Ferric reduction by a CYBDOM protein counteracts increased iron availability in root meristems induced by phosphorus deficiency.

To mobilize sparingly available phosphorus (P) in the rhizosphere, many plant species secrete malate to release P sorbed onto (hydr)oxides of aluminum and iron (Fe). In the presence of Fe, malate can provoke Fe over-accumulation in the root apoplast, triggering a series of events that inhibit root growth. Here, we identified HYPERSENSITIVE TO LOW P1 (HYP1), a CYBDOM protein constituted of a DOMON and a cytochrome b561 domain, as critical to maintain cell elongation and meristem integrity under low P. We demonstrate that HYP1 mediates ascorbate-dependent trans-plasma membrane electron transport and can reduce ferric and cupric substrates in Xenopus laevis oocytes and in planta. HYP1 expression is up-regulated in response to P deficiency in the proximal zone of the root apical meristem. Disruption of HYP1 leads to increased Fe and callose accumulation in the root meristem and causes significant transcriptional changes in roots. We further demonstrate that HYP1 activity overcomes malate-induced Fe accumulation, thereby preventing Fe-dependent root growth arrest in response to low P. Collectively, our results uncover an ascorbate-dependent metalloreductase that is critical to protect root meristems of P-deficient plants from increased Fe availability and provide insights into the physiological function of the yet poorly characterized but ubiquitous CYBDOM proteins.

Sequential removal of cation/H+ exchangers reveals their additive role in elemental distribution, calcium depletion and anoxia tolerance.

Multiple Arabidopsis H+ /Cation exchangers (CAXs) participate in high-capacity transport into the vacuole. Previous studies have analysed single and double mutants that marginally reduced transport; however, assessing phenotypes caused by transport loss has proven enigmatic. Here, we generated quadruple mutants (cax1-4: qKO) that exhibited growth inhibition, an 85% reduction in tonoplast-localised H+ /Ca transport, and enhanced tolerance to anoxic conditions compared to CAX1 mutants. Leveraging inductively coupled plasma mass spectrometry (ICP-MS) and synchrotron X-ray fluorescence (SXRF), we demonstrate CAX transporters work together to regulate leaf elemental content: ICP-MS analysis showed that the elemental concentrations in leaves strongly correlated with the number of CAX mutations; SXRF imaging showed changes in element partitioning not present in single CAX mutants and qKO had a 40% reduction in calcium (Ca) abundance. Reduced endogenous Ca may promote anoxia tolerance; wild-type plants grown in Ca-limited conditions were anoxia tolerant. Sequential reduction of CAXs increased mRNA expression and protein abundance changes associated with reactive oxygen species and stress signalling pathways. Multiple CAXs participate in postanoxia recovery as their concerted removal heightened changes in postanoxia Ca signalling. This work showcases the integrated and diverse function of H+ /Cation transporters and demonstrates the ability to improve anoxia tolerance through diminishing endogenous Ca levels.

Vacuolar sugar transporter EARLY RESPONSE TO DEHYDRATION6-LIKE4 affects fructose signaling and plant growth.

Regulation of intracellular sugar homeostasis is maintained by regulation of activities of sugar import and export proteins residing at the tonoplast. We show here that the EARLY RESPONSE TO DEHYDRATION6-LIKE4 (ERDL4) protein, a member of the monosaccharide transporter family, resides in the vacuolar membrane in Arabidopsis (Arabidopsis thaliana). Gene expression and subcellular fractionation studies indicated that ERDL4 participates in fructose allocation across the tonoplast. Overexpression of ERDL4 increased total sugar levels in leaves due to a concomitantly induced stimulation of TONOPLAST SUGAR TRANSPORTER 2 (TST2) expression, coding for the major vacuolar sugar loader. This conclusion is supported by the finding that tst1-2 knockout lines overexpressing ERDL4 lack increased cellular sugar levels. ERDL4 activity contributing to the coordination of cellular sugar homeostasis is also indicated by 2 further observations. First, ERDL4 and TST genes exhibit an opposite regulation during a diurnal rhythm, and second, the ERDL4 gene is markedly expressed during cold acclimation, representing a situation in which TST activity needs to be upregulated. Moreover, ERDL4-overexpressing plants show larger rosettes and roots, a delayed flowering time, and increased total seed yield. Consistently, erdl4 knockout plants show impaired cold acclimation and freezing tolerance along with reduced plant biomass. In summary, we show that modification of cytosolic fructose levels influences plant organ development and stress tolerance.

Tonoplast cytochrome b561 is a transmembrane ascorbate-dependent monodehydroascorbate reductase: functional characterization of electron currents in plant vacuoles.

Ascorbate (Asc) is a major redox buffer of plant cells, whose antioxidant activity depends on the ratio with its one-electron oxidation product monodehydroascorbate (MDHA). The cytoplasm contains millimolar concentrations of Asc and soluble enzymes that can regenerate Asc from MDHA or fully oxidized dehydroascorbate. Also, vacuoles contain Asc, but no soluble Asc-regenerating enzymes. Here, we show that vacuoles isolated from Arabidopsis mesophyll cells contain a tonoplast electron transport system that works as a reversible, Asc-dependent transmembrane MDHA oxidoreductase. Electron currents were measured by patch-clamp on isolated vacuoles and found to depend on the availability of Asc (electron donor) and ferricyanide or MDHA (electron acceptors) on opposite sides of the tonoplast. Electron currents were catalyzed by cytochrome b561 isoform A (CYB561A), a tonoplast redox protein with cytoplasmic and luminal Asc binding sites. The Km for Asc of the luminal (4.5 mM) and cytoplasmic site (51 mM) reflected the physiological Asc concentrations in these compartments. The maximal current amplitude was similar in both directions. Mutant plants with impaired CYB561A expression showed no detectable trans-tonoplast electron currents and strong accumulation of leaf anthocyanins under excessive illumination, suggesting a redox-modulation exerted by CYB561A on the typical anthocyanin response to high-light stress.

Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters.

A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.

Phospholipid scrambling by a TMEM16 homolog of Arabidopsis thaliana.

Membrane asymmetry is important for cellular physiology and established by energy-dependent unidirectional lipid translocases, which have diverse physiological functions in plants. By contrast, the role of phospholipid scrambling (PLS), the passive bidirectional lipid transfer leading to the break-down of membrane asymmetry, is currently still unexplored. The Arabidopsis thaliana genome contains a single gene (At1g73020) with homology to the eukaryotic TMEM16 family of Ca2+ -activated phospholipid scramblases. Here, we investigated the protein function of this Arabidopsis homolog. Fluorescent AtTMEM16 fusions localized to the ER both in transiently expressing Arabidopsis protoplasts and HEK293 cells. A putative scrambling domain (SCRD) was identified on the basis of sequence conservation and conferred PLS to transfected HEK293 cells, when grafted into the backbone of the non-scrambling plasma membrane-localized TMEM16A chloride channel. Finally, AtTMEM16 'gain-of-function' variants gave rise to cellular phenotypes typical of aberrant scramblase activity, which were reversed by the additional introduction of a 'loss-of-function' mutation into the SCRD. In conclusion, our data suggest AtTMEM16 works as an ER-resident lipid scramblase in Arabidopsis.

Beyond the patch-clamp resolution: functional activity of nonelectrogenic vacuolar NHX proton/potassium antiporters and inhibition by phosphoinositides.

We combined the patch-clamp technique with ratiometric fluorescence imaging using the proton-responsive dye BCECF as a luminal probe. Upon application of a steep cytosol-directed potassium ion (K+ ) gradient in Arabidopsis mesophyll vacuoles, a strong and reversible acidification of the vacuolar lumen was detected, whereas no associated electrical currents were observed, in agreement with electroneutral cation/H+ exchange. Our data show that this acidification was generated by NHX antiport activity, because: it did not distinguish between K+ and sodium (Na+ ) ions; it was sensitive to the NHX inhibitor benzamil; and it was completely absent in vacuoles from nhx1 nhx2 double knockout plants. Our data further show that NHX activity could be reversed, was voltage-independent and specifically impaired by the low-abundance signaling lipid PI(3,5)P2 , which may regulate salt accumulation in plants by acting as a common messenger to coordinately shut down secondary active carriers responsible for cation and anion uptake inside the vacuole. Finally, we developed a theory based on thermodynamics, which supports the data obtained by our novel experimental approach. This work, therefore, represents a proof-of-principle that can be applied to the study of proton-dependent exchangers from plants and animals, which are barely detectable using conventional techniques.

TMEM16E/ANO5 mutations related to bone dysplasia or muscular dystrophy cause opposite effects on lipid scrambling.

Mutations in the human TMEM16E/ANO5 gene are causative for gnathodiaphyseal dysplasia (GDD), a rare bone malformation and fragility disorder, and for two types of muscular dystrophy (MD). Previous studies have demonstrated that TMEM16E/ANO5 is a Ca2+ -activated phospholipid scramblase and that the mutation c.1538C>T (p.Thr513Ile) causing GDD leads to a gain-of-function phenotype. Here, using established HEK293-based functional assays, we investigated the effects of MD-related and further GDD-related amino acid exchanges on TMEM16E/ANO5 function in the same expression system. These experiments also revealed that the gradual changes in HEK293 cell morphology observed upon expression of TMEM16E/ANO5GDD mutants are a consequence of aberrant protein activity. Our results collectively demonstrate that, on the level of protein function, MD mutations are associated to loss-of-function and GDD mutations to gain-of-function phenotypes, confirming conjectures made on the basis of inheritance modes.

Neurite-Enriched MicroRNA-218 Stimulates Translation of the GluA2 Subunit and Increases Excitatory Synaptic Strength.

Local control of protein translation is a fundamental process for the regulation of synaptic plasticity. It has been demonstrated that the local protein synthesis occurring in axons and dendrites can be shaped by numerous mechanisms, including miRNA-mediated regulation. However, several aspects underlying this regulatory process have not been elucidated yet. Here, we analyze the differential miRNA profile in cell bodies and neurites of primary hippocampal neurons and find an enrichment of the precursor and mature forms of miR-218 in the neuritic projections. We show that miR-218 abundance is regulated during hippocampal development and by chronic silencing or activation of neuronal network. Overexpression and knockdown of miR-218 demonstrated that miR-218 targets the mRNA encoding the GluA2 subunit of AMPA receptors and modulates its expression. At the functional level, miR-218 overexpression increases glutamatergic synaptic transmission at both single neuron and network levels. Our data demonstrate that miR-218 may play a key role in the regulation of AMPA-mediated excitatory transmission and in the homeostatic regulation of synaptic plasticity.

The flip side of the Arabidopsis type I proton-pumping pyrophosphatase (AVP1): Using a transmembrane H+ gradient to synthesize pyrophosphate.

Energy partitioning and plant growth are mediated in part by a type I H+-pumping pyrophosphatase (H+-PPase). A canonical role for this transporter has been demonstrated at the tonoplast where it serves a job-sharing role with V-ATPase in vacuolar acidification. Here, we investigated whether the plant H+-PPase from Arabidopsis also functions in "reverse mode" to synthesize PPi using the transmembrane H+ gradient. Using patch-clamp recordings on Arabidopsis vacuoles, we observed inward currents upon Pi application on the cytosolic side. These currents were strongly reduced in vacuoles from two independent H+-PPase mutant lines (vhp1-1 and fugu5-1) lacking the classical PPi-induced outward currents related to H+ pumping, whereas they were significantly larger in vacuoles with engineered heightened expression of the H+-PPase. Current amplitudes related to reverse-mode H+ transport depended on the membrane potential, cytosolic Pi concentration, and magnitude of the pH gradient across the tonoplast. Of note, experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing the Arabidopsis H+-PPase (AVP1) demonstrated Pi-dependent PPi synthase activity in the presence of a pH gradient. Our work establishes that a plant H+-PPase can operate as a PPi synthase beyond its canonical role in vacuolar acidification and cytosolic PPi scavenging. We propose that the PPi synthase activity of H+-PPase contributes to a cascade of events that energize plant growth.

Understanding the Molecular Basis of Salt Sequestration in Epidermal Bladder Cells of Chenopodium quinoa.

Soil salinity is destroying arable land and is considered to be one of the major threats to global food security in the 21st century. Therefore, the ability of naturally salt-tolerant halophyte plants to sequester large quantities of salt in external structures, such as epidermal bladder cells (EBCs), is of great interest. Using Chenopodium quinoa, a pseudo-cereal halophyte of great economic potential, we have shown previously that, upon removal of salt bladders, quinoa becomes salt sensitive. In this work, we analyzed the molecular mechanism underlying the unique salt dumping capabilities of bladder cells in quinoa. The transporters differentially expressed in the EBC transcriptome and functional electrophysiological testing of key EBC transporters in Xenopus oocytes revealed that loading of Na+ and Cl- into EBCs is mediated by a set of tailored plasma and vacuole membrane-based sodium-selective channel and chloride-permeable transporter.

Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia.

Mutations in the human TMEM16E (ANO5) gene are associated both with the bone disease gnathodiaphyseal dysplasia (GDD; OMIM: 166260) and muscle dystrophies (OMIM: 611307, 613319). However, the physiological function of TMEM16E has remained unclear. We show here that human TMEM16E, when overexpressed in mammalian cell lines, displayed partial plasma membrane localization and gave rise to phospholipid scrambling (PLS) as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials. While the activity of wild-type TMEM16E depended on elevated cytosolic Ca2+ levels, a mutant form carrying the GDD-causing T513I substitution showed PLS and large time-dependent ion currents even at low cytosolic Ca2+ concentrations. Contrarily, mutation of the homologous position in the Ca2+-activated Cl- channel TMEM16B paralog hardly affected its function. In summary, these data provide the first direct demonstration of Ca2+-dependent PLS activity for TMEM16E and suggest a gain-of-function phenotype related to a GDD mutation.

Electron current recordings in living cells.

Living cells exploit the electrical properties of matter for a multitude of fundamental physiological processes, such as accumulation of nutrients, cellular homeostasis, signal transmission. While ion channels and transporters (able to couple ions to various substrates) have been extensively studied, direct measurements of electron currents mediated by specific proteins are just at the beginning. Here, we present the various electrophysiological approaches that have allowed recordings of electron currents and highlight the future potential of such experiments.

The signaling lipid phosphatidylinositol-3,5-bisphosphate targets plant CLC-a anion/H+ exchange activity.

Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) is a low-abundance signaling lipid associated with endo-lysosomal and vacuolar membranes in eukaryotic cells. Recent studies on Arabidopsis indicated a critical role of PI(3,5)P2 in vacuolar acidification and morphology during ABA-induced stomatal closure, but the molecular targets in plant cells remained unknown. By using patch-clamp recordings on Arabidopsis vacuoles, we show here that PI(3,5)P2 does not affect the activity of vacuolar H+-pyrophosphatase or vacuolar H+-ATPase. Instead, PI(3,5)P2 at low nanomolar concentrations inhibited an inwardly rectifying conductance, which appeared upon vacuolar acidification elicited by prolonged H+ pumping activity. We provide evidence that this novel conductance is mediated by chloride channel a (CLC-a), a member of the anion/H+ exchanger family formerly implicated in stomatal movements in Arabidopsis H+-dependent currents were absent in clc-a knock-out vacuoles, and canonical CLC-a-dependent nitrate/H+ antiport was inhibited by low concentrations of PI(3,5)P2 Finally, using the pH indicator probe BCECF, we show that CLC-a inhibition contributes to vacuolar acidification. These data provide a mechanistic explanation for the essential role of PI(3,5)P2 and advance our knowledge about the regulation of vacuolar ion transport.

Stepping Out of the Shade: Control of Neuronal Activity by the Scaffold Protein Kidins220/ARMS.

The correct functioning of the nervous system depends on the exquisitely fine control of neuronal excitability and synaptic plasticity, which relies on an intricate network of protein-protein interactions and signaling that shapes neuronal homeostasis during development and in adulthood. In this complex scenario, Kinase D interacting substrate of 220 kDa/ankyrin repeat-rich membrane spanning (Kidins220/ARMS) acts as a multi-functional scaffold protein with preferential expression in the nervous system. Engaged in a plethora of interactions with membrane receptors, cytosolic signaling components and cytoskeletal proteins, Kidins220/ARMS is implicated in numerous cellular functions including neuronal survival, neurite outgrowth and maturation and neuronal activity, often in the context of neurotrophin (NT) signaling pathways. Recent studies have highlighted a number of cell- and context-specific roles for this protein in the control of synaptic transmission and neuronal excitability, which are at present far from being completely understood. In addition, some evidence has began to emerge, linking alterations of Kidins220 expression to the onset of various neurodegenerative diseases and neuropsychiatric disorders. In this review, we present a concise summary of our fragmentary knowledge of Kidins220/ARMS biological functions, focusing on the mechanism(s) by which it controls various aspects of neuronal activity. We have tried, where possible, to discuss the available evidence in the wider context of NT-mediated regulation, and to outline emerging roles of Kidins220/ARMS in human pathologies.

Direct Recording of Trans-Plasma Membrane Electron Currents Mediated by a Member of the Cytochrome b561 Family of Soybean.

Trans-plasma membrane electron transfer is achieved by b-type cytochromes of different families, and plays a fundamental role in diverse cellular processes involving two interacting redox couples that are physically separated by a phospholipid bilayer, such as iron uptake and redox signaling. Despite their importance, no direct recordings of trans-plasma membrane electron currents have been described in plants. In this work, we provide robust electrophysiological evidence of trans-plasma membrane electron flow mediated by a soybean (Glycine max) cytochrome b561 associated with a dopamine ß-monooxygenase redox domain (CYBDOM), which localizes to the plasma membrane in transgenic Arabidopsis (Arabidopsis thaliana) plants and CYBDOM complementary RNA-injected Xenopus laevis oocytes. In oocytes, two-electrode voltage clamp experiments showed that CYBDOM-mediated currents were activated by extracellular electron acceptors in a concentration- and type-specific manner. Current amplitudes were voltage dependent, strongly potentiated in oocytes preinjected with ascorbate (the canonical electron donor for cytochrome b561), and abolished by mutating a highly conserved His residue (H292L) predicted to coordinate the cytoplasmic heme b group. We believe that this unique approach opens new perspectives in plant transmembrane electron transport and beyond.

Functional Interaction between the Scaffold Protein Kidins220/ARMS and Neuronal Voltage-Gated Na+ Channels.

Kidins220 (kinase D-interacting substrate of 220 kDa)/ankyrin repeat-rich membrane spanning (ARMS) acts as a signaling platform at the plasma membrane and is implicated in a multitude of neuronal functions, including the control of neuronal activity. Here, we used the Kidins220(-/-) mouse model to study the effects of Kidins220 ablation on neuronal excitability. Multielectrode array recordings showed reduced evoked spiking activity in Kidins220(-/-) hippocampal networks, which was compatible with the increased excitability of GABAergic neurons determined by current-clamp recordings. Spike waveform analysis further indicated an increased sodium conductance in this neuronal subpopulation. Kidins220 association with brain voltage-gated sodium channels was shown by co-immunoprecipitation experiments and Na(+) current recordings in transfected HEK293 cells, which revealed dramatic alterations of kinetics and voltage dependence. Finally, an in silico interneuronal model incorporating the Kidins220-induced Na(+) current alterations reproduced the firing phenotype observed in Kidins220(-/-) neurons. These results identify Kidins220 as a novel modulator of Nav channel activity, broadening our understanding of the molecular mechanisms regulating network excitability.

Identification of mutations allowing Natural Resistance Associated Macrophage Proteins (NRAMP) to discriminate against cadmium.

Each essential transition metal plays a specific role in metabolic processes and has to be selectively transported. Living organisms need to discriminate between essential and non-essential metals such as cadmium (Cd(2+) ), which is highly toxic. However, transporters of the natural resistance-associated macrophage protein (NRAMP) family, which are involved in metal uptake and homeostasis, generally display poor selectivity towards divalent metal cations. In the present study we used a unique combination of yeast-based selection, electrophysiology on Xenopus oocytes and plant phenotyping to identify and characterize mutations that allow plant and mammalian NRAMP transporters to discriminate between their metal substrates. We took advantage of the increased Cd(2+) sensitivity of yeast expressing AtNRAMP4 to select mutations that decrease Cd(2+) sensitivity while maintaining the ability of AtNRAMP4 to transport Fe(2+) in a population of randomly mutagenized AtNRAMP4 cDNAs. The selection identified mutations in three residues. Among the selected mutations, several affect Zn(2+) transport, whereas only one, E401K, impairs Mn(2+) transport by AtNRAMP4. Introduction of the mutation F413I, located in a highly conserved domain, into the mammalian DMT1 transporter indicated that the importance of this residue in metal selectivity is conserved among NRAMP transporters from plant and animal kingdoms. Analyses of overexpressing plants showed that AtNRAMP4 affects the accumulation of metals in roots. Interestingly, the mutations selectively modify Cd(2+) and Zn(2+) accumulation without affecting Fe transport mediated by NRAMP4 in planta. This knowledge may be applicable for limiting Cd(2+) transport by other NRAMP transporters from animals or plants.