Nga T Nguyen

Mather A Khan

Norma A Castro-Guerrero

Ju-Chen Chia

Olena K Vatamaniuk

Stéphane Mari

Silvia S Jurisson

David G Mendoza-Cozatl

Stephane Mari

Iron Availability Within the Leaf Vasculature Determines the Magnitude of Iron Deficiency Responses in Source and Sink Tissues in Arabidopsis

Keywords: Arabidopsis thaliana, iron deficiency responses, leaf vasculature, phloem-loading cells, source and sink tissues, systemic signaling

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Introduction

Iron (Fe) is an essential element for plants and organisms across all kingdoms. Plants are also the main source of Fe for other organisms, including humans. The majority of life on Earth depends on the capacity of plants to mine Fe from the Earth's crust and make it available for uptake and allocation within plant tissues. Iron is needed in multiple biological processes, including photosynthesis, respiration, DNA replication, and cell cycle control [START_REF] Zhang | Essential functions of iron-requiring proteins in DNA replication, repair and cell cycle control[END_REF][START_REF] López-Millán | Chloroplast Iron Transport Proteins -Function and Impact on Plant Physiology[END_REF]; Despite the importance of Fe regulation in plants, our understanding of the molecular mechanisms that regulate Fe homeostasis in plants have just begun to be fully unraveled. Iron deficiency in humans (i.e. anemia) affects close to 2 billion people around the globe and a better understanding of the Fe homeostatic networks in plants will help developing crops with enhanced Fe content.

Iron uptake in dicot species such as Arabidopsis, relies on what is known as a reducing strategy or Strategy I where Fe, in the form of Fe 3+ -insoluble complexes, is first reduced to Fe 2+ by FRO2 (FERRIC REDUCTION OXIDASE 2) which is the substrate of IRT1 (IRON-REGULATED TRANSPORTER 1), the main Fe 2+ transporter in Arabidopsis roots [START_REF] Eide | A novel iron-regulated metal transporter from plants identified by functional expression in yeast[END_REF][START_REF] Robinson | A ferric-chelate reductase for iron uptake from soils[END_REF][START_REF] Henriques | Knock-out of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects[END_REF][START_REF] Varotto | The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana[END_REF][START_REF] Vert | IRT1, an Arabidopsis Transporter Essential for Iron Uptake from the Soil and for Plant Growth[END_REF]. These two proteins, together with additional proteins such H(+)-ATPases (PLASMA MEMBRANE PROTON ATPASEs/ AHAs), coumarins and coumarin transporters, allow Arabidopsis to thrive in environments with different levels of Fe availability [START_REF] Santi | Dissecting iron deficiency-induced proton extrusion in Arabidopsis roots[END_REF][START_REF] Ziegler | Arabidopsis Transporter ABCG37/PDR9 contributes primarily highly oxygenated Coumarins to Root Exudation[END_REF][START_REF] Rosenkranz | Root exudation of coumarins from soil-grown Arabidopsis thaliana in response to iron deficiency[END_REF][START_REF] Robe | Coumarin accumulation and trafficking in Arabidopsis thaliana: a complex and dynamic process[END_REF]. From the different components that mediate Fe uptake in Arabidopsis, the regulation of IRT1 and FRO2, at transcriptional and post-transcriptional level, is perhaps one of the better studied ones. For instance, the transcription factor FIT has been described as a master regulator of the Fe deficiency responses in roots and is required to induce IRT1 and FRO2 during Fe deficiency [START_REF] Colangelo | The essential basic helix-loop-helix protein FIT1 is required for the iron deficiency response[END_REF]. However, FIT alone is unable to induce the transcription of IRT1 and FRO2. In turn, FIT needs to interact with members of the Ib subgroup of bHLH transcription factors (bHLH38/39/100/101) to promote transcriptional activation of the Fe uptake machinery [START_REF] Yuan | FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis[END_REF][START_REF] Sivitz | Arabidopsis bHLH100 and bHLH101 control iron homeostasis via a FIT-independent pathway[END_REF]. This Ib subgroup of transcription factors is thought to be regulated upstream by members of the IVc subgroup of bHLH transcription factors (bHLH34/104/105/115), which together with the recently discovered Upstream Regulator of IRT1 (URI; bHLH121), completes the transcriptional network required to mount an Fe deficiency response in roots [START_REF] Long | The bHLH Transcription Factor POPEYE Regulates Response to Iron Deficiency in Arabidopsis Roots[END_REF][START_REF] Zhang | The bHLH Transcription Factor bHLH104 Interacts with IAA-LEUCINE RESISTANT3 and Modulates Iron Homeostasis in Arabidopsis[END_REF][START_REF] Selote | Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors[END_REF][START_REF] Li | Two bHLH Transcription Factors, bHLH34 and bHLH104, Regulate Iron Homeostasis in Arabidopsis thaliana[END_REF][START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF][START_REF] Gao | The Transcription Factor bHLH121 Interacts with bHLH105 (ILR3) and Its Closest Homologs to Regulate Iron Homeostasis in Arabidopsis[END_REF]. Notably, bHLH38/39/100/101 are also induced by Fe deficiency in leaves, but FIT is preferentially expressed in roots suggesting that a yet unidentified FIT-like transcription factor may be needed in leaves to properly respond to Fe deficiency [START_REF] Khan | Changes in iron availability in Arabidopsis are rapidly sensed in the leaf vasculature and impaired sensing leads to opposite transcriptional programs in leaves and roots[END_REF].

Other molecular players like BTS and BTS-like proteins also play an important role during Fe deficiency by regulating the turnover of IVc subgroup of bHLH transcription factors and preventing a constitutive activation of the Fe uptake machinery [START_REF] Selote | Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors[END_REF][START_REF] Hindt | BRUTUS and its paralogs, BTS LIKE1 and BTS LIKE2, encode important negative regulators of the iron deficiency response in Arabidopsis thaliana[END_REF]Rodriguez-Celma et al., 2019). FIT and URI are also known to be post-transcriptionally regulated by phosphorylation, and in case of FIT, CPK11 was identified as the kinase responsible for its phosphorylation [START_REF] Gratz | CIPK11-Dependent Phosphorylation Modulates FIT Activity to Promote Arabidopsis Iron Acquisition in Response to Calcium Signaling[END_REF]. Post-transcriptional regulation has also been demonstrated to regulate the activity and cellular localization of IRT1, and an impairment of these mechanisms results in either an inadequate response to Fe deficiency or a deregulated rate of Fe uptake that eventually leads to plant death [START_REF] Barberon | Monoubiquitin-dependent endocytosis of the IRON-REGULATED TRANSPORTER 1 (IRT1) transporter controls iron uptake in plants[END_REF][START_REF] Ivanov | SORTING NEXIN1 Is Required for Modulating the Trafficking and Stability of the Arabidopsis IRON-REGULATED TRANSPORTER1[END_REF]. All these layers of complexity should not be unexpected as the boundaries between Fe deficiency, sufficiency, and excess, are very narrow compared to other nutrients [START_REF] Mendoza-Cózatl | Keep talking: crosstalk between iron and sulfur networks fine-tunes growth and development to promote survival under iron limitation[END_REF].

Regulation of Fe uptake at the root level, particularly the IRT1 post-transcriptional modifications is considered a local response; however, research in Arabidopsis and other plant species have shown that Fe homeostasis is also regulated systemically [START_REF] Vert | IRT1, an Arabidopsis Transporter Essential for Iron Uptake from the Soil and for Plant Growth[END_REF][START_REF] Vert | Dual regulation of the Arabidopsis high-affinity root iron uptake system by local and long-distance signals[END_REF][START_REF] Mendoza-Cózatl | OPT3 Is a Component of the Iron-Signaling Network between Leaves and Roots and Misregulation of OPT3 Leads to an Over-Accumulation of Cadmium in Seeds[END_REF][START_REF] Zhai | OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis[END_REF][START_REF] Gayomba | Local and Systemic Signaling of Iron Status and Its Interactions with Homeostasis of Other Essential Elements[END_REF]. For instance, grafting experiments in sunflower and cucumber plants demonstrated that Fe deficiency in roots could be suppressed by grafting the shoot of an Fe sufficient plant onto a root experiencing Fe deprivation [START_REF] Romera | Role of roots and shoots in the regulation of the Fe efficiency responses in sunflower and cucumber[END_REF]. Similarly, foliar application of Fe was able to suppress Fe deficiency in roots of Arabidopsis, tobacco, pea, tomato, and cucumber plants [START_REF] Enomoto | Longdistance signals positively regulate the expression of iron uptake genes in tobacco roots[END_REF][START_REF] García | Shoot to root communication is necessary to control the expression of iron-acquisition genes in Strategy I plants[END_REF]. These seminal experiments and others suggest that the systemic Fe signaling can override the local root signaling, at least at the transcriptional level. Notably, the molecular nature of this phloem-mobile signal has not been unequivocally identified. In contrast, there is a wealth of information about transporters and molecules required to keep Fe soluble for source-to-sink transport. For instance, nicotianamine (NA) is an Fe chelator synthesized by a family of nicotianamine synthases (NAS1/2/3/4), and the Arabidopsis quadruple mutant nas4x-1 shows severe interveinal chlorosis during Fe deficiency, suggesting that NA is required for long-distance transport of Fe [START_REF] Klatte | The analysis of Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron loading and iron deficiency responses[END_REF][START_REF] Schuler | Nicotianamine functions in the Phloem-based transport of iron to sink organs, in pollen development and pollen tube growth in Arabidopsis[END_REF]. In addition, Fe-NA complexes are transported by a family of transporters known as a Yellow Stripe-Like (YSL) proteins and the double mutant ysl1 ysl3 showed impaired Fe homeostasis and lower Fe in seeds [START_REF] Jean | A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading[END_REF][START_REF] Waters | Mutations in Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 Reveal Their Roles in Metal Ion Homeostasis and Loading of Metal Ions in Seeds[END_REF][START_REF] Chu | Successful reproduction requires the function of Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 metal-nicotianamine transporters in both vegetative and reproductive structures[END_REF][START_REF] Kumar | Iron-Nicotianamine Transporters Are Required for Proper Long Distance Iron Signaling[END_REF]. More recently, the phloemloading transporter OPT3 was identified as a key component of the Fe systemic signaling, and Arabidopsis mutants with residual levels of OPT3 expression (opt3-2 and opt3-3) display a constitutive upregulation of the Fe uptake machinery in roots [START_REF] Mendoza-Cózatl | OPT3 Is a Component of the Iron-Signaling Network between Leaves and Roots and Misregulation of OPT3 Leads to an Over-Accumulation of Cadmium in Seeds[END_REF][START_REF] Zhai | OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis[END_REF]. However, no upstream regulators of OPT3 or YSLs have been identified. Lastly, a family of peptides named IRON MAN were found to be mobile between shoots and roots and have been proposed to also play a role in the Fe systemic signaling [START_REF] Hirayama | The Putative Peptide Gene FEP1 Regulates Iron Deficiency Response in Arabidopsis[END_REF][START_REF] Grillet | IRON MAN is a ubiquitous family of peptides that control iron transport in plants[END_REF], by interacting with BTS and preventing the degradation of bHLH105/115 to preserve Fe homoeostasis [START_REF] Li | IRON MAN interacts with BRUTUS to maintain iron homeostasis in Arabidopsis[END_REF]. In addition, URI/bHLH121 binds to the promoter region of IMA1/FEP3, suggesting that URI may also play a role in regulating the transcriptional activity of these phloem-mobile peptides in leaves [START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF].

In this study, we aimed to disrupt Fe homeostasis in the leaf vasculature to explore further and isolate the role of mature leaves in the regulation of Fe deficiency responses in source and sink tissues within then whole plant.

We achieved this by overexpressing an Arabidopsis Fe transporter (AtZIP5) under the control of a phloem-specific promoter (SUC2p). AtZIP5 belongs to the Zinc/Iron-regulated transporter-like Protein (ZIP) gene family, ubiquitous among species and across kingdoms, including fungi, animals and plants [for a review see [START_REF] Guerinot | The ZIP family of metal transporters[END_REF]]. The Arabidopsis genome harbors 15 ZIP members, twelve of them annotated as ZIP, from 1 to 12, and three IRT transporters (IRT1/2/3). While AtZIP5 expression is downregulated during Fe deficiency, the use of the SUC2p allowed us to maintain a steady expression of AtZIP5 in companion cells, even during Fe deprivation [START_REF] Wintz | Expression Profiles of Arabidopsis thaliana in Mineral Deficiencies Reveal Novel Transporters Involved in Metal Homeostasis[END_REF][START_REF] Dinneny | Cell identity mediates the response of Arabidopsis roots to abiotic stress[END_REF][START_REF] Long | The bHLH Transcription Factor POPEYE Regulates Response to Iron Deficiency in Arabidopsis Roots[END_REF]. Our results show that local disturbances of Fe levels in or around the vasculature of source leaves impacted the timing and magnitude of Fe deficiency responses and that these differences are unique between source (i.e. mature leaves) and sink tissues (i.e. young leaves and roots). We also performed similar experiments in the Arabidopsis mutant nas4x-1, which has residual levels of NA and therefore Fe precipitates in the vasculature of mature leaves, thus limiting Fe transport to sink tissues. In contrast to SUC2p::AtZIP5 lines, nas4x-1 mutants showed exacerbated Fe deficiency responses in sink tissues but dampened responses in mature leaves. Altogether, our data provide further evidence to suggest that Fe sensing in leaves can also occur locally in a leaf-specific manner, and that only mature leaves influence the systemic signaling driving Fe deficiency responses in roots.

Results

AtZIP5 is a plasma membrane transporter expressed in the vasculature.

Arabidopsis ZIP5 (AtZIP5) is a member of the Zinc-regulated Iron-regulated transporter-like Protein family (ZIP), and members of this family have been shown to have the capacity to transport transition metals like iron (Fe) and Zinc (Zn). To test whether AtZIP5 was in fact capable of mobilizing Fe into cells, we expressed AtZIP5 in the Saccharomyces cerevisiae yeast strain fet3 fet4, a yeast mutant impaired in Fe uptake. Figure 1A shows that fet3 fet4 yeast expressing AtZIP5 were able to grow on a medium with limited Fe availability (Figure 1A). Also, yeast cells expressing AtZIP5 accumulated significantly higher levels of Fe compared to cells transformed with an empty vector and grown in medium supplemented with 100 µM of FeCl 3 . Notably, the Fe concentration in ZIP5-expressing cells was comparable to the Fe levels found in wildtype yeast or fet3 fet4 cells expressing AtIRT1 (Figure 1B). Because members of the ZIP family are known to transport other divalent metals, AtZIP5 was also tested for its capacity to transport Zn and Mn in yeast. When expressed in zrt1 zrt2, a yeast mutant defective in Zn transport, AtZIP5 was able to rescue yeast growth under limiting Zn availability; however, AtZIP5 was unable to complement the yeast mutant smf1, defective in Mn transport (Figure S1). These results show that AtZIP5 is a functional Fe/Zn transporter similar to AtIRT1.

Previous cell-specific translatome analyses found that AtZIP5 was preferentially expressed in the vasculature ( [START_REF] Mustroph | Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis[END_REF]; Figure S2A). To experimentally verify this observation, we generated stable transgenic lines expressing the GUS reporter gene under the control of the AtZIP5 promoter region (2.5 kb upstream of the transcription start site). Histochemical analysis of GUS activity in several independent T3 homozygous plants showed that AtZIP5 expression is restricted to the vasculature (Figure 2A-B). Next, we aimed to confirm the cellular localization of AtZIP5 by expressing a translational fusion of the yellow fluorescent protein (YFP) and the coding sequence of AtZIP5 (YFP-AtZIP5) in Nicotiana benthamiana (Figure 2D-F). The plasma membrane marker PIP2A-mCherry was also co-infiltrated with YFP-AtZIP5 and used as a reference to localize plasma membrane proteins. Three days after co-infiltration, fluorescence was examined by confocal microscopy and a strong overlapping fluorescence signal was found between the plasma membrane marker PIP2A-mCherry and YFP-AtZIP5 (Figure 2D-E). The co-localization of PIP2A-mCherry and YFP-AtZIP5 was further corroborated by fluorescence intensity plots showing two overlaping peaks corresponding to two adjacent adjacent cells (Figure 2F), suggesting that in plants, AtZIP5 is a plasma membrane transporter.

Phloem-specific expression of AtZIP5 induces hypersensitivity to Fe deficiency.

Our previous studies have indicated that Fe availability at the whole plant level is primarily sensed in the leaf vasculature followed by a corresponding transcriptional response in roots [START_REF] Khan | Changes in iron availability in Arabidopsis are rapidly sensed in the leaf vasculature and impaired sensing leads to opposite transcriptional programs in leaves and roots[END_REF]. To further advance our understanding of shoot-to-root Fe deficiency responses, we wanted to test the timing and magnitude of the Fe deficiency responses in source and sink tissues of plants overexpressing an Fe transporter in the vasculature.

However, AtZIP5 is induced by Fe excess (Figure S2B-C) and not deficiency [START_REF] Wintz | Expression Profiles of Arabidopsis thaliana in Mineral Deficiencies Reveal Novel Transporters Involved in Metal Homeostasis[END_REF][START_REF] Dinneny | Cell identity mediates the response of Arabidopsis roots to abiotic stress[END_REF][START_REF] Long | The bHLH Transcription Factor POPEYE Regulates Response to Iron Deficiency in Arabidopsis Roots[END_REF] ; therefore, to by-pass this transcriptional regulation while keeping the tissue specificity, we generated independent transgenic lines expressing AtZIP5 under the control of the phloem-specific promoter AtSUC2. Previous whole-genome expression analyses shown that SUC2 expression remains steady when plants over accumulate Fe and during Fe deficiency-like conditions such as Cd exposure [START_REF] Khan | Changes in iron availability in Arabidopsis are rapidly sensed in the leaf vasculature and impaired sensing leads to opposite transcriptional programs in leaves and roots[END_REF]; Figure S3A), thus the SUC2p offered the ideal means to maintain a constitutive vascular-specific expression of AtZIP5 during changes in Fe availability. Two independent T3 homozygous SUC2p::AtZIP5 lines, ZIP5ox 16-10 and ZIP5ox 25-5, were selected for further characterization based on their strong ZIP5 expression detected by RT-PCR (Figure S3B).

To begin the characterization of the SUC2p::AtZIP5 lines (ZIP5ox lines), we first tested whether the overexpression of AtZIP5 in the vasculature had an effect on the ability of plants to grow on Fe limiting conditions. Interestingly, both ZIP5ox lines displayed higher sensitivity to Fe deprivation compared to wildtype (WT, Col-0) (Figure 3B). While roots of ZIP5ox lines were shorter than WT plants, no significant difference in Fe, Zn or Mn accumulation at the whole-plant level were detected between ZIP5ox lines and WT (Figure S4), suggesting that the observed sensitivity to Fe deficiency is not associated with a major distribution of Fe within the plant but rather a more discrete, and perhaps localized disturbance of Fe availability in or around the vasculature.

Constitutive expression of AtZIP5 in companion cells alters the Fe deficiency response in young leaves, mature leaves, and roots

Next, we analyzed Fe deficiency responses in WT and ZIP5ox lines. These experiments were conducted in 4-week-old plants grown hydroponically where young sink leaves can be separated from mature source leaves and roots to better dissect transcriptional responses at a tissue-specific manner (see Figure S5 for a detailed description of leaf selection). Furthermore, to minimize the variation in the magnitude of Fe deficiency responses associated with circadian regulation, all Fe deficiency experiments were started in the morning (T0), more specifically two hours after the light in the growth chamber was turned on (i.e. ZT2). Three well-documented vascular markers for Fe deficiency in leaves (OPT3, bHLH100, and FRO3) (Figure S6 and [START_REF] Khan | Changes in iron availability in Arabidopsis are rapidly sensed in the leaf vasculature and impaired sensing leads to opposite transcriptional programs in leaves and roots[END_REF] were used to quantify the timing and magnitude of the Fe deficiency responses in ZIP5ox lines and WT plants. In some cases, ZIP5ox lines showed a constitutively lower level of expression of these Fe deficiency marker genes, even in Fe replete conditions (Figure 4). For instance, ZIP5ox young leaves had a lower expression level of bHLH100 at T0 (i.e., the beginning of the experiment). Similarly, OPT3 and bHLH100 expression were also lower in mature leaves of ZIP5ox lines compared to WT (Figure 4A-B). These differences were small yet significant and consistent in both ZIP5ox lines.

During Fe deficiency, two major trends were identified in leaves. First, independent of the genotype, OPT3, bHLH100, and FRO3 expression were induced throughout the Fe deficiency experiments (Figure 4A-C), indicating that our Fe deficiency treatment was effective and properly sensed by all genotypes. Second, and related to the magnitude of Fe deficiency responses, we found that young leaves of ZIP5ox plants displayed a reduced response (measured as lower expression levels of OPT3, bHLH100, and FRO3) during Fe deficiency compared to WT young leaves at both 12 hr and 24 hr of Fe deprivation (Figure 4A-C). ZIP5ox mature leaves, on the other hand, had a similar Fe deficiency response than WT at the 12 hr time point, but this response was clearly dampened at the 24 hr time-point. Overall, the expression data from leaves indicate that overexpressing AtZIP5 in the vasculature decreased the magnitude of the Fe deficiency response in leaves.

In roots, the Fe deficiency response was assessed by following the expression of two well-known Fe deficiency markers: FRO2 and IRT1. Similar to leaves, all plant genotypes displayed an increased expression of FRO2 and IRT1 during Fe deprivation, and ZIP5ox lines had lower levels of expression of FRO2 and IRT1 compared to WT. Moreover, the magnitude of the Fe deficiency response in ZIP5ox roots after 12 hrs of Fe deficiency was significantly lower than WT. After 24 hr, both IRT1 and FRO2 reached levels of expression similar to WT suggesting that overexpression of AtZIP5 in the vasculature delayed, but did not inhibit, the full magnitude of Fe deficiency responses in roots.

Nicotianamine deficiency uncouples Fe deficiency responses in young leaves and mature leaves.

To further test the autonomous magnitude of Fe deficiency responses in source and sink tissues, we took advantage of the Arabidopsis nas4x-1 mutant, which has only trace levels of nicotianamine [START_REF] Klatte | The analysis of Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron loading and iron deficiency responses[END_REF][START_REF] Schuler | Nicotianamine functions in the Phloem-based transport of iron to sink organs, in pollen development and pollen tube growth in Arabidopsis[END_REF]. Nicotianamine (NA) is an Fe chelator required for source-to-sink mobilization of Fe and in this case, the nas4x-1 mutant represents an opposite scenario compared to ZIP5ox lines, as the lack of NA results in precipitation of the Fe within the vasculature, thus reducing the levels of Fe available for long-distance transport. To further characterize ZIP5ox lines and nas4x-1 mutant and to explore the distribution of Fe between young and mature leaves, both genotypes were subjected to X-ray fluorescence spectrometry (XRF). While no major changes were observed in young leaves of nas4x-1 or ZIP5ox lines (Figure S7), higher levels of Fe were detected within the vasculature of nas4x-1 mature leaves (Figure 5A). In contrast, ZIP5ox lines showed a consistent lower signal of Fe (shown as pixel intensity) particularly in the vasculature near the edge of the leaf (Figure 5B-C). A similar trend was found for Zn but not for Ca, Cu, K and Mn (Figure S8). The overaccumulation of Fe nas4x-1 in mature leaves was consistent with Fe levels measured by ICP-OES (Figure S9), where young leaves of the nas4x-1 mutant showed lower levels of Fe while mature leaves showed a higher accumulation of Fe. Moreover, shoot-to-root 59 Fe mobilization experiments indicated that nas4x-1 plants have a lower ability to translocate Fe between mature leaves and roots (Figure 5D).

Next, we used the same Fe deficiency markers previously described to assess the Fe deficiency response in young leaves, mature leaves, and roots of nas4x-1 (Figure 6). Interestingly, our results showed striking differences between young and mature leaves. For instance, nas4x-1 young leaves displayed a constitutive Fe deficiency response, where OPT3, bHLH100, and FRO3 were up-regulated at all tested time-points (Figure 6A-C). In nas4x-1 mature leaves however, the expression of all the Fe deficiency markers was consistently lower compared to WT. For instance, at T0, the expression of OPT3 was similar between nas4x-1 and WT; however, the magnitude of Fedeficiency responses in nas4x-1 mature leaves was significantly lower than in WT mature leaves. A similar trend was found for bHLH100 and FRO3 expression (Figure 6B-C); that is, nas4x-1 mature leaves displayed a slightly but significantly higher expression of bHLH100 and FRO3 compared to Col-0 mature leaves at T0. Moreover, under Fe deficiency, nas4x-1 mature leaves had a smaller magnitude of Fe deficiency response at the 12 hr time-point compared to wildtype. At the 24 hr time-point, this difference was reduced between genotypes and the expression levels of both bHLH100 and FRO3 were similar between mature leaves of nas4x-1 and WT.

In addition, and similar to the expression of OPT3 in young leaves, bHLH100 and FRO3 were also constitutively up-regulated in nas4x-1 young leaves and, during Fe deficiency, the magnitude of the transcriptional response was higher compared to WT leaves (Figure 6A-C). A similar response was found in roots, where the expression of FRO2 and IRT1 was higher in nas4x-1 than WT at all tested time-points, and the magnitude of the Fe deficiency response was also higher compared to WT (Figure 6D). Overall, the nas4x-1 results are consistent with a model where sink tissues (i.e., young leaves and roots) are constitutively deprived of Fe due to the lack of NA.

Perhaps more interestingly, we found two additional facts: (1) the typical Fe deficiency response observed in WT plants is far from saturation as the magnitude of the Fe deficiency response in nas4x-1 was higher than WT. These results suggest that Fe deficiency responses are continuously and negatively regulated in WT plants even during Fe deprivation. These negative regulators are yet to be identified. ( 2

Discussion

For decades, systemic shoot-to-root signaling has been known to regulate Fe uptake at the root level in Arabidopsis and other dicot species [START_REF] Romera | Role of roots and shoots in the regulation of the Fe efficiency responses in sunflower and cucumber[END_REF][START_REF] Vert | Dual regulation of the Arabidopsis high-affinity root iron uptake system by local and long-distance signals[END_REF][START_REF] Enomoto | Longdistance signals positively regulate the expression of iron uptake genes in tobacco roots[END_REF][START_REF] García | Shoot to root communication is necessary to control the expression of iron-acquisition genes in Strategy I plants[END_REF]. However, very few details about the molecular mechanisms behind this systemic regulation are known to date. In contrast, several molecular players of the Fe uptake machinery together with their associated transcriptional and post-transcriptional regulators in roots have been described. For instance, recent data have revealed at least two layers of transcriptional regulation controlling the expression of IRT1 and FRO2 during Fe starvation. These two layers include members of the Ib subgroup of bHLH transcription factors (bHLH38/39/100/101), which in coordination with members of the IVc subgroup of bHLH transcription factors (bHLH34/104/105/115), regulate the Fe uptake machinery in roots [START_REF] Yuan | FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis[END_REF][START_REF] Sivitz | Arabidopsis bHLH100 and bHLH101 control iron homeostasis via a FIT-independent pathway[END_REF][START_REF] Long | The bHLH Transcription Factor POPEYE Regulates Response to Iron Deficiency in Arabidopsis Roots[END_REF][START_REF] Zhang | The bHLH Transcription Factor bHLH104 Interacts with IAA-LEUCINE RESISTANT3 and Modulates Iron Homeostasis in Arabidopsis[END_REF][START_REF] Selote | Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors[END_REF][START_REF] Li | Two bHLH Transcription Factors, bHLH34 and bHLH104, Regulate Iron Homeostasis in Arabidopsis thaliana[END_REF]. In addition, local and post-transcriptional regulation of some of these transcription factors, such as URI and FIT, and ubiquitination of IRT1 also contribute to the fine-tuning regulation of Fe homeostasis in roots [START_REF] Barberon | Monoubiquitin-dependent endocytosis of the IRON-REGULATED TRANSPORTER 1 (IRT1) transporter controls iron uptake in plants[END_REF][START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF][START_REF] Gao | The Transcription Factor bHLH121 Interacts with bHLH105 (ILR3) and Its Closest Homologs to Regulate Iron Homeostasis in Arabidopsis[END_REF].

The systemic shoot-to-root regulation of Fe homeostasis, on the other hand, is far less understood compared to roots, but evidence from Arabidopsis and other dicot plants suggest that the systemic signaling dictates, and may even override, the local response to Fe deficiency in roots [START_REF] Romera | Role of roots and shoots in the regulation of the Fe efficiency responses in sunflower and cucumber[END_REF][START_REF] Enomoto | Longdistance signals positively regulate the expression of iron uptake genes in tobacco roots[END_REF][START_REF] García | Shoot to root communication is necessary to control the expression of iron-acquisition genes in Strategy I plants[END_REF]. The entire root system is by definition a sink tissue, as it depends on the energy (i.e. photosynthates) assimilated and translocated from source leaves. In contrast, the shoot system is a more diverse and complex tissue, where sink and source tissues occur synchronously. For instance, young developing leaves are sink tissues that depend on source leaves for an adequate supply of energy and nutrients to sustain growth and development. In a sense, young developing leaves are in continuous demand for Fe and could be considered constitutively Fe deficient during their transition from sink to source leaves. This co-occurrence of source and sink tissues within the shoot system may explain in part why only a few players of the Fe deficiency response in leaves have been identified. After all, it is not uncommon to use the entire shoot system in whole-genome transcriptome analyses, but the shoot system may be displaying different timing and magnitude of transcriptional changes, and even opposite changes, in a leaf-specific manner, thus canceling relevant changes if the entire shoot system is used in whole-genome expression analysis.

Here we aimed to disturb local levels of Fe availability by expressing an Fe transporter (AtZIP5) specifically in companion cells. We were particularly interested in dissecting the responses in a tissue-specific manner, including source leaves (mature leaves) and sink tissues such as young leaves and roots. Our results demonstrate that AtZIP5 is an Fe/Zn transporter preferentially expressed in the vasculature (Figures 1 and2).

AtZIP5, however, is natively up-regulated during Fe excess, not Fe deficiency (Figure S2B-C of the phloem-specific SUC2 promoter, which is expressed only in source leaves and whose transcription is independent of the Fe status of the plant (Figures S3A). Interestingly, ZIP5ox plants displayed hypersensitivity to Fe deficiency in root growth assays (Figure 3). During Fe deficiency, leaves transmit an unknown signal to roots to either up-regulate or repress Fe uptake at the root level. It is possible that expression of AtZIP5 in phloem-loading cells prevented the up-regulation of Fe deficiency responses by altering Fe levels in or around the vasculature in source leaves. This suggestion is supported by the transcriptional responses of Fe deficiency markers in sink tissues, where young leaves and roots show either delayed or dampened responses to Fe deficiency compared to WT plants (Figure 4). In addition, and similar to other ZIP transporters, ZIP5 is able to mobilize both Fe and Zn and although Zn levels were not affected at the whole tissue level (Figure S4B), it is possible that the observed Fe deficiency responses in ZIP5ox plants are also the result of more complex yet unknown interactions between transition elements and their sensing mechanisms [START_REF] Lešková | Metal crossroads in plants: modulation of nutrient acquisition and root development by essential trace metals[END_REF].

To explore the opposite scenario, we took advantage of the Arabidopsis nas4x-1 mutant, which is known to have low mobility of Fe between source and sink tissues due to the residual levels of NA at the whole plant level [START_REF] Klatte | The analysis of Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron loading and iron deficiency responses[END_REF][START_REF] Schuler | Nicotianamine functions in the Phloem-based transport of iron to sink organs, in pollen development and pollen tube growth in Arabidopsis[END_REF]. Thus, nax4x-1 provides an opposite scenario compared to ZIP5ox lines, where Fe is preferentially retained at high levels in mature leaves with only residual transport to young leaves. This scenario was confirmed by elemental imaging using XRF, which demonstrates that Fe is retained in the vasculature of nax4x-1 mature leaves (Figure 5B). In turn, sink tissues such as young leaves and roots display a faster and stronger Fe deficiency response compared to the delayed and dampened response of mature leaves. Notably, this study, together with previous studies, emphasize the need to address Fe deficiency responses in a leaf-specific manner [START_REF] Klatte | The analysis of Arabidopsis nicotianamine synthase mutants reveals functions for nicotianamine in seed iron loading and iron deficiency responses[END_REF][START_REF] Chu | Successful reproduction requires the function of Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 metal-nicotianamine transporters in both vegetative and reproductive structures[END_REF][START_REF] Schuler | Nicotianamine functions in the Phloem-based transport of iron to sink organs, in pollen development and pollen tube growth in Arabidopsis[END_REF][START_REF] Kumar | Iron-Nicotianamine Transporters Are Required for Proper Long Distance Iron Signaling[END_REF]. Moreover, there is now sufficient evidence, including this work, to suggest that each leaf within the shoot system may respond in unique ways (i.e., local Fe sensing in leaves) depending on their access to Fe availability and other environmental cues [START_REF] Schuler | Transcriptome analysis by GeneTrail revealed regulation of functional categories in response to alterations of iron homeostasis in Arabidopsis thaliana[END_REF][START_REF] Hantzis | A Program for Iron Economy during Deficiency Targets Specific Fe Proteins[END_REF]. This may be explained by the fact that young leaves are under development and therefore have a higher demand for Fe; thus, young leaves are more prone to trigger Fe deficiency responses by small changes in Fe levels compared to the changes needed to trigger an equivalent response in mature leaves. Moreover, one of the early responses to Fe deficiency in mature leaves is a reduced mobilization of Fe to young leaves, which may enhance the Fe starvation experienced by sink leaves.

Similar to nax4x-1, the double mutant ysl1 ysl3 also accumulates significant levels of Fe within the vasculature [START_REF] Kumar | Iron-Nicotianamine Transporters Are Required for Proper Long Distance Iron Signaling[END_REF]. YSL1 and YSL3, are metal-nicotianamine transporters preferentially expressed in the vasculature and are thought to be important for source-to-sink transport of Fe as the ysl1 ysl3 mutant shows reduced levels of Fe, Zn and Cu in seeds [START_REF] Waters | Mutations in Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 Reveal Their Roles in Metal Ion Homeostasis and Loading of Metal Ions in Seeds[END_REF]. However, and in contrast to nas4x-1, the ysl1 ysl3 double mutant is unable to induce an Fe deficiency response when Fe availability is reduced. It has been suggested that YSL1/3 may have a role in unloading metal-NA complexes from the vasculature and therefore the increased levels of Fe in the phloem in ysl1 ysl3 may be repressing, constitutively, the Fe deficiency response in roots. The difference between nas4x-1 and ysl1 ysl3 mutants in terms of transcriptional responses during Fe deficiency can also be explained in terms of solubility as ysl1 ysl3 are not impaired in NA synthesis and therefore, NA in the phloem should maintain a proportion of Fe in an available soluble form. In contrast, nas4x-1 has functional YSL1 and YSL3 transporters but the low levels of NA in the vasculature likely favors Fe precipitation, thus reducing the effective concentration of mobile Fe in the phloem. An additional layer of complexity comes from the fact that high levels of Fe lead to the production of reactive oxygen species and these have been found to repress Fe deficiency responses in leaves and roots (Le at la., 2016;[START_REF] Mcinturf | Cadmium interference with iron sensing reveals transcriptional programs sensitive and insensitive to reactive oxygen species[END_REF]. Altogether, these observations illustrate the complexity and hierarchical structure of Fe deficiency responses in plants, which is expected considering the high reactivity of Fe. It is also a reminder that as of today, there is no data about Fe speciation in the phloem sap other than predictions and the identification and quantification of the different soluble forms of Fe is critical to understand the systemic nature of shoot-to-root Fe signaling in plants.

Historically, the identification of molecular mechanisms behind Fe deficiency responses in roots has been more advanced compared to our understanding of Fe deficiency responses in leaves. In hindsight, this should have been expected as the root system is a more homogeneous tissue compared to shoots. In fact, initial experiments that took advantage of the first whole-genome transcriptome technologies, like the Affymetrix microarray chips, were sufficient to identify key transcription factors regulating Fe deficiency responses in roots [START_REF] Colangelo | The essential basic helix-loop-helix protein FIT1 is required for the iron deficiency response[END_REF]Guerinot, 2004, Long et al., 2010). Not surprisingly, similar approaches have not provided the same level of information for leaves. However, it is now clear that Fe deficiency responses in shoots can also be autonomous and leaf specific.

Moreover, and due to the directional movement of nutrients between sink and source tissues, it is likely that source leaves, but not young leaves, play a major role in Fe homeostasis by sensing the Fe levels at the whole plant level to systemically regulate Fe uptake in roots [START_REF] Mendoza-Cózatl | OPT3 Is a Component of the Iron-Signaling Network between Leaves and Roots and Misregulation of OPT3 Leads to an Over-Accumulation of Cadmium in Seeds[END_REF][START_REF] Zhai | OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis[END_REF][START_REF] Khan | Changes in iron availability in Arabidopsis are rapidly sensed in the leaf vasculature and impaired sensing leads to opposite transcriptional programs in leaves and roots[END_REF]. These observations, together with current -omic approaches at a tissue-and cell-specific resolution, offer a renewed opportunity to study Fe homeostasis from a systems level perspective, and may get us all closer -as a field -to a better understanding of the molecular mechanisms behind Fe sensing and Fe homeostasis in plants, at the whole plant level.

Materials and Methods

Plant growth conditions

Surface-sterilized seeds from Arabidopsis Col-0 were geminated on one-quarter Murashige and Skoog (MS) medium after stratification for 2-days in 4 o C/ dark condition. The light setting used throughout the study was 16 h light/ 8 h dark condition with 23°C day/ 21°C night, and the growth chamber humidity was maintained at 60 %.

When seedlings have two true leaves (10-12 days after seed plating), plants were transferred to a hydroponic setup with 50 µM Fe-EDTA as described in [START_REF] Nguyen | Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements[END_REF]. For the phenotypic test on Fe deficiency plates, minimal medium plates were prepared to have the same composition as the hydroponic solution but with 0.7 % phytoagar.

Fe-deficiency plates contained 0.15 mM of ferrozine (Fe chelator) and were not supplemented with Fe. For elemental analysis, roots and leaves were collected from hydroponically grown plants (18-day post transferring seedlings to hydroponic setup) while seeds, seed husk were collected from soil-grown plants. The soil used in this study was growing medium PRO-MIX FLX manufactured by Premier Horticulture LTD. Arabidopsis nas4x-1 mutants were kindly provided by Dr. Petra Bauer (Institute of Botany, Heinrich-Heine University).

Functional complementation assay in yeast

Saccharomyces cerevisiae fet3 fet4, zrt1 zrt2 and smf1 (kindly provided by Dr. David Eide, University of Wisconsin-Madison) were transformed with pYES2 or pAG426GPD (Addgene # 14156) plasmids carrying the coding sequence of either AtZIP5 or AtIRT1 via the lithium acetate method [START_REF] Schiestl | High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier[END_REF].

Transformants were grown in selective media and the presence of the plasmid was verified by standard yeast-colony PCR. Yeast growth complementation assays were performed on selective media (Sunrise Science Products, TN, USA) lacking Fe (Cat# 1551-250), Zn (Cat# 1555-250) or Mn (Cat# 1539-250). In experiments were pYES2 plasmids were used, glucose was replaced by 2% galactose and 1% raffinose to activate the GAL1 promoter, which drives the expression of the transgenes. To determine the Fe concentration in yeast cells, yeast were grown overnight in liquid cultures (-Fe, no iron added, or + Fe, supplemented with 100 µM of FeCl 3 ). Yeast cells were harvested and washed for one time with wash buffer (Tris 20 mM, EDTA 5mM, pH 8) and three times with DI water and brought to constant weight in an oven at 60 ˚C. Dehydrated cells were used for elemental analysis (ICP-OES) to quantify the micronutrient concentrations (Fe, Mn, Zn, Cu) and normalized by dry weight. Wildtype yeast and fet3 fet4 yeast transformed with IRT1 were used as positive controls.

Generation of Arabidopsis stable transgenic lines

To generate the SUC2p::AtZIP5 construct, the AtZIP5 coding sequence was amplified from cDNA of Col-0 plants using PCR and introduced into pENTR/D-TOPO plasmid (Gateway® entry clone) using the manufacturer recommendations. After the sequence was confirmed by Sanger sequencing, the AtZIP5 coding sequence was mobilized to a modified pGreen II 0179 plasmid where 2kb of the SUC2p promoter sequence had been inserted using the restriction enzymes KpnI/BamHI the Gateway cassette containing attLR sites (see Supplementary Table 1 for a detailed list of primers used).

To generate the ZIP5p::GUS construct, 2.5 kb genomic region upstream of ZIP5 transcription start site was amplified from gDNA from the Col-0 ecotype by PCR and introduced into pENTR/D-TOPO. ZIP5 promoter was later recombined into pBGGUS plasmid [START_REF] Kubo | Transcription switches for protoxylem and metaxylem vessel formation[END_REF] by an LR II clonase reaction.

Arabidopsis ecotype Col-0 plants were transformed independently with these constructs using the floral dip method as described in [START_REF] Clough | Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana[END_REF] using Agrobacterium tumefaciens strain GV3101, which carries the pSoup helper plasmid for pGreen II-carrying strains as described in [START_REF] Hellens | pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation[END_REF]. T3 homozygous lines were selected on hygromycin and T3 homozygous were identified by segregation analysis and transferred to soil for seed propagation. (ABRC stock# CD3-686, 35Sp::YFP-Gateway-OCS 3') using an LR reaction. The plasma membrane marker used in this study was a full-length fusion of the aquaporin PIP2A to mCherry that was obtained from the ABRC stock center [START_REF] Nelson | A multicolored set of in vivo organelle markers for colocalization studies in Arabidopsis and other plants[END_REF]. These constructs were transformed into A. tumefaciens strain GV3101 and used transient expression assaya. Leaves of N. benthamiana were co-infiltrated with YFP-AtZIP5 and PIP2A-mCherry as previously described in [START_REF] Wydro | Optimization of transient Agrobacterium-mediated gene expression system in leaves of Nicotiana benthamiana[END_REF]. 3-day post infiltration, the infiltrated area of the leaf was observed using a Leica TCP SP8 confocal microscope (60X magnification).

Transient expression in

Histochemical GUS staining

For GUS staining, leaves and roots of transgenic plants carrying proZIP5::GUS were grown on hydroponics for two weeks under Fe sufficient condition. GUS staining was performed overnight with 2 mM X-Gluc (5-bromo-4-chloro-3-indolyl-β-d-glucuronide) as described in [START_REF] Mendoza-Cózatl | OPT3 Is a Component of the Iron-Signaling Network between Leaves and Roots and Misregulation of OPT3 Leads to an Over-Accumulation of Cadmium in Seeds[END_REF]. Staining patterns were analyzed using Leica M205 stereomicroscope.

Elemental analysis

Inductively coupled plasma -optical emission spectrometry (ICP-OES) was used to determine the elemental composition in yeast cells and plant samples. The protocol was described in [START_REF] Chen | An improved grafting technique for mature Arabidopsis plants demonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis[END_REF]. Briefly, after collection, tissue samples were washed with buffer (Tris 20mM, EDTA 5mM, pH 8) and DI water to eliminate ions bound to the surface. Samples were then dried, weighed, and digested using HNO 3 70 %. Dilution of the digested samples in a homogeneous liquid phase (no debris) was brought to 10 mL and analyzed for Fe, Zn, Mn, and Cu concentrations by ICP-OES.

Gene expression analysis

Eighteen days after transferring to hydroponics, plants were moved to Fe deficient conditions. The Fesufficient hydroponic solution used in this study contains 50 µM of Fe-EDTA; Fe-deficient hydroponic solution had the same composition but without Fe. Roots of plants for Fe-deficiency treatment were gently washed with Fedeficient hydroponic solution for 15 minutes on a shaker before starting the actual treatment. The hydroponic solution used on the treatment day was prepared with Milli-Q water. Young leaves, mature leaves, and roots were collected separately as described in Figure S5. Plant tissues were collected for RNA extraction (Direct-zol™ RNA MiniPrep Plus kit, Zymo Research) and cDNA synthesis (SuperScript III® kit -Invitrogen). Gene expression analysis was performed by semi-quantitative PCR (RT-PCR) or quantitative real-time PCR (qRT-PCR) and normalized using AtACT2 (AT3G18780) expression as described in [START_REF] Khan | Changes in iron availability in Arabidopsis are rapidly sensed in the leaf vasculature and impaired sensing leads to opposite transcriptional programs in leaves and roots[END_REF]. Each biological replicate came from homogenized tissues of five to six plants. Results represent the mean of three biological replicates. Gene expression analysis data represent for three independent experiments.

Fe leaf-to-root transport assay

Hydroponically grown Col-0 and nas4x-1 plants were used in a leaf-to-root transport assay. A big mature leaf (loaded leaf) from each plant was incubated with 0.5 mL buffer solution containing 50 mM MES, 15 µM FeCl 3 (non-radioactive form), 1 mM ascorbic acid, 2 mM sucrose, and 20 µCi 59 FeCl 3 (pH~6). 2 hours after incubation, loaded leaves were carefully removed and washed one time with wash buffer (Tris 20mM, EDTA 5mM) and two times with DI water. Whole roots were removed from plants and gently placed on a tissue paper to remove the hydroponic solution. Fresh weight of the root samples was measured and recorded for normalization. 59 Fe activity in loaded leaves was measured using a dose calibrator. 59 Fe activity in root samples were measured using a liquid scintillation counter as described in [START_REF] Khan | Changes in iron availability in Arabidopsis are rapidly sensed in the leaf vasculature and impaired sensing leads to opposite transcriptional programs in leaves and roots[END_REF].

Synchrotron X-ray fluorescence (XRF) map of Fe distribution in leaves

The first (mature) and the sixth (young) leaves were harvested from 23-24 day-old plants grown hydroponically and leaves were placed in the wet chamber made between two layers of metal-free Kapton film. The spatial distribution of iron in hydrated leaf tissues was imaged via XRF at the F3 station at the Cornell High Energy Synchrotron Source (CHESS) as described in [START_REF] Kumar | Iron-Nicotianamine Transporters Are Required for Proper Long Distance Iron Signaling[END_REF].

  ) Moreover, nas4x-1 plants have less mobile Fe in the phloem, yet vascular-localized Fe deficiency markers such as OPT3, bHLH100, and FRO3 are constitutively repressed in mature leaves, where Fe is deposited around the vasculature. These results suggest that high levels of Fe outside companion cells can override what should have been an Fe deficiency response, triggered by low levels of Fe inside companion cells. It is therefore possible that a portion of the Fe sensing mechanism in mature leaves occurs on the apoplastic side of the vasculature.

  ); therefore, to better control the transcription of AtZIP5, we generated transgenic Arabidopsis plants expressing AtZIP5 under the control Downloaded from https://academic.oup.com/pcp/advance-article/doi/10.1093/pcp/pcac046/6564417 by INRA Avignon user on 25 April 2022

  Nicotiana benthamiana and fluorescence confocal imaging 35Sp::YFP-AtZIP5 fusion was obtained by recombining the AtZIP5 coding sequence into pEarleyGate 104

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. AtZIP5 is preferentially expressed in the vasculature and localized to the plasma membrane. (A-B) GUS staining of ZIP5p::GUS leaves indicate that AtZIP5 expression localizes to the vasculature tissue. (C-E) Transient expression of YFP-AtZIP5 under the control of CaMV 35S promoter in tobacco cells co-infiltrated with the plasma membrane maker PIP2A-mCherry, three-day post infiltration. (E) Overlay image shows AtZIP5 localized to the plasma membrane of the cells. (C) mCherry channel, (D) YFP channel, (E) overlay of C and D, (F) Fluorescence intensity plot showing two peaks corresponding to the 2 plasma membranes of neighboring cells. Overlapping peaks in the two channels indicate colocalization of YFP-ZIP5 with the plasma membrane marker PIP2A-mCherry.

Figure 3 .

 3 Figure 3. Overexpression of AtZIP5 in companion cells promotes increased sensitivity to Fe limitation (A) 10 day-old seedlings of ZIP5ox 16-10 and ZIP5ox 25-5 showed shorter roots compared to WT when grown in Fe deficient media (+Fe, 50 µM Fe-EDTA; Fe, no Fe with 0.15 mM of ferrozine). (B) Quantification of root growth of ZIP5ox plants grown on Fe deficiency plates (n = 6-10 plants; mean ± S.E.). Experiment was repeated three times with similar results. Asterisks indicate a significant difference with a p-value < 0.05.

Figure 4 .

 4 Figure 4. Altered Fe deficiency responses in ZIP5ox lines. (A-C) Relative expression Fe deficiency markers in ZIP5ox lines compared to wildtype . (D) Relative expression of root-specific makers for Fe deficiency responses. 4week-old plants (12 days grown on one-quarter MS medium plates and 18 days grown on hydroponics under Fe

Figure 5 .

 5 Figure 5. Fe precipitation in the leaf vasculature of nas4x-1 results in reduced shoot-to-root Fe transport. (A) Synchrotron X-ray imaging shows that mature leaves of nas4x-1 had a higher signal of Fe around the vasculature compared to Col-0 and ZIP5ox lines. (B) Pixel intensity of XRF images extracted with the 3D surface plot package of Image J shows consistent lower Fe signal in ZIP5ox lines. (C) The lower Fe signal in ZIP5ox lines is more evident in the veins near the edge of the leaf. (D) Leaf-to-root transport assay of radioactive 59 Fe indicated that nas4x-1 transported less Fe downward the roots under Fe sufficient condition. Data show mean ± SE of 5 biological replicates. Asterisks indicate a significant difference with a p-value < 0.05. T-tests were performed using the Minitab statistical package.

Figure 6 .

 6 Figure 6. nas4x-1 exhibits a constitutive Fe deficiency responses in sink tissues. (A-C) Relative expression of Fe deficiency markers in nas4x-1 (D) Relative expression of root specific Fe deficiency makers in nas4x-1. Plant growth conditions and tissue collection were described in Figure 4 and methods section. Data represent three independent experiments, each with three biological replicates. Data show mean ± SE; asterisks indicate significant difference with a p-value < 0.05. T-tests were performed using the Minitab statistical.

Figure 1 Figure 2

 12 Figure 1

Figure 3

 3 Figure 3

Figure 4

 4 Figure 4

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Funding information

This

research was supported in part by the US National Science Foundation (MCB-1818312 and IOS-1734145 to D. M. C.). Radiotracer experiments using 59 Fe were supported through an NSF EPSCoR Track II award (IIA-1430428 to D. G. M-C. and S. S. J.) and by a grant from the Department of Energy (Projects for Interrogations of Biological Systems, DE-SC0002040 to S. S. J.). N. T. N. was supported by the Vietnam Education Foundation Training Program (Exchange visitor program No. G-3-10180). Parts of this research used the F3 Beamline at the Cornell High Energy Synchrotron Source (CHESS) which during the period of research was supported by the National Science Foundation under award DMR-1332208. Parts of this work in the O.K.V. lab was funded by NSF-IOS #1754966 and NSF-IOS #1656321.

ORCID

//www.arabidopsis.org/.

Data Availability

Information about Arabidopsis thaliana genes and proteins that are directly relevant to this study, including ZIP5 (AT1G05300), SUC2 (AT1G22710), NAS1 (AT5G04950), NAS2 (AT5G56080), NAS3 (AT1G09240), NAS4 (AT1G56430), OPT3 (AT4G16370), bHLH100 (AT2G41240), FRO3 (AT1G23020), FRO2 (AT1G01580), and IRT1 (AT4G19690) can be retrieved from The Arabidopsis Information Resource (TAIR) at https://www.arabidopsis.org/. Visualization of whole-genome expression profiling datasets of Arabidopsis wildtype and opt3-2 are available at http://artemis.cyverse.org/david_lab_efp/cgi-bin/efpWeb.cgi

David G. Mendoza-Cozatl http://orcid.org/0000-0002-9616-0791 Nga T. Nguyen https://orcid.org/0000-0003-1016-3068 Olena K. Vatamaniuk: https://0000-0003-2713-3797


Seyyed Mohsen Beladi-Mousavi

Gerardo Salinas

Nikolas Antonatos

Vlastimil Mazanek

Patrick Garrigue

Zdeněk Sofer

Alexander Kuhn
email: kuhn@enscbp.fr

Fine-Tuning the Functionality of Reduced Graphene Oxide via Bipolar Electrochemistry in Freestanding 2D Reaction Layers

Keywords: reduced graphene oxide, bipolar electrochemistry, energy storage, energy conversion, HER, wireless electrochemistry

Graphene has unique characteristics that are appealing for energy-related applications such as ultra-lightweight and high surface-area/electrical-conductivity. However, generating functional graphene sheets is still a very challenging task. Here, a novel approach based on an original bipolar electrochemistry set-up, using a quasi-2D reaction layer, is suggested, which allows a precise control of dispersibility and conductivity of graphene sheets. In this system, a freestanding 2D layer of aqueous solution, containing 2D graphene oxide (GO) sheets, is placed between two platinum feeder electrodes, which are used to apply an electric field. As a result, the GO sheets experience a sufficiently high polarization to cause their transformation into reduced GO (rGO). The degree of reduction can be readily controlled by the field strength and exposure time, resulting in a wide range of rGO with different conductivity/dispersibility features. The partially reduced GO (prGO) sheets with engineered conductivity/dispersibility are used to prepare aqueous composites with a redox-polymer for organic battery applications. Additonally, at higher potentials, Pt nanoparticles are released from the feeder electrodes and attached to rGO sheets. The sheets were used for catalyzing hydrogen evolution reaction with a performance comparable to bulk Pt.

Introduction

Since the discovery of graphene in 2004, this type of two-dimensional (2D) materials have become a cornerstone of contemporary materials science. [START_REF] Novoselov | Electric Field Effect in Atomically Thin Carbon Films[END_REF][START_REF] Meyer | The structure of suspended graphene sheets[END_REF][START_REF] Huang | The Chemistry and Promising Applications of Graphene and Porous Graphene Materials[END_REF][START_REF] Beladi-Mousavi | Atomically Thin 2D-Arsenene by Liquid-Phased Exfoliation: Toward Selective Vapor Sensing[END_REF][START_REF] Li | 2D carbide MXene Ti2CTX as a novel highperformance electromagnetic interference shielding material[END_REF] This is due to their unique mechanical, electrical, thermal, and optical properties, impacting research in different fields such as electronics, energy, biomedicine, and sensing. [START_REF] Stoller | Graphene-Based Ultracapacitors[END_REF][START_REF] Ma | Giant intrinsic photoresponse in pristine graphene[END_REF][START_REF] Reina | Promises, facts and challenges for graphene in biomedical applications[END_REF][START_REF] Banerjee | Slowing DNA Transport Using Graphene-DNA Interactions[END_REF][START_REF] Khan | Graphene based metal and metal oxide nanocomposites: synthesis, properties and their applications[END_REF][START_REF] Nguyen | Doping mechanism directed graphene applications for energy conversion and storage[END_REF] Among suggested approaches for the preparation of graphene, the transformation of graphene oxide (GO) to reduced graphene oxide (rGO) is considered as a promising, commercially feasible technique. [START_REF] Pei | The reduction of graphene oxide[END_REF] GO is a carbon-based nanomaterial in the form of atomically thin 2D sheets, bearing oxygen functions including carboxyl, hydroxyl, or epoxy groups. [START_REF] Dreyer | Harnessing the chemistry of graphene oxide[END_REF][START_REF] Bonanni | On Oxygen-Containing Groups in Chemically Modified Graphenes[END_REF] In contrast to graphene, GO is wellsolubilized in water and a wide range of organic solvents, thus allowing (i) facile fabrication of uniform films on different substrates and networks, [START_REF] Zhu | New Routes to Graphene, Graphene Oxide and Their Related Applications[END_REF][START_REF] Zhu | Graphene and Graphene Oxide: Synthesis, Properties, and Applications[END_REF] and (ii) precise engineering of composite materials in solutions with molecular-level interactions. [START_REF] Beladi-Mousavi | The Metallocene Battery: Ultrafast Electron Transfer Self Exchange Rate Accompanied by a Harmonic Height Breathing[END_REF][START_REF] Beladi-Mousavi | High Performance Poly(viologen)-Graphene Nanocomposite Battery Materials with Puff Paste Architecture[END_REF][START_REF] Beladi-Mousavi | Poly(vinylferrocene)-Reduced Graphene Oxide as a High Power/High Capacity Cathodic Battery Material[END_REF][START_REF] Ambrosi | Graphene and its electrochemistry -an update[END_REF] Such properties place this 2D material in a unique position for different applications, particularly energyrelated devices. [START_REF] Radich | Origin of Reduced Graphene Oxide Enhancements in Electrochemical Energy Storage[END_REF] However, GO is electrically insulating and zero-band gap graphene is obtained only when sp 2 lattices are regenerated.

Several methods including chemical, thermal and electrochemical techniques have been suggested to transform GO into rGO and partially restore the structure and characteristics of graphene. [START_REF] Pei | The reduction of graphene oxide[END_REF] Electrochemical techniques often outperform other systems in terms of toxicity and energy consumption. Typically, a film of GO (or its composites) is initially deposited on an electrode, followed by applying a reductive potential to trigger the GO → rGO transformation. This technique faces three serious limitations: (i) the efficient reduction is only possible if the GO film is moderately thin or highly porous; (ii) an efficient electrochemical reduction often causes significant changes in the morphology of the film, which could largely influence the electrochemical behaviour and the stability of the film; (iii) this method is limited to GO samples that are already deposited on the electrode and it is difficult to isolate the obtained rGO sheets.

Here, we demonstrate an alternative approach to reduce GO sheets dispersed in a 2D solution phase based on bipolar electrochemistry (BPE). This contact-less approach has already been successfully applied on single or double layers of graphene (80×70 mm 2 , attached to a substrate), to precisely modify its peripheries by one or two different metals. [START_REF] Zuccaro | Selective Functionalization of Graphene Peripheries by using Bipolar Electrochemistry[END_REF] However, this system was limited to large graphene sheets deposited on a surface and cannot be used for reduction of GO micro/nanosheets.

In this contribution, we designed a novel BPE setup consisting of a quasi-2D reaction environment to benefit from the aforementioned advantages of electrochemical reduction systems, while avoiding the limitations. BPE, in contrast to conventional electrochemistry, is not using a classic working electrode (WE) that is connected to a power source. It is a wireless system, where the reaction occurs on the desired object (GO as the bipolar electrode in the current study), that is placed between two feeder electrodes without any direct contact with them (Fig. 1a). Upon application of an electric field, the conductive/semiconductive object gets polarized with respect to the surrounding solution, generating a polarization potential gradient along the length of the object. [START_REF] Bouffier | Bipolar (Bio)electroanalysis[END_REF][START_REF] Fosdick | Bipolar Electrochemistry[END_REF][START_REF] Koefoed | Bipolar electrochemistry-A wireless approach for electrode reactions[END_REF][START_REF] Shida | Bipolar Electrochemistry: A Powerful Tool for Electrifying Functional Material Synthesis[END_REF] The two ends of the object experience the highest cathodic/anodic polarization (with an opposite polarity compared to the feeder electrodes). The polarization potential that is established at the extremities of the object, i.e. GO, is responsible for the reductive GO/rGO transformation, following a similar mechanism as for the conventional reduction of GO.

In the current study, the as-prepared rGO sheets are still well-dispersed in the solution.

Notably, we have used a new setup based on a thin 2D reaction layer, in which the 2D bipolar electrodes are trapped in the freestanding layer. This is designed to overcome challenging limitations of conventional 3D BPE setups i.e. the precipitation and/or the random orientation of bipolar electrodes during the reaction and hence unequal and/or short exposure of bipolar electrodes to the desired potential gradient. [START_REF] Roche | Straight-forward synthesis of ringed particles[END_REF][START_REF] Loget | True Bulk Synthesis of Janus Objects by Bipolar Electrochemistry[END_REF][START_REF] Sentic | 3D electrogenerated chemiluminescence: from surface-confined reactions to bulk emission[END_REF] This issue is normally addressed by preparing highly viscous systems e.g., gels, which is often limiting due to the difficulty of dispersing bipolar electrodes in the gel and their extraction after the reaction, as well as the difficulty of the incorporation of the desired salts (e.g. for metal deposition) into the gel structure. [START_REF] Roche | Straight-forward synthesis of ringed particles[END_REF][START_REF] Loget | True Bulk Synthesis of Janus Objects by Bipolar Electrochemistry[END_REF][START_REF] Sentic | 3D electrogenerated chemiluminescence: from surface-confined reactions to bulk emission[END_REF]

Methods

Materials: H 2 SO 4 , NaCl, KCl, Na 2 HPO 4 , KH 2 PO 4 and glycerol were purchased from Sigma-Aldrich.

Polyviologen (PV) was prepared following a method described in a previous study. [START_REF] Beladi-Mousavi | High Performance Poly(viologen)-Graphene Nanocomposite Battery Materials with Puff Paste Architecture[END_REF] GO was prepared based on the Hofmann method. [START_REF] Hofmann | Die Reduktion von Graphitoxyd mit Schwefelwasserstoff[END_REF] Reduction of GO via bipolar electrochemistry: A homemade quasi-2D bipolar electrochemical setup as shown in Fig. 1a with dimensions of 0.6×0.4×0.1 cm was built to prepare prGO and rGO. Two Pt wires (diameter: 0.25 mm) were used as feeder electrodes. In front of each feeder electrode, a membrane was placed to prevent a direct contact between the graphene sheets and the electrodes. Feeder electrodes were connected to a power source (Heinzinger PNC 10000-200 pos) via wrapping wires (Kynar). The GO solution was prepared by mixing 0.4 ml PBS buffer (pH: 7.4), containing 1.5 mg ml -1 GO, with 0.7 ml glycerol followed by 30 min mechanical mixing and 5 min sonication. 60 µl of the obtained solution was suspended as a free-standing quasi-2D film between the two feeder electrodes in the bipolar setup using a micropipette.

X-ray diffraction (XRD): XRD patterns were acquired using a Bruker D8 Discoverer powder diffractometer (Bruker, Germany) in Bragg-Brentano parafocusing geometry and applying Cu K α radiation (λ = 0.15418 nm, U = 40 kV, I =40 mA). The diffraction patterns were collected between 5 o and 90 o of 2θ with a step size of 0.020 o and the acquired data were evaluated using HighScore Plus 3.0e.

High-resolution transmission electron microscopy (HR-TEM): HR-TEM was performed with

an EFTEM Jeol 2200 FS microscope (Jeol, Japan). A 200 keV acceleration voltage was used for measurement. Elemental maps were recorded with a SDD detector X-Max N 80 TS (Oxford Instruments, England). All samples were put on a carbon TEM grid (Ted Pella Inc.) prior to the measurements.

X-ray photoelectron spectroscopy (XPS): XPS measurements were performed in an SPESC

XPS spectrometer with Phoibos 150 hemispherical energy analyser (Specs, Germany) employing a monochromatic aluminum X-ray radiation source (1486.7 eV). Wide-scan surveys of all elements were performed with subsequent high-resolution scans of carbon (C 1s) and oxygen (O 1s). The samples were placed on a Si wafer. For elimination of the sample charging during measurement, an electron gun was utilized (1-5 V). All XPS survey spectra were afterwards analyzed by CasaXPS software. SEM/EDX: SEM coupled with EDS mapping was performed with a Lyra3 GM Tescan microscope. The samples were coated on conductive carbon by drop-casting and drying at 50 ºC for 1h. The measurement was performed using 20 kV acceleration voltage.

Electrical properties:

The conductivity measurements were performed by drop-casting of 5 µl GO solution on interdigital microarray electrodes (dimension: 1cm × 0.5cm, featuring separated 10 µm Pt bands deposited on it), followed by drying at 50 ºC for 3h. The resistance between electrodes was measured using a multimeter.

UV-Vis:

The measurements were performed using Cary 100 UV-Visible Spectrophotometer.

The samples were kept in a dark container between measurements.

Energy storage and conversion:

The electrochemical measurements were performed using a Palmsens4 potentiostat interfaced with a computer with PSTrace software using a threeelectrode system. A 3/1 PV/p(rGO) solution was prepared by mixing 0.2 ml of freshly sonicated (5 min) (p)rGO solution (containing 0.1 mg (p)rGO) with 0.8 ml of PV solution (containing 0.3 mg PV, without considering counterions). After mixing, the prGO composite was stirred for 10 min, while the rGO solution was sonicated for 1h. CV tests were performed using 0.1 KCl solution as an electrolyte, a Pt wire as a counter electrode (CE), Ag/AgCl as reference electrode (RE), and glassy carbon decorated with PV and PV composites as working electrode (WE). WEs were decorated by drop-casting the active materials and drying at 50 ºC for 1h. HER was performed using a three-electrode configuration with 0.5 M H 2 SO 4 as the electrolyte (degassed with argon for 20 min before measurements), Ag/AgCl as RE, a graphite rod as CE, and a glassy carbon electrode decorated with GO and reduced GO as WE.

WEs were prepared via drop-casting of the suspensions followed by drying at 50 ºC for 1h.

Results and Discussion

In the proposed 2D system (Fig. 1a), the reductive reaction on GO sheets as bipolar electrodes are occurring in an ultrathin aqueous layer positioned between two feeder electrodes. The degree of the reduction of GO and thus the conductivity of the resulting partially or totally reduced GO (prGO or rGO) sheets can be precisely controlled by changing the applied potential and the exposure time. In contrast to the conductivity, the dispersibility of the sheets decreases when increasing the reduction state, due to the removal of the oxygen-containing functional groups (Fig. 1b). We have been able to synthesize prGO, which is still welldispersed, but already provides sufficient electrical conductivity as a conductive filler in a composite with redox-active organic polymer (polyviologen), leading to a similar power density as with completely reduced rGO. Furthermore, it was observed that the rGO produced at high voltages contains an extremely low amount of platinum (Pt) nanoparticles (< 1%) released from the platinum feeder electrodes, which can be advantageously used for electrocatalytic reactions. rGO samples with high electrical conductivity, decorated with Pt, were used for studying hydrogen evolution reaction (HER). Remarkably, the electrodes with a C/O ratio ~ 6 showed an overpotential comparable to pure Pt metal (Fig. 1b). These different aspects are discussed one by one in more detail in the following sections. with increasing time and potential of GO reduction. Partially reduced GO (prGO) can be used as dispersible conductive filler to facilitate composite preparation in the solution, while the highly reduced GO (rGO) is decorated with an extremely low amount of Pt (released from Pt feeder electrodes) that can be used for catalysis of electrochemical hydrogen evolution (HER).

GO → prGO → rGO transformation via bipolar electrochemistry

The wireless electrochemical reduction using a bipolar electrochemical setup (Fig. 1a) was performed with free-standing films of aqueous solutions containing GO by applying different potentials [START_REF] Khan | Graphene based metal and metal oxide nanocomposites: synthesis, properties and their applications[END_REF][START_REF] Ambrosi | Graphene and its electrochemistry -an update[END_REF][START_REF] Song | Polymer-Graphene Nanocomposites as Ultrafast-Charge and -Discharge Cathodes for Rechargeable Lithium Batteries[END_REF], 60, and 80V) for 4 min. The novel BPE set-up using a thin reaction space is suggested to better control the dispersibility, and conductivity of the sheets. In this setup, 2D sheets of graphene oxide are placed in a freestanding 2D water-based electrolyte film, which inhibits the precipitation or random orientation of the bipolar objects. Hence, the sheets are homogeneously treated with a constant polarisation potential during the reaction time. This allows a more precise control of the properties of the sheets by varying the potential and the reaction time. Two membranes were placed in front of the Pt feeder electrodes to inhibit direct connection between GO sheets and the electrodes during the experiment. The obtained (p)rGO was washed several times with water and then drop cast on an interdigitated electrode to measure their conductivity (Fig. 2a, -•-blue). A slight increase in the conductivity of the sheets was observed upon increasing the potential from 10 to 40 V, however, a jump in the conductivity of the sheets was observed at 60 V, which was further enhanced at 80 V. We have noticed that at lower concentrations of GO (<5 times, see experimental section), no GO → rGO transformation was observed. This is because the potential range applied in this work does not match the minimum potential necessary (~ 1 kV) to reduce individual small GO particles (5 -10 μm) in the proposed setup. It should be noted that the current setup is designed to reduce the bypass current leading to a more energy efficient GO/rGO transformation, while improving the precipitation problem of the bipolar objects. However, a similar concept can be extrapolated to a more conventional setup if the applied potential and the composition of the electrolyte solution are adjusted.

The successful GO reduction in this study is attributed to the formation of larger colloids of GO as a result of the interconnection of GO sheets at higher concentrations. Their formation is also related to the GO samples used in this work (prepared by the Hofmann method) with a low oxidation degree (32% oxygen content) and hence low solubility (20% precipitation after 10 min in the electrolyte solution). [START_REF] Hofmann | Die Reduktion von Graphitoxyd mit Schwefelwasserstoff[END_REF] Typically, BPE is performed in a highly viscous environment to avoid the rotation of bipolar objects and perform side selective reactions. However, we have chosen a different approach by performing reactions in a freestanding thin layer that is only viscous enough to be stable upon applying the potential, but not too viscous to avoid the free movement of particles. Hence, the bipolar objects are randomly moving in the plane of the liquid layer with an orientation parallel to the liquid film. This enables them to form dynamic 2D clusters. These dynamic clusters of GO have a size which allows establishing a polarization zone within the cluster that is large enough to put entire GO sheets, being located at the  -extremity of the cluster, on a sufficiently negative potential to reduce the oxygen functionalities. As the assembly is dynamic they can be replaced subsequently by not yet reduced GO sheets, until statistically all sheets have undergone a reduction.

Reoxidation at the opposite extremity of the cluster is not occurring, because water oxidation is easier than oxidation of graphene.

The photos of the GO solutions reduced at different potentials are presented in Fig. 2b. The color of the solution changes from light brown to dark brown and finally black indicating the transformation of GO to prGO and finally rGO. Note that each of these solutions is the mixture of 10 individual experiments and the reported resistances are average values.

As a control experiment, hydroquinone (HQ) was added to the GO solution and a similar analysis was performed (Fig. 2a, -■-red). It is expected that the addition of this redox-active organic molecule facilitates GO reduction because HQ has a lower oxidation potential in comparison to water. [START_REF] Loget | Electric field-induced chemical locomotion of conducting objects[END_REF] In this case, the resistance values and the corresponding color already change at 10 V, due to the lower required driving force.

Structural analysis

The crystalline structure of the GO sample before, during (at 40 V, i.e., prGO), and after electrochemical reduction (at 100 V, i.e., rGO) was measured via X-ray powder diffraction (XRD) analysis (Fig. 3a). GO shows the characteristic carbon peak (001) at 2θ = 10.9° corresponding to a d-spacing of 0.81 nm, while prGO and rGO show broad peaks due to deterioration of crystallinity of GO sheets with higher values, indicating a smaller layer distance after the GO/rGO transformation due to the removal of functional oxygen groups. [START_REF] Beladi-Mousavi | High Performance Poly(viologen)-Graphene Nanocomposite Battery Materials with Puff Paste Architecture[END_REF] In addition, high-resolution transmission electron microscopy (HR-TEM) and selected area electron diffraction (SAED) were performed on a typical lacey carbon substrate to characterize the crystallinity of a representative selection of the obtained sheets (Fig. 3b and S1). The HR-TEM image and energy dispersive X-ray spectroscopy (EDX) map of rGO appears to be highly electron transparent. The sheets are slightly wrinkled and counting the periodic stripped edges of sheets confirmed < 3 layers of rGO partially covering each other. [START_REF] Navik | Curcumin-assisted ultrasound exfoliation of graphite to graphene in ethanol[END_REF] The SAED pattern (Fig. 3b, inset) shows a hexagonal array of diffraction peaks, in agreement with the crystal structure of graphene sheets. The spots are labeled via (hkl) Miller-Bravais notation. [START_REF] Wilson | Graphene Oxide: Structural Analysis and Application as a Highly Transparent Support for Electron Microscopy[END_REF] X-ray photoelectron spectroscopy (XPS) of GO at different oxidation states was performed to obtain more details about the elimination of oxygencontaining functional groups during the wireless electrochemical reduction process. As shown in Fig. 3c, the C 1s spectrum of GO exhibits peaks at 284.5 (C=C), 285.4 (C-C), 286.3 (C-O), 287.9 (C=O), and 290.9 (O-C=O) eV, attributed to the sp 2 carbon, sp 3 carbon, the epoxy, carbonyl, and carboxyl functional groups, respectively. After electrochemical treatment of the sample at 40 V for 4 min (prGO), the peak intensity of oxygen-containing functional groups significantly decreases, while a higher intensity of sp 3 carbon compared to sp 2 carbon is still observed, indicating removal of oxygen groups and uncompleted regeneration of sp 2 carbons. This is attributed to the removal of the oxygen-containing functional groups and a partial regeneration of the conjugated π system. Applying a higher potential, i.e. 100 V (rGO), results in a significant enhancement of the sp 2 carbon (C=C) peak, in comparison to all other peaks, indicating an efficient GO → rGO transformation. [START_REF] Park | Colloidal Suspensions of Highly Reduced Graphene Oxide in a Wide Variety of Organic Solvents[END_REF]

Conductivity and dispersibility

The conductivity and the carbon/oxygen (C/O) ratio of GO samples after electrochemical treatments at different potentials (20, 40, 60 80, 100 V) and treatment times (1, 2 4 min) were measured (Fig. 4). The resistance and C/O ratio values are the average of 10 independent experiments. The GO suspensions were washed several times with distilled water before all analyses. As shown in Fig. 4a, applying a potential decreases the resistance of the samples, which is in agreement with the gradual color changes from brown to black as a function of the applied potential (Fig. S2). The resistance of samples significantly decreases at 40 V, particularly for the 4 min experiment, which appears to be a critical potential for the proposed wireless reduction of GO via bipolar electrochemistry. Such intermediate phase shifts in the conductivity are also observed in other reduction mechanisms, which is attributed to the growth of enough graphitic domains to allow efficient percolative electronic transport. [START_REF] Lipatov | Lowtemperature thermal reduction of graphene oxide: In situ correlative structural, thermal desorption, and electrical transport measurements[END_REF] At higher potentials (60, 80, and 100 V), even lower resistances down to 7 Ω cm -2 (4 min, 100V) were recorded, however, the relative resistance changes beyond 40 V were smaller. In order to better understand the origin of these changes, the ratio between the atomic contents of carbon and oxygen in samples produced at different potentials was measured via SEM/EDX (Fig. S3-S4). In samples with 1 or 2 min treatments, a steady increase of the C/O ratio with a small jump at 80 V is observed, while in the case of 4 min treatment, a much higher jump is recorded (Fig. 4b). This C/O ratio phase shift, which occurs at a higher potential than the conductivity phase shift is attributed to the massive removal of oxygen-containing functional groups. [START_REF] Lipatov | Lowtemperature thermal reduction of graphene oxide: In situ correlative structural, thermal desorption, and electrical transport measurements[END_REF] Remarkably, the final C/O ratio in the samples with 4 min electrochemical treatment (Fig. 4b) is significantly higher than for other samples, which is in agreement with their conductivity. Note that, while other GO reduction methods, e.g. thermal treatment, might result in C/O ratios > 8 (reduction at > 500 ºC), the electrochemical approach is more economical in terms of energy efficiency and the electrical conductivity achieved via both systems is comparable even if the C/O ratios obtained with the proposed method are lower [START_REF] Pei | The reduction of graphene oxide[END_REF]. As for the purpose of this work the critical factor is the electrical conductivity, the electrochemical approach appears to be a satisfying alternative. Subsequently, the dispersibility of the obtained prGO and rGO in an aqueous solution is compared with the original GO suspension. This is important regarding the preparation of hierarchical nanostructures and macroscopic composite materials via supramolecular selfassembly mechanisms to generate new properties and functionalities for various technological applications such as (opto)electronics, catalysis and energy conversion. [START_REF] Beladi-Mousavi | Poly(vinylferrocene)-Reduced Graphene Oxide as a High Power/High Capacity Cathodic Battery Material[END_REF][START_REF] Yuan | Self-Assembled Graphene-Based Architectures and Their Applications[END_REF] The stability of samples in distilled water is evaluated by their UV adsorption at different time intervals (Fig. S5). As shown in Fig. 5a, pristine GO solutions showed the lowest precipitation after 48 h, compared to GO solutions that were electrochemically treated for 4 min (see Fig. S6 for 1 and 2 min treatments). It is evident that increasing the potential results in the formation of prGO and rGO suspensions that are less stable in water. This is explained by the removal of their oxygen-containing functional groups (Fig. 3), resulting in lower solubility in polar solvents. [START_REF] Beladi-Mousavi | The Metallocene Battery: Ultrafast Electron Transfer Self Exchange Rate Accompanied by a Harmonic Height Breathing[END_REF][START_REF] Shao | Facile and controllable electrochemical reduction of graphene oxide and its applications[END_REF] The dispersibility is also studied as a function of the duration of the electrochemical treatment at different potentials (Fig. 5b, and S5). While in all cases lower dispersibility is observed for higher potentials, longer treatments i.e., 4 min resulted in even lower dispersibility. Notably, a considerable change in the dispersibility of prGO was observed at 40 V (Fig. 5b, red), which correlates well with the enhanced conductivity observed at the same potential (Fig. 4a,red). their UV absorption with standard deviations of 4.3, 5.6, 13.9, 13.5, 14.9, and 18.9, respectively; the samples were washed and sonicated before this measurement. b) Dispersibility after 48h of pristine GO electrochemically treated for 1, 2, and 4 min at different potentials [START_REF] Ambrosi | Graphene and its electrochemistry -an update[END_REF][START_REF] Song | Polymer-Graphene Nanocomposites as Ultrafast-Charge and -Discharge Cathodes for Rechargeable Lithium Batteries[END_REF]60. 80 and 100 V).

Applications

Energy storage

Single sheets of graphene are ideal fillers to provide both electrical and ionic conductivity in battery materials. [START_REF] Ju | Unveiling the dimensionality effect of conductive fillers in thick battery electrodes for high-energy storage systems[END_REF] This is due to graphene's high electrical conductivity and the lower amount of required conductive fillers if single sheets of graphene are efficiently distributed among active materials. [START_REF] Song | Polymer-Graphene Nanocomposites as Ultrafast-Charge and -Discharge Cathodes for Rechargeable Lithium Batteries[END_REF] However, the low solubility and immediate restacking of graphene sheets after the exfoliation process make them impractical for large-scale applications. Hence, in many studies, GO is initially used as the filler, which is followed by a thermal GO → rGO transformation step of the final electrode. Nevertheless, this is only possible if the active material can tolerate temperatures above 350 °C. [START_REF] Sengupta | Thermal reduction of graphene oxide: How temperature influences purity[END_REF] Electrochemical methods are also suggested to reduce GO at the surface of electrodes via applying cathodic potentials. [START_REF] Viinikanoja | Electrochemical reduction of graphene oxide and its in situ spectroelectrochemical characterization[END_REF] Nonetheless, GO is electrically insulating, and electrochemical reduction is only efficient for thin layers. In the current study, composites of a redox-active organic polymer i.e., polyviologen (PV) and prGO (reduced at 40V) or rGO (reduced at 100V) are prepared via mixing of their aqueous solutions with a PV/(p)rGO ratio of 3. PV is a dicationic polymer (PV ++ ), which can be reversibly reduced to the radical cation state at cathodic potentials (Fig. 6a). As shown in our previous studies, a self-assembly between PV ++ and fillers (GO/rGO) via electrostatic and/or π-π interaction is expected. [START_REF] Beladi-Mousavi | High Performance Poly(viologen)-Graphene Nanocomposite Battery Materials with Puff Paste Architecture[END_REF] The obtained composites are drop cast on current collectors and the electrodes are charged and discharged at different scan rates while discharge capacities are recorded. Cyclic voltammetry (CV) curves at 20 mV s -1 , exhibiting a redox signal at ~ -0.3V (vs. Ag/AgCl) are shown in Fig. 6b (see other scan rates in Fig. S7). The reduction current has a lower peak value, but the peak is very broad, while the oxidation current exhibits a higher and sharper peak. However, integration of both peaks leads to very similar values, indicating that the charge transfer process is completely reversible. The areal capacity of the PV alone or PV composite with GO is significantly lower than the PV composites with electrically conductive prGO and rGO.

Remarkably, the capacity of composites in the presence of prGO or rGO are very similar. This demonstrates the efficiency of prGO to provide enough electrical conductivity in the battery materials, while this filler is much more dispersible in aqueous solutions compared to rGO samples. This becomes even more evident when taking into account the easy composite preparation procedure by simple mixing of prGO and PV, while the rGO composite needs a harsh time-consuming sonication shortly before experiments for a similar performance. A comparable trend in the behaviour of the composites is observed at different scan rates (Fig. 6c). mV cm -2 . c) The areal capacity (mC cm -2 ) of PV, PV-GO, PV-prGO (40V), and PV-rGO (100V) at different scan rates [START_REF] Khan | Graphene based metal and metal oxide nanocomposites: synthesis, properties and their applications[END_REF][START_REF] Ambrosi | Graphene and its electrochemistry -an update[END_REF]50,75, and 100 mV s -1 ) obtained from the discharge curves of their CVs. Ag/AgCl reference electrode, Pt wire counter electrode, glassy carbon decorated with PV, and PV composites as the working electrode.

Electrocatalysis

Pt is widely used in electrolyzers for water splitting due to its excellent catalytic characteristics, however, the high price and limited resources of Pt are seriously limiting factors. Various materials e.g., transition metal oxides are proposed to replace Pt, however, these materials can barely provide comparable activities and are often produced through expensive and time-consuming procedures. An attractive alternative method could be to elaborate electrodes using extremely low quantities of Pt while keeping its electrocatalytic performance. The wireless electrochemical reduction of GO proposed in this study involves two Pt feeder electrodes. It should be noted that in principle these Pt electrodes can be replaced by other appropriate materials such as carbon. However, in the present work we used Pt on purpose in order to verify whether it might be possible to trigger the release of extremely small quantities of this noble metal from the feeder electrodes, which then can act as catalytically active modifiers of the rGO sheets. HR-TEM of rGO reduced at 100 V indicates the presence of micro/nano-sized Pt particles (down to 0.6 wt%), mapped by the corresponding EDX analysis (Fig. 7a and S8-S9). The obtained rGO/Pt samples were washed with distilled water and drop cast on a glassy carbon current collector and their performance for HER was assessed in 0.5 M H 2 SO 4 . The linear sweep voltammetry (LSV) curves of rGO, produced at different potentials (20-100V), are compared with the LSV of pristine GO and a bulk Pt electrode (Fig. 7b). It should be noted that the currents are normalized with respect to the geometric surface area of the electrodes. Overpotential values at a current density of 10 mA cm -2 are compared for the different samples (Fig. 7c). Significantly lower overpotential values were recorded for samples that were synthesized at higher potentials in the bipolar modification experiments. However, a significant jump in the overpotential value was observed at 80 V. This is attributed to (i) the adsorption of Pt particles on the surface of rGO sheets, detached from feeder electrodes at higher potentials and (ii) the high electrical conductivity of the rGO sheets and thus of the final electrode. Notably, the performance of the rGO with homogenously distributed Pt particles (Fig. S9) that are produced at 80 and 100V have overpotential values of 161 and 94 mV (at 10 mA cm -2 ), respectively, which are comparable to those measured for a bulk Pt electrode (63.5 mV). This indicates an excellent performance of these electrodes, even in the presence of only 0.6 wt % of Pt, which is an order of magnitude lower than some literature values. [START_REF] Browne | Flexible Pt/Graphene Foil Containing only 6.6 wt % of Pt has a Comparable Hydrogen Evolution Reaction Performance to Platinum Metal[END_REF] Fine tuning of the Pt release from the feeder electrodes might even allow a further decrease of the relative Pt content, while keeping a similar catalytic performance. In order to evaluate the stability of the rGO/Pt film, a chronopotentiometry experiment at 10 mA cm -2 was performed for 2000 seconds (Fig. 7d).

Despite minor fluctuations, no significant degradation was observed at this time scale.

Conclusions

We have prepared rGO sheets with different oxidation states using an original BPE set-up based on a quasi-2D reaction space. This system allows overcoming typical limitations of the conventional 3D BPE setups, such as the precipitation and/or the random rotation of the plane of bipolar electrodes with respect to the electric field lines during the reaction and hence unequal and/or short exposure of bipolar electrodes to the desired polarization potential. The approach results in a single-step preparationdirectly in an aqueous solution without any contact with the feeder electrodes-of highly conductive yet dispersible sheets without the presence of any surfactant. Moreover, the proposed 2D setup enables a precise control of the degree of oxidation, and thus a fine-tuning of several physico-chemical properties of the sheets, by simply changing the electric field strength and reaction time. This mild and green approach doesn't require any toxic ingredients and produces high-quality (p)rGO sheets with adjustable electrical and chemical properties in a very short time (< 4 min) using a simple setup. As a proof-of-concepts, we have demonstrated the tunable performance of prGO sheets by using them as conductive fillers for organic batteries, and as catalysts for HER. Notably, as prepared prGO sheets, in contrast to typical carbon-based conductive fillers, are welldispersed in aqueous solution, thus allowing a facile preparation of composites with organic redox polymers, having an energy storage performance comparable to highly reduced rGO.

Regarding HER measurements, Pt/rGO sheets with very low Pt loading (< 1 wt%) could be synthesized in a set of proof-of-principle experiments at high potentials, exhibiting an HER activity comparable to the one of pure Pt metal in terms of overpotential and current density. Such experiments might be pushed further in the future to reach still a similar catalytic performance, but with even lower percentages of metal. The proposed straightforward synthesis process therefore opens up promising perspectives for the controlled elaboration of designer nano-and micromaterials with various functionalities. Further expansion of such systems based on wireless electrochemistry might result in novel approaches for the preparation of graphene-based composites.

Fig. 1 .

 1 Fig. 1. a) Schematic overview of the bipolar electrochemical setup. Length: 0.6 cm, Width:

Fig. 2 .

 2 Fig. 2. Wireless reduction of GO. a) Resistance of GO synthesized at different potentials (10,

Fig. 3 .

 3 Fig. 3. Structural analysis. a) XRD patterns of GO, prGO, and rGO. b) HR-TEM of rGO and

Fig. 4 .

 4 Fig. 4. The resistance and the carbon content of samples during the GO → prGO → rGO transformation. a) Resistance (kΩ cm -2 ) of GO and the electrochemically treated samples at

Fig. 5 .

 5 Fig. 5. Dispersibility for different reduced states. a) Dispersibility of pristine GO and samples

Fig. 6 .

 6 Fig. 6. The energy storage performance of PV composites. a) Reversible redox reactions of PV (PV ++ ↔ PV •+ ). b) CV curves of PV, PV-GO, PV-prGO (40V), and PV-rGO (100V) at 20

Fig. 7 .

 7 Fig. 7. The hydrogen evolution performance of rGO decorated with Pt. a) HR-TEM of thin

Acknowledgements

The work has been funded by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement n° 741251, ERC Advanced grant ELECTRA). Z.S. was supported by Czech Science Foundation (GACR No. 20-16124J).

Credit author contribution statement

The manuscript has been read and approved by all named authors.

Declaration of competing interest

There are no conflicts to declare.

Appendix A. Supplementary data

Supplementary data to this article can be found online