMA1 and NYC1) were downregulated, indicating limited Fe availability in plastids. In contrast, expression of FRO3, encoding a ferric reductase involved in Fe import into the mitochondria, was maintained, and Fe-dependent enzymes in the mitochondria were unaffected in nramp3 nramp4 Together, these data show that a failure to mobilize Fe stores during germination triggered Fe deficiency responses and strongly affected plastids, but not mitochondria.

Phosphatidylinositol 3-phosphate-binding protein AtPH1 controls the localization of the metal transporter NRAMP1 in Arabidopsis.

"Too much of a good thing" perfectly describes the dilemma that living organisms face with metals. The tight control of metal homeostasis in cells depends on the trafficking of metal transporters between membranes of different compartments. However, the mechanisms regulating the location of transport proteins are still largely unknown. Developing Arabidopsis thaliana seedlings require the natural resistance-associated macrophage proteins (NRAMP3 and NRAMP4) transporters to remobilize iron from seed vacuolar stores and thereby acquire photosynthetic competence. Here, we report that mutations in the pleckstrin homology (PH) domain-containing protein AtPH1 rescue the iron-deficient phenotype of nramp3nramp4 Our results indicate that AtPH1 binds phosphatidylinositol 3-phosphate (PI3P) in vivo and acts in the late endosome compartment. We further show that loss of AtPH1 function leads to the mislocalization of the metal uptake transporter NRAMP1 to the vacuole, providing a rationale for the reversion of nramp3nramp4 phenotypes. This work identifies a PH domain protein as a regulator of plant metal transporter localization, providing evidence that PH domain proteins may be effectors of PI3P for protein sorting.

Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant.

To improve seed iron (Fe) content and bioavailability, it is crucial to decipher the mechanisms that control Fe storage during seed development. In Arabidopsis (Arabidopsis thaliana) seeds, most Fe is concentrated in insoluble precipitates, with phytate in the vacuoles of cells surrounding the vasculature of the embryo. NATURAL RESISTANCE ASSOCIATED-MACROPHAGE PROTEIN3 (AtNRAMP3) and AtNRAMP4 function redundantly in Fe retrieval from vacuoles during germination. When germinated under Fe-deficient conditions, development of the nramp3nramp4 double mutant is arrested as a consequence of impaired Fe mobilization. To identify novel genes involved in seed Fe homeostasis, we screened an ethyl methanesulfonate-mutagenized population of nramp3nramp4 seedlings for mutations suppressing their phenotypes on low Fe. Here, we report that, among the suppressors, two independent mutations in the VACUOLAR IRON TRANSPORTER1 (AtVIT1) gene caused the suppressor phenotype. The AtVIT1 transporter is involved in Fe influx into vacuoles of endodermal and bundle sheath cells. This result establishes a functional link between Fe loading in vacuoles by AtVIT1 and its remobilization by AtNRAMP3 and AtNRAMP4. Moreover, analysis of subcellular Fe localization indicates that simultaneous disruption of AtVIT1, AtNRAMP3, and AtNRAMP4 limits Fe accumulation in vacuolar globoids.

Aquaporins Contribute to ABA-Triggered Stomatal Closure through OST1-Mediated Phosphorylation.

Stomatal movements in response to environmental stimuli critically control the plant water status. Although these movements are governed by osmotically driven changes in guard cell volume, the role of membrane water channels (aquaporins) has remained hypothetical. Assays in epidermal peels showed that knockout Arabidopsis thaliana plants lacking the Plasma membrane Intrinsic Protein 2;1 (PIP2;1) aquaporin have a defect in stomatal closure, specifically in response to abscisic acid (ABA). ABA induced a 2-fold increase in osmotic water permeability (Pf) of guard cell protoplasts and an accumulation of reactive oxygen species in guard cells, which were both abrogated in pip2;1 plants. Open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6), a protein kinase involved in guard cell ABA signaling, was able to phosphorylate a cytosolic PIP2;1 peptide at Ser-121. OST1 enhanced PIP2;1 water transport activity when coexpressed in Xenopus laevis oocytes. Upon expression in pip2;1 plants, a phosphomimetic form (Ser121Asp) but not a phosphodeficient form (Ser121Ala) of PIP2;1 constitutively enhanced the Pf of guard cell protoplasts while suppressing its ABA-dependent activation and was able to restore ABA-dependent stomatal closure in pip2;1. This work supports a model whereby ABA-triggered stomatal closure requires an increase in guard cell permeability to water and possibly hydrogen peroxide, through OST1-dependent phosphorylation of PIP2;1 at Ser-121.

Rhizobium metallidurans sp. nov., a symbiotic heavy metal resistant bacterium isolated from the Anthyllis vulneraria Zn-hyperaccumulator.

A Gram-stain-negative, aerobic, rod-shaped, non-spore-forming bacterium (ChimEc512(T)) was isolated from 56 host seedlings of the hyperaccumulating Anthyllis vulneraria legume, which was on an old zinc mining site at Les Avinières, Saint-Laurent-Le-Minier, Gard, South of France. On the basis of 16S rRNA gene sequence similarities, strain ChimEc512(T) was shown to belong to the genus Rhizobium and to be most closely related to Rhizobium endophyticum CCGE 2052(T) (98.4%), Rhizobium tibeticum CCBAU 85039(T) (98.1%), Rhizobium grahamii CCGE 502(T) (98.0%) and Rhizobium mesoamericanum CCGE 501(T) (98.0%). The phylogenetic relationships of ChimEc512(T) were confirmed by sequencing and analyses of recA and atpD genes. DNA-DNA relatedness values of strain ChimEc512(T) with R. endophyticum CCGE 2052(T), R. tibeticum CCBAU 85039(T), R. mesoamericanum CCGE 52(T), Rhizobium grahamii CCGE 502(T), Rhizobium etli CCBAU 85039(T) and Rhizobium radiobacter KL09-16-8-2(T) were 27, 22, 16, 18, 19 and 11%, respectively. The DNA G+C content of strain ChimEc512(T) was 58.9 mol%. The major cellular fatty acid was C18 : 1ω7c, characteristic of the genus Rhizobium . The polar lipid profile included phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol and phosphatidylcholine and moderate amounts of aminolipids, phospholipid and sulfoquinovosyl diacylglycerol. Although ChimEc512(T) was able to nodulate A. vulneraria, the nodC and nifH genes were not detected by PCR. The rhizobial strain was tolerant to high concentrations of heavy metals: up to 35 mM Zn and up to 0.5 mM Cd and its growth kinetics was not impacted by Zn. The results of DNA-DNA hybridizations and physiological tests allowed genotypic and phenotypic differentiation of strain ChimEc512(T) from species of the genus Rhizobium with validly published names. Strain ChimEc512(T), therefore, represents a novel species, for which the name Rhizobium metallidurans sp. nov. is proposed, with the type strain ChimEc512(T) ( =DSM 26575 = CIP 110550(T)).

Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response to abscisic acid.

Eukaryotic anion/proton exchangers of the CLC (chloride channel) family mediate anion fluxes across intracellular membranes. The Arabidopsis thaliana anion/proton exchanger AtCLCa is involved in vacuolar accumulation of nitrate. We investigated the role of AtCLCa in leaf guard cells, a specialized plant epidermal cell that controls gas exchange and water loss through pores called stomata. We showed that AtCLCa not only fulfilled the expected role of accumulating anions in the vacuole during stomatal opening but also mediated anion release during stomatal closure in response to the stress hormone abscisic acid (ABA). We found that this dual role resulted from a phosphorylation-dependent change in the activity of AtCLCa. The protein kinase OST1 (also known as SnRK2.6) is a key signaling player and central regulator in guard cells in response to ABA. Phosphorylation of Thr(38) in the amino-terminal cytoplasmic domain of AtCLCa by OST1 increased the outward anion fluxes across the vacuolar membrane, which are essential for stomatal closure. We provide evidence that bidirectional activities of an intracellular CLC exchanger are physiologically relevant and that phosphorylation regulates the transport mode of this exchanger.

The metal transporter PgIREG1 from the hyperaccumulator Psychotria gabriellae is a candidate gene for nickel tolerance and accumulation.

Nickel is an economically important metal and phytotechnologies are being developed to limit the impact of nickel mining on the environment. More than 300 plant species are known to hyperaccumulate nickel. However, our knowledge of the mechanisms involved in nickel accumulation in plants is very limited because it has not yet been possible to study these hyperaccumulators at the genomic level. Here, we used next-generation sequencing technologies to sequence the transcriptome of the nickel hyperaccumulator Psychotria gabriellae of the Rubiaceae family, and used yeast and Arabidopsis as heterologous systems to study the activity of identified metal transporters. We characterized the activity of three metal transporters from the NRAMP and IREG/FPN families. In particular, we showed that PgIREG1 is able to confer nickel tolerance when expressed in yeast and in transgenic plants, where it localizes in the tonoplast. In addition, PgIREG1 shows higher expression in P. gabriellae than in the related non-accumulator species Psychotria semperflorens. Our results designate PgIREG1 as a candidate gene for nickel tolerance and hyperaccumulation in P. gabriellae. These results also show how next-generation sequencing technologies can be used to access the transcriptome of non-model nickel hyperaccumulators to identify the underlying molecular mechanisms.

The metal hyperaccumulators from New Caledonia can broaden our understanding of nickel accumulation in plants.

While an excess of metals such as zinc, cadmium or nickel (Ni) is toxic for most plants, about 500 plant species called hyperaccumulators are able to accumulate high amounts of these metals. These plants and the underlying mechanisms are receiving an increasing interest because of their potential use in sustainable biotechnologies such as biofortification, phytoremediation, and phytomining. Among hyperaccumulators, about 400 species scattered in 40 families accumulate Ni. Despite this wide diversity, our current knowledge of the mechanisms involved in Ni accumulation is still limited and mostly restricted to temperate herbaceous Brassicaceae. New Caledonia is an archipelago of the tropical southwest pacific with a third of its surface (5500 km(2)) covered by Ni-rich soils originating from ultramafic rocks. The rich New Caledonia flora contains 2145 species adapted to these soils, among which 65 are Ni hyperaccumulators, including lianas, shrubs or trees, mostly belonging to the orders Celastrales, Oxalidales, Malpighiales, and Gentianales. We present here our current knowledge on Ni hyperaccumulators from New Caledonia and the latest molecular studies developed to better understand the mechanisms of Ni accumulation in these plants.

The Arabidopsis ABA-activated kinase OST1 phosphorylates the bZIP transcription factor ABF3 and creates a 14-3-3 binding site involved in its turnover.

BACKGROUND: Genetic evidence in Arabidopsis thaliana indicates that members of the Snf1-Related Kinases 2 family (SnRK2) are essential in mediating various stress-adaptive responses. Recent reports have indeed shown that one particular member, Open Stomata (OST)1, whose kinase activity is stimulated by the stress hormone abscisic acid (ABA), is a direct target of negative regulation by the core ABA co-receptor complex composed of PYR/PYL/RCAR and clade A Protein Phosphatase 2C (PP2C) proteins. METHODOLOGY/PRINCIPAL FINDINGS: Here, the substrate preference of OST1 was interrogated at a genome-wide scale. We phosphorylated in vitro a bank of semi-degenerate peptides designed to assess the relative phosphorylation efficiency on a positionally fixed serine or threonine caused by systematic changes in the flanking amino acid sequence. Our results designate the ABA-responsive-element Binding Factor 3 (ABF3), which controls part of the ABA-regulated transcriptome, as a genuine OST1 substrate. Bimolecular Fluorescence Complementation experiments indicate that ABF3 interacts directly with OST1 in the nuclei of living plant cells. In vitro, OST1 phosphorylates ABF3 on multiple LXRXXpS/T preferred motifs including T451 located in the midst of a conserved 14-3-3 binding site. Using an antibody sensitive to the phosphorylated state of the preferred motif, we further show that ABF3 is phosphorylated on at least one such motif in response to ABA in vivo and that phospho-T451 is important for stabilization of ABF3. CONCLUSIONS/SIGNIFICANCE: All together, our results suggest that OST1 phosphorylates ABF3 in vivo on T451 to create a 14-3-3 binding motif. In a wider physiological context, we propose that the long term responses to ABA that require sustained gene expression is, in part, mediated by the stabilization of ABFs driven by ABA-activated SnRK2s.

Phospho-site mapping, genetic and in planta activation studies reveal key aspects of the different phosphorylation mechanisms involved in activation of SnRK2s.

Snf1-related protein kinases 2 (SnRK2s) are major positive regulators of drought stress tolerance. The kinases of this family are activated by hyperosmotic stress, but only some of them are also responsive to abscisic acid (ABA). Moreover, genetic evidence has indicated the ABA-independence of SnRK2 activation in the fast response to osmotic stress. Although phosphorylation was demonstrated to be crucial for the activation or activity of the kinases of both subgroups, different phosphorylation mechanisms were suggested. Here, using one kinase from each subgroup (SnRK2.6 and SnRK2.10), two phosphorylation sites within the activation loop were identified by mass spectrometry after immunoprecipitation from Arabidopsis cells treated by ABA or osmolarity. By site-directed mutagenesis, the phosphorylation of only one of the two sites was shown to be necessary for the catalytic activity of the kinase, whereas both sites are necessary for the full activation of the two SnRK2s by hyperosmolarity or ABA. Phosphoprotein staining together with two-dimensional PAGE followed by immunoblotting indicated distinct phosphorylation mechanisms of the two kinases. While SnRK2.6 seems to be activated through the independent phosphorylation of these two sites, a sequential process occurs in SnRK2.10, where phosphorylation of one serine is required for the phosphorylation of the other. In addition, a subgroup of protein phosphatases 2C which interact and participate in the regulation of SnRK2.6 do not interact with SnRK2.10. Taken together, our data bring evidence for the involvement of distinct phosphorylation mechanisms in the activation of SnRK2.6 and SnRK2.10, which may be conserved between the two subgroups of SnRK2s depending on their ABA-responsiveness.

Protein phosphatases 2C regulate the activation of the Snf1-related kinase OST1 by abscisic acid in Arabidopsis.

The plant hormone abscisic acid (ABA) orchestrates plant adaptive responses to a variety of stresses, including drought. This signaling pathway is regulated by reversible protein phosphorylation, and genetic evidence demonstrated that several related protein phosphatases 2C (PP2Cs) are negative regulators of this pathway in Arabidopsis thaliana. Here, we developed a protein phosphatase profiling strategy to define the substrate preferences of the HAB1 PP2C implicated in ABA signaling and used these data to screen for putative substrates. Interestingly, this analysis designated the activation loop of the ABA activated kinase OST1, related to Snf1 and AMPK kinases, as a putative HAB1 substrate. We experimentally demonstrated that HAB1 dephosphorylates and deactivates OST1 in vitro. Furthermore, HAB1 and the related PP2Cs ABI1 and ABI2 interact with OST1 in vivo, and mutations in the corresponding genes strongly affect OST1 activation by ABA. Our results provide evidence that PP2Cs are directly implicated in the ABA-dependent activation of OST1 and further suggest that the activation mechanism of AMPK/Snf1-related kinases through the inhibition of regulating PP2Cs is conserved from plants to human.

Phosphorylation of the Arabidopsis AtrbohF NADPH oxidase by OST1 protein kinase.

The plant hormone abscisic acid (ABA) triggers production of reactive oxygen species (ROS) in guard cells via the AtrbohD and AtrbohF NADPH oxidases, leading to stomatal closure. The ABA-activated SnRK2 protein kinase open stomata 1 (OST1) (SRK2E/SnRK2.6) acts upstream of ROS in guard cell ABA signaling. Here, we report that OST1 phosphorylates Ser13 and Ser174 on AtrbohF. In addition, substitution of Ser174 to Ala results in a approximately 40% reduction in the phosphorylation of AtrbohF by OST1. We also show that OST1 physically interacts with AtrbohF. These results provide biochemical evidence suggesting that OST1 regulates AtrbohF activity.

Dictyostelium Dock180-related RacGEFs regulate the actin cytoskeleton during cell motility.

Cell motility of amoeboid cells is mediated by localized F-actin polymerization that drives the extension of membrane protrusions to promote forward movements. We show that deletion of either of two members of the Dictyostelium Dock180 family of RacGEFs, DockA and DockD, causes decreased speed of chemotaxing cells. The phenotype is enhanced in the double mutant and expression of DockA or DockD complements the reduced speed of randomly moving DockD null cells' phenotype, suggesting that DockA and DockD are likely to act redundantly and to have similar functions in regulating cell movement. In this regard, we find that overexpressing DockD causes increased cell speed by enhancing F-actin polymerization at the sites of pseudopod extension. DockD localizes to the cell cortex upon chemoattractant stimulation and at the leading edge of migrating cells and this localization is dependent on PI3K activity, suggesting that DockD might be part of the pathway that links PtdIns(3,4,5)P(3) production to F-actin polymerization. Using a proteomic approach, we found that DdELMO1 is associated with DockD and that Rac1A and RacC are possible in vivo DockD substrates. In conclusion, our work provides a further understanding of how cell motility is controlled and provides evidence that the molecular mechanism underlying Dock180-related protein function is evolutionarily conserved.

A versatile strategy to define the phosphorylation preferences of plant protein kinases and screen for putative substrates.

Most signaling networks are regulated by reversible protein phosphorylation. The specificity of this regulation depends in part on the capacity of protein kinases to recognize and efficiently phosphorylate particular sequence motifs in their substrates. Sequenced plant genomes potentially encode over than 1000 protein kinases, representing 4% of the proteins, twice the proportion found in humans. This plethora of plant kinases requires the development of high-throughput strategies to identify their substrates. In this study, we have implemented a semi-degenerate peptide array screen to define the phosphorylation preferences of four kinases from Arabidopsis thaliana that are representative of the plant calcium-dependent protein kinase and Snf1-related kinase superfamily. We converted these quantitative data into position-specific scoring matrices to identify putative substrates of these kinases in silico in protein sequence databases. Our data show that these kinases display related but nevertheless distinct phosphorylation motif preferences, suggesting that they might share common targets but are likely to have specific substrates. Our analysis also reveals that a conserved motif found in the stress-related dehydrin protein family may be targeted by the SnRK2-10 kinase. Our results indicate that semi-degenerate peptide array screening is a versatile strategy that can be used on numerous plant kinases to facilitate identification of their substrates, and therefore represents a valuable tool to decipher phosphorylation-regulated signaling networks in plants.

Constitutive activation of a plasma membrane H(+)-ATPase prevents abscisic acid-mediated stomatal closure.

Light activates proton (H(+))-ATPases in guard cells, to drive hyperpolarization of the plasma membrane to initiate stomatal opening, allowing diffusion of ambient CO(2) to photosynthetic tissues. Light to darkness transition, high CO(2) levels and the stress hormone abscisic acid (ABA) promote stomatal closing. The overall H(+)-ATPase activity is diminished by ABA treatments, but the significance of this phenomenon in relationship to stomatal closure is still debated. We report two dominant mutations in the OPEN STOMATA2 (OST2) locus of Arabidopsis that completely abolish stomatal response to ABA, but importantly, to a much lesser extent the responses to CO(2) and darkness. The OST2 gene encodes the major plasma membrane H(+)-ATPase AHA1, and both mutations cause constitutive activity of this pump, leading to necrotic lesions. H(+)-ATPases have been traditionally assumed to be general endpoints of all signaling pathways affecting membrane polarization and transport. Our results provide evidence that AHA1 is a distinct component of an ABA-directed signaling pathway, and that dynamic downregulation of this pump during drought is an essential step in membrane depolarization to initiate stomatal closure.