1

1

Direct recording of trans-plasma membrane electron currents mediated 2

by a member of the cytochrome b561 family of soybean 3

4

5

6

Cristiana Picco1†, Joachim Scholz-Starke1†, Margherita Festa1†, Alex Costa2, Francesca Sparla3, 7

Paolo Trost3* and Armando Carpaneto1* 8

9

1. Institute of Biophysics - CNR, Via De Marini 6, 16149 Genova, Italy 10

2. Department of Biosciences, University of Milan, Via G. Celoria 26, 20133 Milan, Italy, 11

Institute of Biophysics, Consiglio Nazionale delle Ricerche, Via G. Celoria 26, 20133 Milan, 12

Italy 13

3. Department of Pharmacy and Biotechnology (FaBiT) - University of Bologna, Via Irnerio 42, 14

40126 Bologna, Italy 15

16

† These authors contributed equally to this work. 17

18

*Corresponding authors: Paolo Trost and Armando Carpaneto 19

Email: paolo.trost@unibo.it ; phone: +390512091329; fax: +39051242576 ; 20

Email: carpaneto@ge.ibf.cnr.it ; phone: +390106475559; fax: +39010647500 21

22

Running title: Plant cytochrome b561 electron current recordings 23

24

Key words 25

cytochrome b561, Xenopus oocytes, ascorbate, iron 26

27

Plant Physiology Preview. Published on August 17, 2015, as DOI:10.1104/pp.15.00642

Copyright 2015 by the American Society of Plant Biologists

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One sentence summary: 28

Electron currents mediated by a soybean cytochrome b561 protein can be detected and 29

functionally characterized using a classical electrophysiological approach. 30

31

Abstract 32

Trans-plasma-membrane electron transfer is achieved by b-type cytochromes of different families, 33

and plays a fundamental role in diverse cellular processes involving two interacting redox couples 34

that are physically separated by a phospholipid bilayer, such as iron uptake and redox signalling. 35

Despite their importance, no direct recordings of trans-plasma-membrane electron currents have 36

been described in plants. In this work, we provide robust electrophysiological evidence of trans-37

plasma-membrane electron flow mediated by a soybean CYBDOM, a member of the cytochrome 38

b561 family, which localizes to the plasma membrane in transgenic Arabidopsis plants and 39

CYBDOM cRNA-injected Xenopus oocytes. In oocytes, two-electrode voltage-clamp experiments 40

showed that CYBDOM-mediated currents were activated by extracellular electron acceptors in a 41

concentration- and type-specific manner. Current amplitudes were voltage-dependent, strongly 42

potentiated in oocytes pre-injected with ascorbate, the canonical electron donor for cytochromes 43

b561, and abolished by mutating a highly conserved histidine residue (H292L) predicted to 44

coordinate the cytoplasmic heme b group. We believe that this novel approach opens new 45

perspectives in plant trans-membrane electron transport and beyond. 46

47

48

Introduction 49

In biological membranes, the presence of ion channels and transporters enables the selective 50

movement of ions and charged/polar metabolites across phospholipid bilayers. The development 51

of electrophysiological techniques allowed to study the functional properties of these proteins 52

directly. After the first recordings of ionic currents from protoplasts enzymatically isolated from 53

wheat and Vicia faba leaves more than 30 years ago (Moran et al., 1984; Schroeder et al., 1984), 54

there was an incredible progress in the understanding of the biophysical structures and the 55

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physiological roles of a plethora of plant channels and transporters (Hedrich, 2012). However, for 56

the intriguing class of plant trans-membrane electron transporters, no direct current recordings are 57

available. 58

59

Cells and sealed plasma membrane (PM) vesicles isolated from different plant species and organs 60

have long been known to constitutively reduce externally added, hydrosoluble electron acceptors 61

like ferricyanide (FC) or other suitable artificial dyes, often in the absence of any inductive stimulus 62

(Lüthje et al., 2013 and refs therein). Reduction of the external electron acceptors was typically 63

associated with PM depolarization, supporting the view that the electrons required for the redox 64

activity were provided by a cytoplasmic reductant via a trans-membrane electron transport system 65

whose molecular identity remained elusive (Lüthje et al., 2013). By their activity, these electron 66

transport systems provide a connection between cytoplasmic and apoplastic redox couples that 67

are physically separated by the plasma membrane. 68

69

Cytochromes b561 (CYB561) and related proteins containing a CYB561-core domain are 70

membrane-spanning proteins encoded in angiosperms by gene families of about 15 members 71

(Tsubaki et al., 2005; Preger et al., 2009; Asard et al., 2013). The CYB561 core domain is 72

constituted by a bundle of four trans-membrane alpha-helices with two hemes b facing opposite 73

sides of the membrane (Asard et al., 2013). The simplest members of the CYB561 family consist of 74

a single CYB561-core domain plus two additional trans-membrane helices with no obvious 75

catalytic role (Asard et al., 2013). Plants often contain four isoforms of these CYB561 sensu strictu, 76

one of these (CYB561B1, for a systematic nomenclature see Asard et al., 2013) has been 77

localized on the tonoplast (Griesen et al., 2004; Preger et al., 2004) or the plasma membrane, 78

depending on the plant species investigated (Nanasato et al., 2005). A second isoform from 79

Arabidopsis (At_CYB561B2), for which the crystal structure has recently been solved (Lu et al., 80

2014), is apparently chloroplastic (Dutta et al., 2014). The few CYB561 proteins characterized to 81

date catalyze trans-membrane electron transport between cytoplasmic ascorbate (ASC) and extra-82

cytoplasmic electron acceptors that can be either monodehydroascorbate (MDHA), for extra-83

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cytoplasmic ASC regeneration, or a ferrichelate, for ferrous ion mobilization (Asard et al., 2013). In 84

animal systems as well, MDHA and ferrichelates are suitable electron acceptors for different 85

CYB561 isoforms. CYB561 of chromaffin granules in mammals reduces intravesicular MDHA to 86

sustain ASC-dependent catecholamine biosynthesis (Njus and Kelley, 1993) and a similar MDHA 87

reductase activity was demonstrated in several CYB561 plant isoforms (Horemans et al., 1994; 88

Nakanishi et al., 2009; Asard et al., 2013). On the other hand, the activity of plant CYB561 with 89

ferrichelates (Bérczi et al., 2007) recalls the activity of a duodenal CYB561 isoform (Dcytb) of 90

mammals that performs the ascorbate-dependent reduction of ferric chelates derived from the diet, 91

allowing the absorption of ferrous iron by divalent metal transporters (McKie et al., 2001). In 92

several instances, both in plants and animals, single CYB561 isoforms proved capable of reducing 93

both MDHA and ferrichelates, leaving some uncertainty about their specific role in vivo (Asard et 94

al., 2013). 95

96

Besides CYB561s sensu strictu, the CYB561 protein family includes CYB561-related proteins in 97

which an additional domain, known as DOMON (Iyer et al., 2007) and sometimes present in two 98

copies, extends towards the extracytoplasmic space. These proteins have been named CYBDOMs 99

(Asard et al., 2013). The hydrophilic DOMON domain binds an additional heme b group (Preger et 100

al., 2009), such that all CYBDOMs contain two hemes in the CYB561 core plus one heme for each 101

extracellular DOMON (Asard et al., 2013). Very little is known on the physiological role of 102

CYBDOMs, for which angiosperms often contain around 10 encoding genes. 103

104

Here we show the electron transport capability of a soybean CYBDOM localized at the plasma 105

membrane (systematic name Gm_CYBDOMF21 (Asard et al., 2013)), the first plant CYBDOM ever 106

described, and demonstrate the feasibility of electrophysiological techniques to measure electron 107

currents generated by trans-membrane electron transport systems expressed in Xenopus oocytes. 108

The electron currents mediated by soybean CYBDOM proved to be stimulated by the membrane 109

potential and to depend on both internal ASC concentration and external ferricyanide 110

concentration. Albeit less efficiently, extracellular ferricyanide could be substituted by a ferrichelate 111

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(FeNTA). This work paves the way for further experiments, aimed at elucidating the molecular 112

mechanism underlying these elusive electron transport systems present in particular large number 113

in plants. 114

115

116

Materials and Methods 117

DNA constructs and plant expression 118

The cDNA of Glycine max CYBDOM (Glyma09g01390.1) was subcloned into the commercial 119

pMOS vector (GE Healthcare) by PCR using the following pair of primers both containing BamHI 120

restriction sites (underlined): 5’-GTAACTGGATCCACAGGAAGATGG-3’ and 5’-121

AGCCAAGGATCCAGTATCTATGAG-3’. The cDNA sequence was PCR-amplified and cloned into 122

pGEMHE (Liman et al., 1992) for oocyte expression and into pSAT6-EGFP-N1 (Tzfira et al., 2005) 123

for transient Arabidopsis protoplast transformation. The CYBDOM::EGFP fragment was subcloned 124

into the pTLN plasmid (Lorenz et al., 1996) for oocyte expression of the fusion protein. The single 125

point mutation H249L was generated in the pTLN-CYBDOM-EGFP plasmid using specific primers 126

and the QuickChange site-directed mutagenesis kit (Agilent Technologies Italia, Cernusco sul 127

Naviglio, Italy). Mutagenesis was confirmed by subsequent DNA sequencing. 128

For the generation of Arabidopsis thaliana plants (Col-0 ecotype) stably expressing the CYBDOM-129

EGFP fusion, the CYBDOM coding sequence was cloned upstream the EGFP coding sequence in 130

a modified pBI121 vector (Bregante et al., 2008) under the control of a double CaMV35S promoter. 131

The EGFP coding sequence was inserted into the BamHI and SacI sites of the modified pBI121 132

vector. Subsequently, the CYBDOM sequence, excised from the pGEMHE-CYBDOM plasmid 133

using BamHI, was transferred into the BamHI-linearized pBI121-EGFP plasmid. The obtained 134

construct was inserted into Agrobacterium tumefaciens strain GV3101 that was used to generate 135

transgenic Arabidopsis plants using the floral-dip method (Clough and Bent, 1998). Ten 136

independent antibiotic-resistant transgenic Arabidopsis lines were selected on MS1/2 (MS, M0222 137

elements including Vitamins, Duchefa, http://www.duchefa-biochemie.nl/) agar (0.8% w/v) medium 138

supplemented with 50 μg/mL of kanamycin. Five independent lines were analyzed for CYBDOM-139

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EGFP localization. 140

Isolation of Arabidopsis mesophyll protoplasts and transient expression assays were performed as 141

described previously (Costa et al., 2012). 142

143

Oocyte expression 144

In vitro transcription of NheI-linearized pGEMHE-CYBDOM and MluI-linearized pTLN-CYBDOM-145

EGFP DNA templates was done using mMESSAGE mMACHINE T7 or SP6 kits (Life 146

Technologies, Monza, Italy), respectively, and the integrity of the transcribed cRNA was confirmed 147

by agarose gel analysis. 148

Oocytes were surgically removed from adult female Xenopus laevis frogs, treated with collagenase 149

(1 mg/ml) for 30 min at RT under constant shaking and washed twice with standard solution (in 150

mM): 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2 and 5 HEPES, pH 7.5). Isolated oocytes were injected with 151

transcribed cRNA at 40 ng per oocyte or with an equivalent volume of nuclease-free water, using a 152

Drummond "Nanoject” microinjector. Oocytes were then incubated at 18°C in standard solution 153

supplemented with 0.1 mg/ml gentamycin. 154

155

Confocal fluorescence microscopy 156

Confocal microscopy analyses of leaves of Arabidopsis transgenic plants expressing the 157

CYBDOM-EGFP chimeric proteins were performed using a Leica SP2 (Leica, Germany, 158

http://www.leica-microsystems.com) laser scanning confocal imaging system. For EGFP detection, 159

excitation was set at 488 nm and detection between 515 and 530 nm. For chlorophyll and FM4-64 160

detection, excitation was set at 488 nm and detection ranges were 650-750 nm and 575-625 nm, 161

respectively. For FM4-64 staining, leaf discs were dipped for 5 minutes in a water-based solution in 162

which FM4-64 was diluted to a final concentration of 17 μM (Bolte et al., 2004). After staining the 163

leaves were immediately imaged. The lambda spectrum records were carried out following the 164

application notes of the Leica SP2 microscope. Image post-acquisition analyses were performed 165

with the ImageJ bundle software. 166

Confocal microscopy analyses of Xenopus oocytes expressing CYBDOM-EGFP chimeric proteins 167

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were performed using a Leica SP2 equipped with a 40X oil-immersion objective. Both EGFP and 168

FM4-64 were excited at 488 nm and emissions were monitored at 505-512 nm and 645-655 nm, 169

respectively. Confocal images were obtained by scanning the laser scan line axially (x-z scan) 170

along a 12–μm section (0.5 μm steps) within the oocyte (see for details (Naso et al., 2009)). All 171

measurements were averaged from more than six independent scans in several oocytes (N ≥ 8) 172

harvested from more than two donor frogs. 173

174

Colorimetric ferric reductase activity assay 175

Ferric reductase activity assays (McKie et al., 2001; Vargas et al., 2003) were conducted on 176

individual Xenopus oocytes placed in 96-well plates. Oocytes were incubated for 15 hours in the 177

dark in 50 μl incubation buffer (standard solution containing 100 μM FeNTA and 1 mM ferrozine). 178

Formation of ferrous iron was calculated from the optical density of the incubation buffer at 562 nm. 179

Optical density was measured using a FLUOstar Omega micro-plate reader. Experiments were 180

performed three times using at least 15 oocytes harvested from different frogs. 181

182

Two-electrode voltage-clamp recordings 183

Whole-cell membrane currents were measured at room temperature using a home-made two-184

electrode voltage-clamp amplifier. Briefly, oocytes are impaled with two glass microelectrodes, one 185

to measure the membrane potential (termed voltage electrode), the second (termed the current 186

electrode) to pass sufficient current in order to maintain the membrane voltage at the desired value 187

(voltage clamp), using a feedback circuit. The amount of current is determined from the difference 188

between the actual membrane potential and the desired value (command voltage). Microelectrodes 189

filled with 3 M KCl solution had a pipette resistance of 0.2-0.4 MOhm, corresponding to a tip 190

diameter of a few micrometers. Membrane currents elicited by exposure to extracellular electron 191

acceptors were generally recorded at a holding potential of -20 mV. Current-voltage relationships 192

were determined from steady-state membrane currents recorded in response to 25-ms voltage 193

pulses ranging from +40 mV to -100 mV in steps of 10 mV, from a holding potential of -20 mV. By 194

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convention, negative currents are inward currents, due to either the influx of cations or the efflux of 195

anions. 196

The ionic bath solution was the standard solution adjusted to pH 5.5 substituting 10 mM Hepes 197

with equimolar concentration of MES.. The BTP/MES solution contained 200 mM 2-(N-198

morpholino)ethanesulfonic acid (MES), 20 mM bis-tris-propane (BTP), pH 5.5. A gravity-driven 199

perfusion system was used for continuous superfusion of oocytes during voltage-clamp recordings 200

and for switching between different bath solutions. Ferric compounds were prepared as stock 201

solutions and diluted appropriately in bath solution, potassium ferricyanide (FeCN) at 100 mM, 202

ferric nitrilotriacetate (FeNTA), ferric citrate (FeCIT) and ferric EDTA (FeEDTA) at 30 mM. 203

Ferrocyanide was prepared as stock solution at 100 mM and diluted at 100 μM in the standard 204

solution at pH 5.6; 20 μM DTT was added in the final solution to avoid production of ferricyanide 205

from ferrocyanide. 206

For ascorbate injection experiments, ascorbate stock solution (100 mM) was made freshly and 207

adjusted to pH 7.0 with KOH. After the baseline recording series, the oocyte was removed from the 208

voltage-clamp setup and injected with 50 nl of ascorbate stock solution using the microinjector, 209

resulting in a final concentration of injected ascorbate of 10 mM (considering a volume of 210

approximately 500 nl for a mean oocyte diameter of 1 mm). Upon injection, the oocyte volume 211

transiently increased, but recovered to its original size within minutes. Injected oocytes were 212

allowed to recover for 30 minutes at RT before voltage-clamp measurements were resumed. 213

214

215

Results 216

217

Plasma membrane localization of CYBDOM in leaves of transgenic Arabidopsis plants 218

219

Localization of soybean CYBDOM was studied in Arabidopsis following different strategies. First, 220

Arabidopsis thaliana plants were stably transformed with a CYBDOM::EGFP construct under the 221

control of a double 35S promoter. The EGFP fluorescence signal detected in leaves of CYBDOM-222

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EGFP transgenic plants nicely matched the boundaries of leaf epidermal cells (Fig. 1, A, C and D) 223

and stomatal guard cells (Fig. 1, E, G and H). Spectral analysis confirmed that the observed 224

fluorescence was due to EGFP and not to cell wall autofluorescence (data not shown). Moreover, 225

the spatial distribution of the EGFP signal matched with the plasma membrane marker FM4-64 226

(Fig. 1, I-L). 227

In an alternative experimental strategy, a CYBDOM-EGFP fusion construct was used for transient 228

transformation of isolated Arabidopsis mesophyll protoplasts (Supplementary Fig. 1A). Again, the 229

CYBDOM-EGFP signal perfectly matched the FM4-64 signal at the plasma membrane. The EGFP 230

fusion of the tonoplast protein AtTPC1 (Boccaccio et al., 2014), used as a negative control, did not 231

match the FM4-64 distribution (Supplementary Fig. 1B), in spite of the close proximity of tonoplast 232

and plasma membrane in these cells. 233

234

Plasma membrane localization and ferric reductase activity of CYBDOM in Xenopus oocytes 235

236

For functional studies, we used Xenopus laevis oocytes microinjected with CYBDOM or CYBDOM-237

EGFP cRNA. Confocal imaging showed strong EGFP signals at the surface of oocytes expressing 238

a CYBDOM-EGFP fusion protein (Fig. 2A). Colocalization studies with the plasma membrane 239

marker FM4-64 confirmed that the fusion protein was targeted to the plasma membrane also in this 240

heterologous expression system (Fig. 2B-C). A colorimetric assay was used to probe the capability 241

of CYBDOM to reduce an external electron acceptor like ferric nitrilotriacetate (NTA) in the 242

presence of ferrozine as a Fe2+ chelator. In CYBDOM cRNA-injected oocytes, a 27-fold increase of 243

the reductase activity was observed compared to control oocytes (Fig. 2D). 244

245

Ascorbate-dependent electron transfer mediated by CYBDOM in Xenopus oocytes 246

247

We used two-electrode voltage-clamp recordings for direct measurements of CYBDOM-mediated 248

trans-membrane electron transport in Xenopus oocytes. Exposure of CYBDOM cRNA-injected 249

oocytes to the electron acceptor ferricyanide (FeCN; 100 μM) reversibly elicited small inward 250

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currents at -20 mV (Fig. 2E, lower trace), while none of the control oocytes showed downward 251

deflections of the current level (Fig. 2E, upper trace). Similar experiments were performed at 252

different voltages between +40 and -100 mV. FeCN-dependent currents were completely absent in 253

control oocytes, while CYBDOM cRNA-injected oocytes showed an increase of the current 254

amplitude with negative-going potentials (Fig. 2F). 255

256

In order to investigate the responsiveness of soybean CYBDOM to cytosolic ascorbate, the 257

canonical electron donor of the CYB561 family, FeCN-elicited currents were measured twice on 258

the same oocyte (Fig. 3A): first, in baseline conditions using the endogenous cytosolic ascorbate 259

pool (black traces) and successively, after an ascorbate injection raising the intracellular ascorbate 260

concentration by 10 mM (red traces). The increase of cytosolic ascorbate strongly affected 261

CYBDOM-dependent inward currents, in particular at elevated FeCN concentrations. Plots of the 262

current amplitudes against the applied FeCN concentration revealed Michaelis-Menten-like 263

behaviour in both experimental conditions (Fig. 3B). Notably, raising the intracellular ascorbate 264

concentration caused a 7-fold increase of the apparent maximum current at saturating [FeCN] (Fig. 265

3C). In parallel, the apparent affinity constant for FeCN changed from 4.3 μM in baseline 266

conditions to 26.0 μM after ascorbate injection (Fig. 3D). Taking advantage of the higher current 267

amplitudes recorded after raising the intracellular ascorbate concentration, experiments described 268

hereafter were conducted on oocytes pre-injected with 10 mM ascorbate. 269

270

First, we investigated the current responses of CYBDOM-expressing oocytes challenged with 271

alternative electron acceptors. Applied at a concentration of 500 μM, ferric nitrilotriacetate (FeNTA) 272

evoked an approximately 8-fold smaller response than FeCN, while exposure to ferric citrate 273

(FeCIT) and ferric EDTA (FeEDTA) had no significant effect on the current level (Fig. 4A, B). Since 274

reduced electron acceptors produced during the reaction were rapidly washed away by the 275

perfusion system, differences in electron currents could not be ascribed to differences in the redox 276

potential of the iron compounds. Moreover, none of the electron acceptors elicited measurable 277

current deflections in control oocytes (Fig. 4A). As negative control, we also tested the effect of 278

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ferrocyanide in oocytes pre-injected with ascorbate. As shown in Fig. 4C, 100 mM ferrocyanide did 279

not elicit measurable currents. 280

The characteristics of inward currents in CYBDOM-expressing oocytes, i.e. strong potentiation by 281

intracellular injection of the electron donor ascorbate, strict dependence on an extracellular 282

electron acceptor like FeCN and no detectable signal induced by ferrocyanide were fully 283

compatible with trans-membrane electron transport mediated by CYBDOM. Further experiments 284

using bath solutions with altered ionic composition were conducted to exclude the possible 285

contribution of alternative ion fluxes. FeCN-induced current amplitudes recorded in a modified bath 286

solution - containing only the large, membrane-impermeable ions BTP and MES - were identical to 287

those recorded in standard bath solution (Fig. 4D). Raising the extracellular pH from 5.5 to 7.5 288

determined smaller FeCN-evoked currents over a range of [FeCN] (Fig. 4E). A closer inspection 289

using dose-response analyses (Fig. 4F) revealed that the apparent maximum current at saturating 290

[FeCN] was about 36% higher at pHext 5.5 compared to pHext 7.5, while the apparent affinity 291

constant for FeCN was not significantly affected (Fig. 4G). The observed effects likely reflect a 292

pHext-dependent modulation of CYBDOM activity. 293

294

The conserved histidine residue H249 is necessary for CYBDOM-mediated electron transfer 295

296

The two hemes b groups of CYB561 core domains are coordinated by four conserved histidines 297

located at the N-terminal end of four consecutive alpha-helices (Asard et al., 2013), as shown in 298

Fig. 5A. Based on multiple protein sequence alignments (Fig. 5B), H249 of CYBDOM was 299

identified as a putative ligand of the cytoplasmic heme of the protein. The orthologous histidine of 300

other CYB561 isoforms was demonstrated to be essential for proper heme ligation, though 301

dispensable for correct protein folding (Bérczi and Zimányi, 2014 and refs therein). We used site-302

directed mutagenesis to target H249 in CYBDOM. Confocal imaging of oocytes expressing a 303

CYBDOMH249L-EGFP fusion, together with FM4-64 staining, confirmed that the H249L mutation did 304

neither affect the plasma membrane targeting (Fig. 5C) nor the surface expression level of the 305

protein (Fig. 5D). Strikingly however, the mutation completely abolished FeCN-evoked inward 306

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currents in Xenopus oocytes, both in baseline conditions using the endogenous cytosolic 307

ascorbate pool and after an ascorbate injection raising the intracellular ascorbate concentration 308

(Fig. 5E, F), strongly suggesting that the cytoplasmic heme group coordinated by the H249 residue 309

is necessary for trans-membrane electron transport mediated by CYBDOM. 310

311

312

Discussion 313

In the present study, we provide evidence that the soybean family of CYB561 proteins contains at 314

least one plasma-membrane-associated isoform, named CYBDOM. Heterologous expression in 315

Xenopus laevis oocytes confirmed the PM localization of CYBDOM and disclosed its trans-316

membrane electron transport activity. 317

318

CYBDOM cRNA-injected oocytes acquired the capability to reduce an external electron acceptor, 319

as determined by a simple colorimetric assay. Under two-electrode voltage-clamp, extracellular 320

application of the electron acceptor elicited inward currents, consistent with trans-membrane 321

electron transport. In support of this hypothesis, we provide multiple lines of evidence: first, 322

activation of inward currents was dependent on extracellular application of the electron acceptor 323

FeCN in a concentration-dependent manner and was fully reversible upon washout; second, 324

inward current amplitudes increased strongly in CYBDOM-expressing oocytes pre-injected with 325

ascorbate, the canonical electron donor for CYB561 proteins; third, inward current amplitudes were 326

similar in bath solutions containing only membrane-impermeable ions; fourth, inward currents, but 327

not CYBDOM expression on the PM, were totally abolished in the mutant H249L, predicted to lack 328

the cytoplasmic heme (Bérczi and Zimányi, 2014). This latter result also represents the first 329

experimental demonstration that electron transport in CYBDOM proteins depends on the hemes of 330

their CYB561-core domain. We therefore conclude that inward currents reflected the CYBDOM-331

mediated movement of electrons from the intracellular electron donor ascorbate to the extracellular 332

electron acceptor FeCN or, less efficiently, the ferrichelate FeNTA. Moreover, electron transport 333

was stimulated by the membrane potential and by acidic external pH. 334

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335

This work presents the first direct recordings of the activity of a plant trans-plasma membrane 336

redox protein (CYBDOM). Prior to soybean CYBDOM, there were only two examples of trans-337

membrane electron transport studied by electrophysiological techniques. The first was the 338

characterization of NADPH oxidase in human phagocytes (Schrenzel et al., 1998; DeCoursey et 339

al., 2003; Petheo and Demaurex, 2005; Ahluwalia, 2008), which took advantage of the high activity 340

of this electron transport system in specialized blood cells like eosinophils and neutrophils. Current 341

amplitudes in the picoampere range were recorded in human eosinophils applying the patch-clamp 342

technique in the inside-out patch configuration and were unambiguously associated to electrons 343

moving from cytoplasmic NADPH to extracellular oxygen (Petheo and Demaurex, 2005). The 344

activity of a Drosophila CYBDOM named SDR2 represents the second example of electron current 345

recordings in Xenopus oocytes (Picco et al., 2014). Comparing the functional properties of the two 346

transporters we found that soybean CYBDOM, while being expressed at similar levels in oocytes 347

(Supplementary Fig. 2A), presented significantly lower current amplitudes (Supplementary Fig. 2B) 348

and higher FeCN affinity (Supplementary Fig. 2C) than Drosophila SDR2. Interestingly, the lower 349

current amplitude is consistent with the negative shift of CYBDOM voltage-dependence 350

(Supplementary Fig. 2D), which, in turn, nicely correlates with the more negative resting voltage in 351

plant cells, -110/-150 mV (Hedrich, 2012), compared to animal cells, -40/-100 mV (Hille, 2001). 352

353

All features of soybean CYBDOM are consistent with this protein being a component of a 354

constitutive redox system of plant cells and may be related to different physiological functions that 355

need to be tested in planta. The existence of constitutive redox systems in plant plasma 356

membranes has been proposed more than 30 years ago (Craig and Crane, 1981), but their 357

molecular nature remained obscure. Trans-plasma membrane redox activity in plants was typically 358

revealed by the reduction of external ferricyanide and found associated to membrane potential 359

depolarization and acidification of the external medium, the latter resulting from the activation of 360

the plasma membrane H+-ATPase (Sijmons et al., 1984; Marrè et al., 1988; Lüthje et al., 2013). In 361

some cases, constitutive reduction of external ferricyanide was based on NAD(P)H as internal 362

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14

reductant (Menckhoff and Lüthje, 2004), in others ascorbate was shown to be a suitable electron 363

donor, and cytochromes b to be involved the trans-membrane electron transfer (Asard et al., 364

1992). Interestingly, CYBDOM is an ASC-dependent cytochrome b localized on the plasma 365

membrane, its activity is electrogenic and may thus depolarize the membrane (see Supplementary 366

Fig. 3 for membrane depolarization in Xenopus oocytes). In the context of a plant cell, CYBDOM-367

dependent membrane depolarization would stimulate the activity of the H+-ATPase, while pump 368

activation hyperpolarizes the plasma membrane and acidifies the apoplast, both conditions that 369

would in turn activate the depolarizing activity of CYBDOM. Such reciprocal interplay between H+ 370

pump and plasma membrane redox systems has long been hypothesized, based on the 371

observations that reduction of external ferricyanide by plant cells activated the H+ pump and that H+ 372

pump activation by fusicoccin stimulated ferricyanide reduction (Marrè et al., 1988 and references 373

therein). 374

375

Besides regulating the membrane potential, a cooperation between proton pump and PM redox 376

systems would in principle result in energy dissipation. This possibility was investigated in wild 377

watermelon (Citrullus lanatus) where a plasma membrane isoform of CYB561 was proposed to 378

form, together with apoplastic ASC and ASC oxidase, a novel pathway for excess light energy 379

dissipation (Nanasato et al., 2005). On the other hand, based on its reducing activity toward 380

ferrichelates, CYBDOM may also play a role in iron homeostasis, reminiscent of DcytB in animal 381

systems (McKie et al., 2001). In any case, whichever the physiological electron acceptor(s), 382

CYBDOM is clearly a strong candidate as a component of the constitutive redox system of plant 383

plasma membranes. 384

385

The technical approach proposed in our work can be applied to the functional characterization of 386

further members of the large family of CYB561 proteins and generally to any other trans-387

membrane electron transporter, opening new perspectives in this fundamental field of plant life. 388

389

390

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15

Acknowledgements 391

We thank Anna Moroni and Alessandro Vitale (University of Milan, Italy) and Gerhard Thiel 392

(University of Darmstadt, Germany) for critical comments on the manuscript. The technical 393

assistance of Paolo Guastavino, Francesca Quartino, Damiano Magliozzi e Alessandro Barbin 394

(IBF-CNR, Italy) was highly appreciated. A. Carpaneto was supported by the Italian “Progetti di 395

Ricerca di Interesse Nazionale” (PRIN2010CSJX4F) and by Compagnia di San Paolo Research 396

Foundation (ROL 291). F. Sparla and P. Trost were supported by Ministero dell’Istruzione, 397

dell’Università e della Ricerca (PRIN2009). This work was also supported by Ministero 398

dell’Istruzione, dell’Università e della Ricerca (FIRB) 2010 (RBFR10S1LJ_001) grant to A. Costa. 399

400

401

Bibliography 402 403 Ahluwalia J (2008) Characterisation of electron currents generated by the human neutrophil 404 NADPH oxidase. Biochem Biophys Res Commun 368: 656–661 405 Asard H, Barbaro R, Trost P, Bérczi A (2013) Cytochromes b561: ascorbate-mediated trans-406 membrane electron transport. Antioxid Redox Signal 19: 1026–1035 407 Asard H, Horemans N, Caubergs RJ (1992) Transmembrane electron transport in ascorbate-408 loaded plasma membrane vesicles from higher plants involves a b-type cytochrome. FEBS Lett 409 306: 143–146 410 Bérczi A, Su D, Asard H (2007) An Arabidopsis cytochrome b561 with trans-membrane 411 ferrireductase capability. FEBS Lett 581: 1505–1508 412 Bérczi A, Zimányi L (2014) The trans-membrane cytochrome b561 proteins: structural information 413 and biological function. Curr Protein Pept Sci 15: 745–760 414 Boccaccio A, Scholz-Starke J, Hamamoto S, Larisch N, Festa M, Gutla PVK, Costa A, 415 Dietrich P, Uozumi N, Carpaneto A (2014) The phosphoinositide PI(3,5)P2 mediates activation of 416 mammalian but not plant TPC proteins: functional expression of endolysosomal channels in yeast 417 and plant cells. Cell Mol Life Sci 71: 4275–4283 418 Bolte S, Talbot C, Boutte Y, Catrice O, Read ND, Satiat-Jeunemaitre B (2004) FM-dyes as 419 experimental probes for dissecting vesicle trafficking in living plant cells. J Microsc 214: 159–173 420 Bregante M, Yang Y, Formentin E, Carpaneto A, Schroeder JI, Gambale F, Schiavo F Lo, 421 Costa A (2008) KDC1, a carrot Shaker-like potassium channel, reveals its role as a silent 422 regulatory subunit when expressed in plant cells. Plant Mol Biol 66: 61–72 423 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated 424 transformation of Arabidopsis thaliana. Plant J 16: 735–743 425 Costa A, Gutla PVK, Boccaccio A, Scholz-Starke J, Festa M, Basso B, Zanardi I, Pusch M, 426 Schiavo FL, Gambale F, et al (2012) The Arabidopsis central vacuole as an expression system 427 for intracellular transporters: functional characterization of the Cl-/H+ exchanger CLC-7. J Physiol 428 590: 3421–3430 429 Craig T, Crane FL (1981) A transplasmamembrane electron transport system in plant cells. Plant 430 Physiol Suppl 67: 99 431 DeCoursey TE, Morgan D, Cherny VV (2003) The voltage dependence of NADPH oxidase 432 reveals why phagocytes need proton channels. Nature 422: 531–534 433 Dutta S, Teresinski HJ, Smith MD (2014) A split-ubiquitin yeast two-hybrid screen to examine the 434 substrate specificity of atToc159 and atToc132, two Arabidopsis chloroplast preprotein import 435

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16

receptors. PloS One 9: e95026 436 Griesen D, Su D, Bérczi A, Asard H (2004) Localization of an ascorbate-reducible cytochrome 437 b561 in the plant tonoplast. Plant Physiol 134: 726–734 438 Hedrich R (2012) Ion channels in plants. Physiol Rev 92: 1777–1811 439 Hille B (2001) Ion Channels of Excitable Membranes, Third Edition, 3rd Edition edition. Sinauer 440 Associates, Sunderland, Mass 441 Horemans N, Asard H, Caubergs RJ (1994) The Role of Ascorbate Free Radical as an Electron 442 Acceptor to Cytochrome b-Mediated Trans-Plasma Membrane Electron Transport in Higher Plants. 443 Plant Physiol 104: 1455–1458 444 Iyer LM, Anantharaman V, Aravind L (2007) The DOMON domains are involved in heme and 445 sugar recognition. Bioinforma Oxf Engl 23: 2660–2664 446 Liman ER, Tytgat J, Hess P (1992) Subunit stoichiometry of a mammalian K+ channel 447 determined by construction of multimeric cDNAs. Neuron 9: 861–871 448 Lorenz C, Pusch M, Jentsch TJ (1996) Heteromultimeric CLC chloride channels with novel 449 properties. Proc Natl Acad Sci 93: 13362–13366 450 Lu P, Ma D, Yan C, Gong X, Du M, Shi Y (2014) Structure and mechanism of a eukaryotic 451 transmembrane ascorbate-dependent oxidoreductase. Proc Natl Acad Sci U S A 111: 1813–1818 452 Lüthje S, Möller B, Perrineau FC, Wöltje K (2013) Plasma membrane electron pathways and 453 oxidative stress. Antioxid Redox Signal 18: 2163–2183 454 Marrè MT, Moroni A, Albergoni FG, Marrè E (1988) Plasmalemma redox activity and h extrusion: 455 I. Activation of the h-pump by ferricyanide-induced potential depolarization and cytoplasm 456 acidification. Plant Physiol 87: 25–29 457 McKie AT, Barrow D, Latunde-Dada GO, Rolfs A, Sager G, Mudaly E, Mudaly M, Richardson 458 C, Barlow D, Bomford A, et al (2001) An iron-regulated ferric reductase associated with the 459 absorption of dietary iron. Science 291: 1755–1759 460 Menckhoff M, Lüthje S (2004) Transmembrane electron transport in sealed and NAD(P)H-loaded 461 right-side-out plasma membrane vesicles isolated from maize (Zea mays L.) roots. J Exp Bot 55: 462 1343–1349 463 Moran N, Ehrenstein G, Iwasa K, Bare C, Mischke C (1984) Ion channels in plasmalemma of 464 wheat protoplasts. Science 226: 835–838 465 Nakanishi N, Rahman MM, Sakamoto Y, Takigami T, Kobayashi K, Hori H, Hase T, Park S-Y, 466 Tsubaki M (2009) Importance of the conserved lysine 83 residue of Zea mays cytochrome b(561) 467 for ascorbate-specific transmembrane electron transfer as revealed by site-directed mutagenesis 468 studies. Biochemistry 48: 10665–10678 469 Nanasato Y, Akashi K, Yokota A (2005) Co-expression of cytochrome b561 and ascorbate 470 oxidase in leaves of wild watermelon under drought and high light conditions. Plant Cell Physiol 46: 471 1515–1524 472 Naso A, Dreyer I, Pedemonte L, Testa I, Gomez-Porras JL, Usai C, Mueller-Rueber B, 473 Diaspro A, Gambale F, Picco C (2009) The role of the C-terminus for functional heteromerization 474 of the plant channel KDC1. Biophys J 96: 4063–4074 475 Njus D, Kelley PM (1993) The secretory-vesicle ascorbate-regenerating system: a chain of 476 concerted H+/e(-)-transfer reactions. Biochim Biophys Acta 1144: 235–248 477 Petheo GL, Demaurex N (2005) Voltage- and NADPH-dependence of electron currents generated 478 by the phagocytic NADPH oxidase. Biochem J 388: 485–491 479 Picco C, Scholz-Starke J, Naso A, Preger V, Sparla F, Trost P, Carpaneto A (2014) How are 480 cytochrome b561 electron currents controlled by membrane voltage and substrate availability? 481 Antioxid Redox Signal 21: 384–391 482 Preger V, Scagliarini S, Pupillo P, Trost P (2004) Identification of an ascorbate-dependent 483 cytochrome b of the tonoplast membrane sharing biochemical features with members of the 484 cytochrome b561 family. Planta 220: 365–375 485 Preger V, Tango N, Marchand C, Lemaire SD, Carbonera D, Di Valentin M, Costa A, Pupillo 486 P, Trost P (2009) Auxin-responsive genes AIR12 code for a new family of plasma membrane b-487 type cytochromes specific to flowering plants. Plant Physiol 150: 606–620 488 Schrenzel J, Serrander L, Bánfi B, Nüsse O, Fouyouzi R, Lew DP, Demaurex N, Krause KH 489 (1998) Electron currents generated by the human phagocyte NADPH oxidase. Nature 392: 734–490 737 491

www.plantphysiol.orgon March 22, 2019 - Published by Downloaded from Copyright © 2015 American Society of Plant Biologists. All rights reserved.

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Schroeder JI, Hedrich R, Fernandez JM (1984) Potassium-selective single channels in guard cell 492 protoplasts of Vicia faba. Nature 312: 361–362 493 Sijmons PC, Lanfermeijer FC, de Boer AH, Prins HB, Bienfait HF (1984) Depolarization of Cell 494 Membrane Potential during Trans-Plasma Membrane Electron Transfer to Extracellular Electron 495 Acceptors in Iron-Deficient Roots of Phaseolus vulgaris L. Plant Physiol 76: 943–946 496 Tsubaki M, Takeuchi F, Nakanishi N (2005) Cytochrome b561 protein family: expanding roles 497 and versatile transmembrane electron transfer abilities as predicted by a new classification system 498 and protein sequence motif analyses. Biochim Biophys Acta 1753: 174–190 499 Tzfira T, Tian G-W, Lacroix B, Vyas S, Li J, Leitner-Dagan Y, Krichevsky A, Taylor T, 500 Vainstein A, Citovsky V (2005) pSAT vectors: a modular series of plasmids for autofluorescent 501 protein tagging and expression of multiple genes in plants. Plant Mol Biol 57: 503–516 502 Vargas JD, Herpers B, McKie AT, Gledhill S, McDonnell J, van den Heuvel M, Davies KE, 503 Ponting CP (2003) Stromal cell-derived receptor 2 and cytochrome b561 are functional ferric 504 reductases. Biochim Biophys Acta 1651: 116–123 505 506 507 508

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Figure 1: A CYBDOM-EGFP fusion localizes to the plasma membrane of leaf cells in transformed Arabidopsis plants. A, EGFP fluorescence (green) from a single focal plane of a representative Arabidopsis leaf expressing the chimeric CYBDOM-EGFP protein. The fluorescence signal was detected as a continuous and uniform signal at the periphery of the epidermal cells. B, Chlorophyll autofluorescence of the same cells shown in a. C, Bright field image of the same cells shown in a. D, Overlay image of A-C, in which the match of EGFP fluorescence with the boundaries of leaf epidermal cells can be easily appreciated. E, EGFP fluorescence from a single focal plane of stomatal guard cells expressing the chimeric CYBDOM-GFP protein. As in the case of the epidermal cells the fluorescence was detected as a continuous and uniform signal at the periphery of the cells. F, Chlorophyll autofluorescence of the same stomata shown in e. G, Bright field of the same the same stomata shown in e. H, Overlay image of E-G. I, EGFP fluorescence from a single focal plane of an Arabidopsis leaf expressing the chimeric CYBDOM-GFP protein. J, FM4-64 (plasma membrane marker, red) fluorescence in the same epidermal cells shown in I. K, The EGFP signal of CYBDOM-EGFP (green) co-localizes with FM4-64 (red) as revealed by co-localization of the two fluorescence signals (orange). I, Plot profile of EGFP (green trace) and FM4-64 (red trace) fluorescence along the white line shown in K.

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Figure 2: External ferricyanide elicits inward currents in CYBDOM-expressing Xenopus oocytes. A, Expression of a CYBDOM-EGFP fusion construct in Xenopus oocytes. Confocal microscopy images taken in transmission light (left) and in the EGFP fluorescence channel (right; green signal). B, Confocal images showing the border of an oocyte expressing CYBDOM-EGFP (left; green signal) and stained with the plasma membrane marker FM4-64 (middle; red signal); right: merged signals. C, Fluorescence intensity profiles of the EGFP (green) and FM4-64 signals (red) along the z-axis of the oocyte border (as in A). D, Ferric reductase activity in non-injected (n = 22) and CYBDOM cRNA-injected oocytes (n = 17) determined by the ferrozine-based colorimetric assay (see Methods section for details). Experiment was repeated twice with similar results. E, Membrane current traces recorded in a water-injected oocyte (upper trace) and in a CYBDOM cRNA-injected oocyte (lower trace), in response to exposure (horizontal bar) to 100 μM ferricyanide (FeCN) in standard bath solution (pH 5.5), at a holding potential of -20 mV. F, I-V relationships of steady-state membrane currents elicited by 100 μM FeCN in control (n = 7; empty symbols) and CYBDOM-expressing oocytes (n = 7; filled symbols). Membrane currents were recorded in response to 25-ms voltage pulses ranging from +40 mV to -100 mV in steps of 10 mV, from a holding potential of -20 mV.

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Figure 3: Amplification of CYBDOM-mediated inward currents in Xenopus oocytes by ascorbate injection. A, Current responses of a CYBDOM-expressing oocyte upon FeCN application (arrowhead) at increasing concentrations in standard bath solution (pH 5.5), at a holding potential of -20 mV. Black traces were recorded in baseline conditions using the endogenous pool of cytosolic ascorbate, red traces were recorded from the same oocyte after injection of 10 mM ascorbate. B, Dose-response curves showing the dependence of current amplitudes in A on the applied [FeCN], in baseline conditions (black symbols) and after injection of 10 mM ascorbate (red symbols). Data points were fitted with a Michaelis-Menten function (continuous lines). C, D, Summary plots of the apparent maximum current at saturating [FeCN] (IFmax

app; C) and the apparent affinity constant for FeCN (KF

app; D) in baseline conditions, using the endogenous pool of cytosolic ascorbate (Asc [cyt]), and after injection of 10 mM ascorbate (Asc [+10mM]). Mean values ± sem (n = 10 different oocytes).

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Figure 4: CYBDOM transport activity depends on the type of electron acceptor and is driven by electron trans-membrane movement. A, Current recordings in a control oocyte (upper traces) and in a CYBDOM-expressing oocyte (lower traces), pre-injected with 10 mM ascorbate, upon application (arrow) of ferricyanide (FeCN), ferric nitrilotriacetate (FeNTA), ferric citrate (FeCIT) and ferric EDTA (FeEDTA) in standard bath solution (each at 500 μM). Holding potential at -20 mV. B, Current amplitudes recorded in CYBDOM-expressing oocytes in response to alternative electron acceptors (as shown in a) were normalized to the FeCN amplitude. Mean values ± sem (n = 8 different oocytes for FeNTA, n = 7 for FeCIT, n = 5 for FeEDTA). C, Left: Current recordings in a CYBDOM-expressing oocyte (pre-injected with 10 mM ascorbate) upon exposure (horizontal bar) to 100 μM FeCN (upper traces) and 100 μM ferrocyanide (lower traces). Holding potential at -20 mV. Right: Mean current amplitudes (± sem) recorded from 5 different oocytes. D, Left: Membrane current recordings in a CYBDOM-expressing oocyte (pre-injected with 10 mM ascorbate) upon exposure to 100 μM FeCN (horizontal bar) in standard bath solution (Std) and successively in a modified bath solution (BTP/MES). Holding potential at -20 mV. Right: Summary plot of current amplitudes in standard and modified bath solution. Mean values ± sem (n = 5 different oocytes). E, Current responses of a CYBDOM-expressing oocyte (pre-injected with 10 mM ascorbate) upon exposure to FeCN (horizontal bar) at concentrations of 3, 10, 30, 100 and 500 μM (as in Figure 3A), recorded in standard bath solution adjusted to pH 5.5 (upper traces) or to pH 7.5 (lower traces). Holding potential at -20 mV. F, Dose-response curves showing the dependence of current amplitudes on the applied [FeCN] at pH 5.5 (filled symbols) or pH 7.5 (empty symbols). Data points were fitted with a Michaelis-Menten function (continuous lines). G, Summary plot of the ratio between the values determined at pH 5.5 and at pH 7.5, for the apparent maximum current at saturating [FeCN] (Imax) and the apparent affinity constant for FeCN (KF), both derived from Michaelis-Menten fits (see F). Mean values ± sem (n = 5 different oocytes).

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Figure 5: CYBDOM-mediated inward currents are abolished by mutation of a conserved histidine residue. A, Predicted CYBDOM secondary structure showing the positions of the four conserved histidines (H) predicted to coordinate two heme groups (black vertical bars) within the CYB561 core domain (blue cylinders). The H249 residue mutated into leucine (L) is highlighted in red. B, Protein sequence alignment of selected members of the CYB561 family. The highly conserved histidine residue corresponding to position 249 in GmCYBDOM is highlighted. Further sequences are from Arabidopsis thaliana (At; AAM61586.1), Zea mays (Zm; XP_008678372.1), Oryza sativa japonica (Osj; BAD05824.1), Triticum aestivum (Ta; CBH32553.1), Caenorhabditis elegans (Ce; NP_001023900.1), Drosophila melanogaster (Dm; NP_611079.2; Picco et al., 2014), Bos taurus (Bt; NP_777262.1), Mus musculus (Mm; NP_031831.2), Homo sapiens (Hs; NP_001017917.1). C, Confocal images of a Xenopus oocyte expressing CYBDOMH249L-EGFP (left; green signal) and stained with the plasma membrane marker FM4-64 (middle; red signal); right: merged signals. Scale bar 75 μm. Regions of interest ROI1 and ROI2 were used for fluorescence quantification in panel D. D, Ratio of fluorescence intensities (F) of ROI1 and ROI2 in the EGFP channel (image in c) in oocytes expressing wild-type CYBDOM (n = 8) or CYBDOMH249L (n = 8). E, Membrane current recordings in oocytes expressing wild-type CYBDOM (left traces) or CYBDOMH249L (right traces), in response to application of 100 μM FeCN (arrowhead), in baseline conditions using Asc[cyt] (black traces) and after injection of 10 mM ascorbate (red traces). Holding potential at -20 mV. F, Mean inward current amplitudes recorded in oocytes expressing wild-type CYBDOM (n = 8) or CYBDOMH249L (n = 8), in baseline conditions using Asc[cyt] and after injection of 10 mM ascorbate (Asc [+10mM]).

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Horemans N, Asard H, Caubergs RJ (1994) The Role of Ascorbate Free Radical as an Electron Acceptor to Cytochrome b-MediatedTrans-Plasma Membrane Electron Transport in Higher Plants. Plant Physiol 104: 1455-1458

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Iyer LM, Anantharaman V, Aravind L (2007) The DOMON domains are involved in heme and sugar recognition. Bioinforma Oxf Engl23: 2660-2664

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Liman ER, Tytgat J, Hess P (1992) Subunit stoichiometry of a mammalian K+ channel determined by construction of multimericcDNAs. Neuron 9: 861-871

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lorenz C, Pusch M, Jentsch TJ (1996) Heteromultimeric CLC chloride channels with novel properties. Proc Natl Acad Sci 93:13362-13366

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lu P, Ma D, Yan C, Gong X, Du M, Shi Y (2014) Structure and mechanism of a eukaryotic transmembrane ascorbate-dependentoxidoreductase. Proc Natl Acad Sci U S A 111: 1813-1818

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lüthje S, Möller B, Perrineau FC, Wöltje K (2013) Plasma membrane electron pathways and oxidative stress. Antioxid Redox Signal18: 2163-2183

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Marrè MT, Moroni A, Albergoni FG, Marrè E (1988) Plasmalemma redox activity and h extrusion: I. Activation of the h-pump byferricyanide-induced potential depolarization and cytoplasm acidification. Plant Physiol 87: 25-29

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

McKie AT, Barrow D, Latunde-Dada GO, Rolfs A, Sager G, Mudaly E, Mudaly M, Richardson C, Barlow D, Bomford A, et al (2001) Aniron-regulated ferric reductase associated with the absorption of dietary iron. Science 291: 1755-1759

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Menckhoff M, Lüthje S (2004) Transmembrane electron transport in sealed and NAD(P)H-loaded right-side-out plasma membranevesicles isolated from maize (Zea mays L.) roots. J Exp Bot 55: 1343-1349

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Moran N, Ehrenstein G, Iwasa K, Bare C, Mischke C (1984) Ion channels in plasmalemma of wheat protoplasts. Science 226: 835-838

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Nakanishi N, Rahman MM, Sakamoto Y, Takigami T, Kobayashi K, Hori H, Hase T, Park S-Y, Tsubaki M (2009) Importance of theconserved lysine 83 residue of Zea mays cytochrome b(561) for ascorbate-specific transmembrane electron transfer as revealedby site-directed mutagenesis studies. Biochemistry 48: 10665-10678 www.plantphysiol.orgon March 22, 2019 - Published by Downloaded from

Copyright © 2015 American Society of Plant Biologists. All rights reserved.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Nanasato Y, Akashi K, Yokota A (2005) Co-expression of cytochrome b561 and ascorbate oxidase in leaves of wild watermelonunder drought and high light conditions. Plant Cell Physiol 46: 1515-1524

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Naso A, Dreyer I, Pedemonte L, Testa I, Gomez-Porras JL, Usai C, Mueller-Rueber B, Diaspro A, Gambale F, Picco C (2009) Therole of the C-terminus for functional heteromerization of the plant channel KDC1. Biophys J 96: 4063-4074

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Njus D, Kelley PM (1993) The secretory-vesicle ascorbate-regenerating system: a chain of concerted H+/e(-)-transfer reactions.Biochim Biophys Acta 1144: 235-248

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Petheo GL, Demaurex N (2005) Voltage- and NADPH-dependence of electron currents generated by the phagocytic NADPHoxidase. Biochem J 388: 485-491

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Picco C, Scholz-Starke J, Naso A, Preger V, Sparla F, Trost P, Carpaneto A (2014) How are cytochrome b561 electron currentscontrolled by membrane voltage and substrate availability? Antioxid Redox Signal 21: 384-391

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Preger V, Scagliarini S, Pupillo P, Trost P (2004) Identification of an ascorbate-dependent cytochrome b of the tonoplast membranesharing biochemical features with members of the cytochrome b561 family. Planta 220: 365-375

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Preger V, Tango N, Marchand C, Lemaire SD, Carbonera D, Di Valentin M, Costa A, Pupillo P, Trost P (2009) Auxin-responsivegenes AIR12 code for a new family of plasma membrane b-type cytochromes specific to flowering plants. Plant Physiol 150: 606-620

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Schrenzel J, Serrander L, Bánfi B, Nüsse O, Fouyouzi R, Lew DP, Demaurex N, Krause KH (1998) Electron currents generated bythe human phagocyte NADPH oxidase. Nature 392: 734-737

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

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