# Coumarin-facilitated iron transport: an IRT1 independent strategy for iron acquisition in Arabidopsis thaliana.

Kevin Robe, Max J.J. Stassen, Shunsuke Watanabe, Javier Espadas, Philippe Gonzalez, Alice Rossille, Meijie Li, Sonia Hem, Aurélien Roux, Véronique Santoni, et al.

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Kevin Robe, Max J.J. Stassen, Shunsuke Watanabe, Javier Espadas, Philippe Gonzalez, et al. Coumarin-facilitated iron transport: an IRT1 independent strategy for iron acquisition in Arabidopsis thaliana. Plant Communications, 2025, 6 (9), pp.101431. 10.1016/j.xplc.2025.101431. hal-05456016

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Coumarin-facilitated iron transport: an IRT1 independent strategy for iron acquisition in *Arabidopsis thaliana*.

Kevin Robe, Max J.J. Stassen, Shunsuke Watanabe, Javier Espadas, Philippe Gonzalez, Alice Rossille, Meijie Li, Sonia Hem, Aurélien Roux, Véronique Santoni, Joseph Chamieh, Christian Dubos, Esther Izquierdo

PII: S2590-3462(25)00193-2

DOI: https://doi.org/10.1016/j.xplc.2025.101431

Reference: XPLC 101431

To appear in: PLANT COMMUNICATIONS

Received Date: 20 November 2024

Revised Date: 28 April 2025

Accepted Date: 23 June 2025

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Please cite this article as: Robe, K., Stassen, M.J.J., Watanabe, S., Espadas, J., Gonzalez, P., Rossille, A., Li, M., Hem, S., Roux, A., Santoni, V., Chamieh, J., Dubos, C., Izquierdo, E., Coumarin-facilitated iron transport: an IRT1 independent strategy for iron acquisition in *Arabidopsis thaliana*, *Plant Communications* (2025), doi: https://doi.org/10.1016/j.xplc.2025.101431.

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© 2025 The Author(s). Published by Elsevier Inc. on behalf of CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, and Chinese Society for Plant Biology.
1 Coumarin-facilitated iron transport: an IRT1 independent strategy for iron
2 acquisition in *Arabidopsis thaliana*.

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4 Kevin Robe¹, Max J J Stassen¹, Shunsuke Watanabe¹, Javier Espadas², Philippe

5 Gonzalez³, Alice Rossille¹, Meijie Li¹, Sonia Hem¹, Aurélien Roux², Véronique Santoni¹,

6 Joseph Chamieh³, Christian Dubos¹,*, Esther Izquierdo¹,*

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8 ¹ IPSIM, Univ Montpellier, CNRS, INRAE, Institut Agro, Montpellier, France

9 ² Department of Biochemistry, University of Geneva, Geneva, Switzerland

10 ³ IBMM, Univ Montpellier, CNRS, ENSCM, Montpellier, France.

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12 * Corresponding authors: ester.izquierdo-alegre@inrae.fr, christian.dubos@inrae.fr

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15 **Short summary:** Iron, an essential micronutrient for plant growth and development, is poorly
16 available in alkaline soils. As a response, non-grass species such as *Arabidopsis thaliana* secrete
17 into the rhizosphere catechol coumarins whose exact function is such soils remains enigmatic.

18 We found that secreted catechol coumarins form stable complexes with iron that are taken up
19 by plant roots, improving plant iron nutrition.

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**Abstract**

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Iron (Fe) is an essential micronutrient for plant growth and development. Despite its importance, Fe uptake in alkaline soils is challenging for most plants because of its poor bioavailability. Plants have evolved two main strategies to acquire Fe. Grass species release phytosiderophores (PS) into the rhizosphere and take up Fe as Fe(III)-PS complexes via specific transporters (Strategy II). Non-grass species, such as *Arabidopsis thaliana*, reduce Fe(III) to Fe(II) at the root surface and take up Fe(II) into the root via the high-affinity transporter IRT1 (Strategy I). In addition, they also secrete catechol coumarins like fraxetin into the rhizosphere to improve Fe acquisition. Although the importance of catechol coumarins in Fe reduction has been clearly demonstrated in acidic soils, their functions in alkaline condition remains enigmatic. In the present work, we first showed that at circumneutral pH, the catechol coumarin fraxetin forms stable complexes with Fe(III). We further demonstrated that fraxetin can significantly improve-Fe nutrition, even in mutant plants lacking IRT1 and in the presence of the strong Fe(II) chelator ferrozine, suggesting that the plant can bypass the conventional Fe(II)-dependent uptake pathway. These findings support that Fe-coumarin complexes are taken up by plant roots in a manner similar to that of Fe(III)-PS complexes in grass species, and therefore challenge the actual paradigm for plant Fe uptake pointing toward a more unified and flexible model in which Strategy I plants can also employ Fe(III)-chelating mechanisms, similar to that of Strategy II.

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**Key words:** *Arabidopsis thaliana*, Coumarins, Fraxin, Iron, Nutrition

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## 45 Introduction

Iron (Fe) is a major micronutrient required for plant growth and development whose availability affects crop productivity and the quality of their derived products (Briat et al., 2015). Although Fe is one of the most abundant elements in soil, plants often suffer from Fe deficiency (Chen and Barak, 1982), which most often occurs in alkaline soils, where Fe solubility is low. In aerated soils, Fe solubility decreases by 1000 times for each pH unit increase between 4 and 9 (Lindsay, 1979). The poor Fe bioavailability in soil has led plants to evolve strategies for Fe mobilisation and uptake, the so-called Strategy I and Strategy II (Chao and Chao, 2022; Gao and Dubos, 2021; Huang et al., 2024).

Grass species (Strategy II plants) release phytosiderophores of the mugineic acid family (MA) to solubilize Fe. MA form stable complexes with both Fe(III) (ferric iron) and Fe(II) (ferrous iron). MA-Fe complexes are then taken up into the plant root by epidermis-localized transporters of the YS1 (YELLOW STRIPE 1) family (Curie et al., 2001; Weber et al., 2006; Xuan et al., 2006). When expressed in the Fe uptake-defective yeast mutant *fet3fet4*, the maize (*Zea mays*) ZmYS1 can restore the growth of yeast grown in a MA-containing medium, even in the presence of the Fe(II) chelator BPDS (bathophenanthroline disulfonic acid) (Meda et al., 2007). Interestingly, despite the physiological importance of phytosiderophore uptake at neutral and alkaline pH, it was shown that the uptake of phytosiderophore-Fe complexes into maize roots was a pH sensitive mechanism that can be explained by the nature of ZmYS1; a proton-coupled symporter (Schaaf et al., 2004). It is noteworthy that other microelements such as copper (Cu), zinc (Zn), nickel (Ni) and cobalt (Co) also form complexes with phytosiderophores and are taken up as such by the plant (Xuan et al., 2006).

In non-grass species (Strategy I), efficient Fe uptake from soil is ensured by a reduction-based mechanism, at least in acidic soils (Kobayashi et al., 2012; Brumbarova et al., 2015). This process involves the acidification of the rhizosphere through the release of protons, the
reduction of Fe(III) to Fe(II) by a membrane localized reductase and the subsequent transport
of Fe(II) across the cell plasma membrane by high affinity Fe transporters. In *Arabidopsis
thaliana*, these three steps rely on the activity of AHA2 (ATPase2), FRO2 (FERRIC
REDUCTION OXIDASE2) and IRT1 (IRON-REGULATED TRANSPORTER1),
respectively (Robinson et al., 1999; Vert et al., 2002).

At alkaline pH, FRO2 activity is negligible (Terés et al., 2019) and as a consequence, Fe(III) cannot be effectively reduced by FRO2, leading to low Fe uptake. To cope with the inactivity of FRO2 under high pH, Fe-mobilising coumarins (i.e., catechol coumarins such as fraxetin and sideretin in *Arabidopsis thaliana*) are secreted in the rhizosphere by the ABCG transporter PDR9 (PLEIOTROPIC DRUG RESISTANCE 9) (Fourcroy et al., 2014). Coumarins are specialized metabolites that derive from the phenylpropanoid pathway (Rodríguez-Celma et al., 2013; Fourcroy et al., 2014; Schmidt et al., 2014). In *Arabidopsis thaliana*, F6'H1 (FERULOYL-COA 6-HYDROXYLASE 1), a 2-oxoglutarate-dependent dioxygenase enzyme, is responsible for the ortho-hydroxylation of feruloyl-CoA into 6-hydroxyferuloyl-CoA, the common precursor of coumarins, that is then converted into scopoletin by COSY (COUMARIN SYNTHASE) (Vanholme et al., 2019; Rodríguez-Celma et al., 2013; Schmid et al., 2014). Because scopoletin lacks catechol groups, it cannot bind to Fe and thus is not directly involved in Fe acquisition (Rajniak et al., 2018). The two main catechol coumarins fraxetin and sideretin are biosynthesized by S8H (SCOPOLETIN 8-HYDROXYLASE), a 2-oxoglutarate-dependent dioxygenase (Siwinska et al., 2018; Tsai et al., 2018), and CYP82C4, a P450-dependent monooxygenase, respectively. Both genes have been shown to be upregulated by Fe deficiency (Murgia et al., 2011; Rajniak et al., 2018). While alkaline pH favours the biosynthesis of fraxetin, the main catechol coumarin secreted at acidic pH is sideretin. The importance of catechol coumarin secretion for Fe nutrition has been demonstrated in several studies (Schmid et al., 2014; Rajniak et al., 2018; Tsai et al., 2018, Paffrath et al., 2023). It has been shown that
catechol coumarins can reduce Fe(III) to Fe(II) and form stable complexes with Fe(III).

However, the relative importance of each phenomenon for the plant and the precise function of
these metabolites in Fe nutrition is still not fully understood, especially in alkaline soils, where
the reduction of Fe by coumarins is negligible (Paffrath et al., 2023). One might formulate three
hypotheses on the role of catechol coumarins in plant Fe nutrition in high pH soils: (i) catechol
coumarins form stable complexes with Fe(III) that are translocated into the roots through the
activity of a yet unknown mechanism at the root epidermis cells, or (ii) the Fe(III) is reduced
by the catechol coumarins and directly taken up by IRT1, or (iii) the Fe(III) solubilized by
coumarins is used as substrate for the FRO2 reductase (Rodríguez-Celma and Schmidt, 2013).

These three hypotheses are not mutually exclusive and may co-exist, although the latter one
seems less likely in high pH soils, as FRO2 activity is negligible. The uptake of catechol
coumarins by Strategy I plants grown in Fe deficient conditions has already been reported (Robe
et al., 2021a); however, the biological function of this phenomenon was still to be decrypted.
These recent results led us to consider coumarins as putative siderophores, with strong
similarities to MA.

In the present work we show that, at circumneutral pH, the catechol coumarin fraxetin forms
stable complexes with Fe(III). Our data support that Fe-coumarin complexes are taken up by
plant roots that in turn significantly improve Fe nutrition, even in mutants lacking the high
affinity Fe(II) transporter IRT1 and in the presence of the strong Fe(II) chelator ferrozine.

Remarkably, this novel mechanism of coumarin-mediated Fe uptake in Arabidopsis strikingly
recalls the uptake of Fe-phytosiderophore complexes by grasses. Taken together, these data
further confirm that the boundaries between Strategy I and Strategy II plants are much narrower
than what is classically described.

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# Results

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## Coumarin uptake in the presence of iron

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The uptake and glycosylation of coumarins by Arabidopsis roots under Fe deficient conditions on agar plate were previously shown (Robe et al., 2021a). However, this mechanism has never been assessed for plants grown in soil, where large amounts of insoluble iron hydroxides are expected to accumulate. This was assayed using *f6'h1* mutant plants grown close to WT plants in potting soil supplemented with different calcium oxide (CaO) concentrations (i.e. 0%, 0.2%, 0.4% and 0.6%). CaO was used to increase the pH of the soil and thus to stimulate the production of coumarins such as fraxetin and esculetin (Figure 1A, Rajniak et al., 2018). Since, in roots, coumarins are mostly present in their glycosylated form, only coumarin glucosides were quantified in this tissue. As no commercial standards are available for sideretin glucosides, we used mass spectrometry (MS) to unequivocally identify the coumarins that are produced and taken up (Figure 1B). Because CaO addition strongly reduces root length, we can exclude any contamination of *f6'h1* roots by the co-cultivated WT roots. In WT plants, scopolin and fraxin accumulation was increased in a CaO concentration dependent manner whereas the accumulation of sideretin glucoside followed the opposite trend. As expected, none of the studied coumarins were identified in the roots of the *f6'h1* mutant grown alone for all the CaO concentration tested. However, MS analysis of *f6'h1* roots co-cultivated with WT plants confirmed that fraxetin and the non-catechol coumarin scopoletin can be taken up by Arabidopsis root when grown in soil. Esculin content in this experiment was very low and it could not be quantified by MS. Sideretin glucosides were detected in *f6'h1* roots co-cultivated with the WT and not in *f6'h1* roots grown alone, showing that sideretin can also be taken up by plant roots (Figure 1B). To study the kinetics of the uptake process, a time course experiment was conducted using *f6'h1* seedlings grown on control media and transferred to a medium
containing synthetic fraxetin. Samples were harvested 1, 3, 7, 24, 48 and 72 hours after transfer and fraxin accumulation was followed by HPLC (Figure 1C). One hour after transfer, fraxin could already be detected in the roots even though the levels were still low, indicating that both the uptake and the glycosylation, which converts fraxetin to fraxin, are fast processes. Then, the accumulation of fraxin in the roots increased regularly over the first 48 hours of the experiment and started to decrease 72 hours after the transfer. Spectral imaging was used to investigate the localization of scopolin, fraxin and esculin after the uptake of their corresponding aglycone form by *f6'h1* mutants in the presence of poorly available Fe. For this purpose, *f6'h1* mutant was grown in a medium containing 25 µM of iron chloride (FeCl₃) at pH7 and catechol or non-catechol coumarins. Spectral imaging confirmed that seedlings were able to take up coumarins in the presence of Fe, glycosylate them, and store the glucosides in the vacuoles (Figure 1D). Similarly to the accumulation pattern observed in response to Fe deficiency (Robe et al., 2021a), fraxin and esculin showed intense accumulation in the cortex and to a lesser extent in the epidermis and endodermis, whereas scopolin accumulated predominantly in the endodermis, with weaker signals in the cortex and minimal presence in the epidermis. The absence of fraxetin and escultin in root hairs (Figure 1D) suggests that coumarins are either not taken up by the trichoblasts or that once taken up they are secreted back into the rhizosphere.

## Identification and characterization of Fe-catechol coumarins complexes

Previous studies reported the formation of Fe-coumarin complexes, but their characterization remained incomplete (Rajniak et al., 2018, Baune et al., 2020). In order to investigate Fe-coumarin complexes at circumneutral to alkaline pH, we prepared scopoletin, escultin and fraxetin solutions and mixed them to FeCl₃ in an ammonium bicarbonate buffer at pH 7. Each mixture was analyzed by direct infusion in positive and negative ESI modes. In positive ESI
mode, only free coumarins showed efficient ionisation and we could barely detect Fe complexes. In negative ESI mode, Fe(III) complexes with fraxetin and esculetin were identified as singly charged species (Figure 2A-B). Fe complexes can be easily recognized by the characteristic isotopic profile of Fe (Tsednee et al., 2016): the relative abundance of naturally occurring stable Fe isotopes is approximately $ ^{54}Fe $ (5.8%), $ ^{56}Fe $ (91.7%), $ ^{57}Fe $ (2.2%) and $ ^{58}Fe $ (0.3%). When fraxetin/FeCl$_3$ mixtures were infused, the main species detected was the 1:2 stoichiometry complex [Fe$^{III}$(fraxetin)$_2$-4H$^+$]⁻ (m/z 467.99), but we could also observe the 1:3 [Fe$^{III}$(fraxetin)$_3$-4H$^+$]⁻ (m/z 676.03) and the 2:4 [Fe$^{III}_2$(fraxetin)$_4$-8H$^+$]⁻ (m/z 936.98) (Figure 2A). The analysis of esculetin/FeCl$_3$ solutions led to the identification of [Fe$^{III}$(esculetin)$_2$-4H$^+$]⁻ (m/z 407.96) (Figure 2B). No Fe complexes with scopoletin were observed. It is noteworthy that even without Fe addition small quantities of Fe complexes with fraxetin were detected (Figure S1), most probably due to Fe contamination of the infusion system. To confirm that Fe-fraxetin complexes were formed before the introduction in MS system, fraxetin was incubated with isotopically labelled $ ^{57}Fe^{3+} $. In this experiment we observed the expected 1 Da MS shift for species containing 1 Fe ([Fe$^{III}$(fraxetin)$_2$-4H$^+$]⁻ (m/z 468.98) and [Fe$^{III}$(fraxetin)$_3$-4H$^+$]⁻ (m/z 677.02) and 2 Da MS shift for species containing 2 Fe ([Fe$^{III}_2$(fraxetin)$_4$-8H$^+$]⁻ (m/z 938.97)), as well as the modification of the isotopic profile for all the main species identified as Fe-fraxetin complexes (Figure S2). The decrease of free coumarin signal intensity after Fe addition was much higher for fraxetin (90%) than for esculetin (25%) (Figure 2A and 2B compared to Figure S1), showing that fraxetin is a stronger Fe chelator than esculetin at circumneutral pH. Fraxetin is mostly produced in alkaline soil (Figure 1B), suggesting that Fe-fraxetin complexes are more stable under this condition. Interestingly, when Fe was added we could detect significant amounts of oxidized free fraxetin (fraxetin semiquinone, m/z 206.0221) (Figure 2A), while in the absence of Fe only the reduced form of fraxetin was detected (m/z 207.0351) (Figure S1). This observation suggests that, while most of the solubilized Fe(III) forms stable chelates with
195 fraxetin, the redox dissociation of this complex may give rise to a small fraction of soluble Fe²⁺
196 and free oxidized fraxetin.

197 The stability of Fe-fraxetin complexes at different pH was assessed by MS (Figure S3). While
198 no significant differences in signal intensity of the complex (m/z 467.99) were observed at pH
199 8 and 7, it slightly decreased at pH 6, 50% of the complex was lost at pH 5, and almost no
200 complex remained at pH 3. The loss of the complex signal resulted in an increase of free fraxetin
201 signal, thus confirming that the Fe-fraxetin complex dissociates at acidic pH (Figure S3).

202 Contrary to a previous work (Baune et al., 2020), all the Fe-coumarin complexes detected in
203 our study contained mainly Fe(III). Singly charged species of Fe(II) complexes would contain
204 one more proton than Fe(III) ones and consequently are 1Da heavier. The isotopic profiles of
205 467.99 and 676.01 m/z peaks were very similar to theoretical ones (Table S1) making the
206 presence of +1Da signals belonging to Fe(II) complexes very unlikely. The ESI source is an
207 artificial environment that induces ionisation and does not reflect the real charge of the species
208 in their physiological state. Moreover, not all the species can be ionized with the same yield.

209 To further elucidate if fraxetin chelates Fe(III) or Fe(II), we analyzed Fe-fraxetin complexes by
210 capillary electrophoresis, which is a milder technique that keeps molecules in their native state.

211 Figure 2C shows the electrophoretic mobility distributions of fraxetin and a fraxetin/FeCl₃
212 mixture obtained by transforming the temporal experimental electropherogram at pH 7 into a
213 mobility distribution (Chamieh et al., 2015). Fraxetin had an average electrophoretic mobility
214 (μ<sub>ep</sub>) of -16.42 · 10<sup>-5</sup> cm<sup>2</sup> s<sup>-1</sup> V<sup>-1</sup>, while the Fe(III)-Fraxetin complex had a value of μ<sub>ep</sub> = -27.02
215 · 10<sup>-5</sup> cm<sup>2</sup> s<sup>-1</sup> V<sup>-1</sup> (Figure 2C). By using the latter μ<sub>ep</sub> values and the hydrodynamic radii values
216 of fraxetin, (R<sub>h</sub> = 0.49 nm obtained by Taylor Dispersion Analysis), and of Fe(III)-Fraxetin 1:3
217 complex (R<sub>h</sub> = 0.77 nm obtained by modelling), the effective charge of both species could be
218 calculated and was found to be -0.8 and -2.9 for fraxetin and the Fe(III)-fraxetin complex,
219 respectively. The fraxetin effective charge value is in accordance with the pK<sub>a</sub> = 6.61 value
determined in this work (Figure S4). The effective charge value of -2.9 obtained for the Fe-
fraxetin complex indicates that the main species existing at pH 7 is the 1:3 stoichiometry
Fe(III)-fraxetin complex (Figures 2C and S5, Table S2) where one Fe³⁺ atom is coordinated by
three fraxetin molecules, each having lost two protons, resulting in a global charge of -3. Other
stoichiometry complexes might also be present but at very low concentrations. The higher MS
intensity observed for the 467.98 *m/z* peak corresponding to the 1:2 complex may be due to a
better ionisation yield of this species and/or to *in source* dissociation of the 1:3 complex.

In analogy with phytosiderophores, we wondered if coumarins could form complexes with
other transition metals. We investigated the interaction of fraxetin with the divalent cations
Ni²⁺, Zn²⁺, Mn²⁺ and Cu²⁺ and the trivalent cation Al³⁺. The identification of metal-coumarin
complexes relied, as for Fe, on metal-specific isotopic signatures of the metal complex spectra
(Table S3). We could detect low signals corresponding to fraxetin complexes with Zn, Al, Ni,
Mn and Cu in a metal-ligand stoichiometry of 1:2 (Figure S6). However, for all of them, most
of the fraxetin remained as free ligand, showing that the chelation efficiency of these metals by
fraxetin is very low compared to Fe. These results demonstrate that fraxetin is a strong and
highly specific Fe chelator compared to other transition metals.

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237 **Fe-coumarin complexes can rescue *irt1* and *bhlh121* mutants, defective in Fe²⁺ uptake**

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Some studies already reported that *f6'h1* mutant growth defects could be complemented by
 growing WT plants in close vicinity to them or by adding exogenous coumarins to the culture
 medium (Tsai et al., 2018; Vanholme et al., 2019). Nevertheless, how coumarins can
 complement *f6'h1* is still an open question. The stability of Fe(III)-fraxetin complexes
 characterized in this work, but also recently reported (Kang et al., 2023), supports the appealing
 hypothesis that once secreted, catechol coumarins may re-enter the root as Fe-coumarin
 complexes, similarly to MAs in grass species. To test this hypothesis, we used *irt1* and *bhlh121*
mutants. The *irt1* mutant is unable to take up Fe²⁺ ions from the soil, leading to strong chlorosis of the plant (Vert et al., 2002). The recently described *bhlh121* mutant is unable to activate the Fe deficiency responses, including the biosynthesis of coumarins (Kim et al., 2019; Gao et al., 2020a). These two mutants display strong chlorosis and cannot grow correctly unless high concentration of Fe is provided (Vert et al., 2002; Kim et al., 2019; Gao et al., 2020a). We first investigated the effect of different fraxetin concentrations on *bhlh121* mutant phenotype. For this purpose, *bhlh121* mutant was grown for one week on Hoagland medium containing 75 µM Fe-EDTA and then transferred to a Hoagland medium containing poorly available Fe. As expected, when *bhlh121* mutant was grown in the presence of 25 µM FeCl₃ at pH 7, plants displayed strong chlorosis phenotype, with necrotic spots in younger leaves (Figure 3A). As little as 5 µM fraxetin supply to the medium partially rescued the phenotype. At this concentration, no necrotic spots were detectable even though leaves were still chlorotic. When the concentration of fraxetin was increased, the *bhlh121* chlorotic phenotype progressively disappeared, in a dose dependent manner. Optimal growth was observed for fraxetin concentrations ranging from 70 µM to 100 µM (Figures 3A and S7A). We also confirmed by spectral imaging that fraxetin was taken up by the root and glycosylated into fraxin in *bhlh121* mutant (Figure S7B).

Even though *bhlh121* chlorotic phenotype was rescued by external addition of fraxetin, we could not exclude that this effect was due to the uptake of fraxetin-reduced Fe²⁺ ions by additional ferrous iron transport systems. To confirm that coumarins directly enter the root bound to Fe(III), we used the strong Fe(II) chelator ferrozine (3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4',4"-disulfonic acid) to chelate all the free Fe²⁺ ions that could have been reduced by fraxetin. The ability of fraxetin to rescue the *bhlh121* and the *irt1* mutant Fe-deficiency phenotypes was not reduced when ferrozine was also present in the culture medium and plants displayed the same phenotype as those grown without ferrozine (Figure 3B-C). Moreover, the
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presence of ferrozine did not affect fraxetin uptake (Figure S8). These results indicate that the complementation of *bhlh121-2* and *irt1* mutant phenotypes by fraxetin does not depend on Fe²⁺ ions and is thus most probably due to the direct uptake of Fe(III)-fraxetin complexes by plant roots.

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The Fe-deficiency phenotype of *bhlh121*, *irt1* and other Fe acquisition mutants such as *fro2* and *f6'h1*, directly grown on Hoagland medium containing poorly available Fe, was rescued by the addition of fraxetin and to a lesser extent of esculetin (Figure 3D-F). In contrast, scopoletin, that lacks the catechol moiety, was unable to complement any mutant (Figure 3D-F). It is noteworthy that *irt1* produces catechol coumarins in both Fe sufficient and deficient growth conditions, contrary to *bhlh121* mutant (Figure S9).

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Since ferritin accumulation is a proxy of Fe content in plants, we performed Western Blot on *irt1* seedlings grown on media containing poorly available Fe and different coumarins. *irt1* mutant accumulated very low levels of ferritins in leaves. Ferritins accumulation increased when *irt1* mutant was grown with fraxetin but not in the presence of scopoletin or esculetin, confirming that addition of fraxetin increased Fe content in the symplasm of the plants (Figure S10). Since esculetin could only partially complement the phenotype of *irt1* and *bhlh121* mutants and its uptake did not result in ferritins accumulation, it is likely that the main role of this coumarin is not Fe transport into the roots. Moreover, the secretion of esculetin is rather limited (Ziegler et al., 2017; Tsai et al., 2018; Terés et al., 2019) and its specific role in plant Fe nutrition, if any, is still to be determined.

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## Uptake of Fe(III)-fraxetin is not restricted to Arabidopsis

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Several eudicots such as tomato (*Solanum lycopersicum*) are able to take up fraxetin into their roots (Robe et al., 2021a). To test if the uptake of Fe-coumarin complexes observed for Arabidopsis can also rescue Fe acquisition mutants in other Strategy I plants, we grew the
T3238fer tomato mutant, unable to activate the Fe deficiency response, with or without fraxetin (Ling et al., 2002). After three weeks of growth in Hoagland medium containing 75 µM Fe-EDTA, plants were transferred to Hoagland medium containing poorly available Fe supplemented or not with 100 µM fraxetin. One week after transfer in alkaline condition, T3238fer mutants were chlorotic. However, T3238fer mutants grown with fraxetin did not display a chlorotic phenotype (Figure S11A), strongly suggesting that Fe(III)-fraxetin complexes are also taken up in tomato. Additionally, HPLC analysis of root extracts showed that the T3238fer mutant can glycosylate the uptaken fraxetin, converting it to fraxin (Figure S11B). These observations indicate that fraxetin can prevent chlorosis in other eudicots than *Arabidopsis thaliana*. The existence of S8H orthologues in several eudicot species strongly suggests that the uptake of Fe(III)-fraxetin complexes in eudicot plants is a widespread strategy for Fe acquisition (Rajniak et al., 2018).

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## Catechol coumarins uptake is an active mechanism

Several specialized metabolites such as alkaloids have been reported to passively diffuse in plant roots once in the soil (Selmar et al., 2015). According to their octanol-water partition coefficient (Log K<sub>ow</sub>) values (~1.5) (Mercer et al., 2013), the coumarins studied here are also predicted to passively diffuse through the plant plasma membrane (Limmer and Burken, 2014; Selmar et al., 2015). However, at pH 7, fraxetin was shown to be deprotonated and therefore negatively charged (Figures 2C and S4) which strongly impairs its diffusion capacity. Also, the inability of rice and maize to take up fraxetin (Robe et al., 2021a) suggests that the uptake of catechol coumarins is not passive but requires a specific molecular mechanism only present in Strategy I plants. To test this hypothesis, we studied fraxetin diffusion in liposomes at pH 7 using confocal microscopy. Liposomes were incubated one hour in the presence of 1 mM fraxetin before observation. We could not detect fraxetin in liposomes, indicating that fraxetin
cannot diffuse through lipidic membranes (Figure 4A). We further investigated fraxetin lipophilicity by using Fe chelating molecules previously described as being capable of diffusing through lipid membranes (hinokitiol) or not (deferiprone) (Grillo et al., 2017). The three ligands rapidly bind Fe to form colored complexes. Unlike membrane permeable Hinokitiol-Fe neutral complexes, the negatively charged Fraxetin-Fe complexes predominantly partitioned into water over polar solvent (Figure 4B). Similar observation was made for Fe chelates with deferiprone (used as model for hydrophilic chelates), showing that both ligands (i.e., fraxetin and deferiprone) form hydrophilic complexes. This result further support that, due to their biophysical properties, Fraxetin-Fe complexes are not likely to passively diffuse through lipid membranes.

We also investigated the uptake of Fe-mobilizing coumarins using sodium orthovanadate ($Na_3VO_4$), an inhibitor of plasma membrane ATPases (pATPase), that inhibits ABC transporter activities and the formation of proton gradient across the plasma membrane (Kang et al., 2011).

For this purpose, *f6'h1* mutant was grown in hydroponic condition and supplemented with two catechol coumarins (fraxetin and esculetin) in the presence or not of 50 µM orthovanadate during 3 hours or 3 days. Their corresponding glucosides (i.e., fraxin and esculin, respectively) were quantified by HPLC. These analyses revealed that the orthovanadate treatment almost abolished root uptake of fraxetin and esculetin, since fraxin and esculin content was reduced by about 70% after 3 hours of treatment (Figure 4C). Importantly, after 3 days, nearly no fraxin or esculin was detected in roots by HPLC (Figure S12), demonstrating that orthovanadate prevents fraxetin and esculetin uptake and not their glycosylation. It was previously shown that orthovanadate treatment does not interfere with vacuolar loading of glycosylated coumarins (Robe et al., 2021a). After 3 hours, non-glycosylated coumarins could be detected in hydroponically grown roots (Figure 4C) and, as for their glycosylated counterparts, their levels were highly affected by orthovanadate treatment. The active uptake of fraxetin was confirmed
using the sulfonylurea glibenclamide, another inhibitor of ATP-dependent transport (Forestier et al., 2003), and the proton uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Schaaf et al. 2004) that similarly to orthovanadate, inhibited fraxetin uptake (Figure S13A-B).

These results demonstrate that the uptake of catechol coumarins by Arabidopsis roots cannot be explained by passive diffusion and relies on a yet unknown ATP dependent mechanism.

Due to the similarities between MAs and coumarins uptake, we speculated that AtYSL (YELLOW STRIPE LIKE), the closest homologues of ZmYS1, might be involved in the uptake of fraxetin. Among the AtYSL family, AtYSL1, AtYSL2 and AtYSL3 are the closest homologue to ZmYS1, and have thus been selected for further analysis (Chen et al., 2018). To test the hypothesis that these AtYSL could mediate Fraxetin-Fe uptake, we analysed *ysl1-2*, *ysl2-1*, *ysl3-1* and *ysl1-2 ysl3-1* mutants grown on Hoagland agar plates for a week and then transferred to a medium containing poorly available Fe (25 µM FeCl₃) and supplemented or not with fraxetin. Uptaken fraxetin was then quantified by HPLC. None of the tested *ysl* mutants showed reduced fraxetin uptake compared to WT (Figure S14), suggesting that AtYSL1, AtYSL2 and AtYSL3 transporters are not involved in fraxetin-Fe uptake, even though functional redundancy cannot be excluded.

## Fraxetin uptake increase the iron content in plant tissues

To confirm that fraxetin is selectively taken up by the root in the form of Fe(III)-fraxetin complexes, we measured the metal content of leaves and roots of *bhlh121* and *irt1* mutants grown with fraxetin supplementation. The analysis revealed that Fe content increased in both roots and shoots of *irt1* and *bhlh121* mutants grown with fraxetin (Figure 5). Remarkably, the Fe content increase due to fraxetin was totally abolished when orthovanadate was also present in the culture media. Since we have shown that fraxetin uptake is also abolished by orthovanadate (Figures 4B and S12), these results strongly suggest that a yet unknown ATP
dependent transport system takes up Fe and fraxetin jointly. To evaluate the contribution of the Fe-fraxetin uptake to the overall plant Fe acquisition we compared the metal contents of WT plants grown in the presence or in the absence of exogenously supplied fraxetin. The addition of fraxetin resulted in an increase of the Fe content in WT plants similar to that observed for *irt1* mutant (Figure S15A-B), showing that, at circumneutral pH, Fe-fraxetin uptake plays a major role in plant Fe nutrition. It is noteworthy that the application of fraxetin did not result in an increase of other non-ferric metals, in accordance with the limited capacity of fraxetin to form complexes with non-ferric metals.

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## 379 Discussion

### 380 Uptake of Fe(III)-coumarin complexes as an alternative iron uptake system

Coumarins have emerged as key metabolites for iron acquisition in non-grass species (Fourcroy et al., 2014; Schmidt et al., 2014; Rajniak et al., 2018; Tsai et al., 2018); however, the mechanisms by which these compounds improve iron nutrition were still unclear. Several studies show that catechol coumarins are involved in Fe reduction and/or Fe chelation (Fourcroy et al., 2016; Sisó-Terraza et al., 2016; Rajniak et al., 2018; Tsai et al., 2018, Paffrath et al., 2023). We recently showed that the catechol coumarin fraxetin is taken up by several non-grass species, suggesting that it might be a widespread phenomenon occurring within the plant kingdom (Robe et al., 2021a). Whether this uptake is accompanied by an uptake of Fe was still an open question (Rodríguez-Celma and Schmidt, 2013; Tsai et al., 2018). By complementing mutants impaired in Fe(II) uptake (i.e., *irt1* and *bhlh121*) with synthetic fraxetin, we provided strong genetic evidences that Arabidopsis has the ability to directly take up Fe(III)-Fraxetin from the growth media. Interestingly, other Fe(III)-chelates (i.e., Fe(III)-EDDHA, Fe-ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid), and Fe(III)-HSAL, Fe-hydrothermal sulfuric acid lignin) failed to complement such mutants (i.e., *irt1*) supporting that
specific uptake transport for Fe(III)-Fraxetin complexes might be at play (Lieu et al., 2023; Paffrath et al., 2023). The *fro2* mutant was also complemented by exogenous fraxetin at pH7, showing that the function of coumarins at high pH is not limited to providing chelated Fe(III) for FRO2, as had been recently suggested (Paffrath et al. 2023). Complementation assays conducted in the presence of ferrozine confirmed this observation. In addition, we showed that at pH 7 fraxetin and Fe can interact *in vitro* and form highly specific and stable Fe(III)-fraxetin complexes with a 1:3 metal:ligand stoichiometry, similar to those observed for grasses MAs. Since both fraxetin and fraxetin-mediated Fe uptake are abolished in the presence of orthovanadate, our results strongly suggest that fraxetin participates to the plant Fe nutrition by forming stable chelates with Fe(III) that are directly taken up by the plant root system (Figure 6) through a specific ATP dependent mechanism.

406

## 407 Several ways to re-enter the root after secretion

While secretion of catechol coumarins through the PDR9 transporter has been clearly demonstrated, the uptake mechanism remains much more elusive (Fourcroy et al., 2014). Here, we provide evidences of a long time suspected parallel Fe acquisition system in non-grass species. This IRT1/FRO2 independent system was already proposed by Vansuyt et al., 2007, who showed that iron from pyoverdine, a bacterial siderophore, was not incorporated through the classical IRT1 transporter. Similar to the Fraxetin-Fe complexes, several Fe-ligands complexes have already been proposed to be taken up by plants. Tipton and Buell (1970) proposed that benzoxazinoids, which are secreted into the rhizosphere by maize roots and strongly bind iron, can function as phytosiderophores. Similarly, Honda et al., (2020) provided evidence that mimosine functions as a siderophore in the leguminous nitrogen-fixing tree *Leucaena leucocephala*. Interestingly, the peanut AhYSL1 mediates the uptake of maize's
419

siderophores, indicating that at least some strategy I plants can use strategy II siderophores (Xiong et al., 2013).

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Several ways to enter the root might be considered for Fe-coumarins; passive diffusion across the plasma membrane or active transport through a specific transporter. The negative charge of the Fe-Fraxetin complex and the phase partitioning results presented here exclude the diffusion hypothesis. Since orthovanadate treatment strongly impaired the uptake of catechol coumarins and fraxetin uptake induced Fe accumulation, an active mechanism for the uptake appears much more likely. ABC transporters are plausible candidates, as they are known to transport a wide diversity of ions and metabolites and are inhibited by orthovanadate. Alternatively, the inhibition of catechol coumarins uptake by orthovanadate could also be due to the inhibition of plasma membrane-localized ATPases, such as AHA2, which would disrupt the proton gradient necessary for transport. The inhibitory effect of CCCP on coumarin uptake further supports this idea, even though an indirect effect arising from disrupted ATP synthesis cannot be excluded.

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Overall, since the inhibitors used have a wide range of effects, especially in *in vivo* systems, and do not exclusively inhibit ATP-dependent processes, the conclusions that can be drawn from these experiments will require extensive investigations to fully conclude on the exact nature of the molecular entities responsible for the import of Fe-coumarin complexes.

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Nonetheless, the data provided here suggest the possibility of a proton-coupled transport mechanism for Fe-coumarin complexes, similar to the Fe-MA uptake mediated by ZmYS1.

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Considering the substrate of YSL (nicotianamine bound to different metals) and the inability of ZmYS1 closest homologues to transport fraxetin, the AtYSL family seems unlikely to be involved in Fe-fraxetin uptake. AtNRAMP1 activity in *irt1* and *bhlh121* mutants could potentially explain the positive effect of fraxetin addition to *irt1* and *bhlh121*, even though the *in planta* role of AtNRAMP1 in Fe uptake is not clearly demonstrated. Moreover, fraxetin addition still complements *irt1* and *bhlh121* mutants in the presence of ferrozine. However,

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443
since NRAMP1 transports Fe(II), this hypothesis would require Fe(III) to be reduced by
fraxetin, and this activity has been shown to be negligible at neutral pH (Paffrat et al., 2023).

Extensive studies will be necessary to identify and characterize the mechanism that is at play
for the uptake of Fe-coumarin complexes. Indeed, it is likely that the uptake mechanism for
non-catechol coumarins such as scopoletin differs from that of fraxetin, occurring most
probably independently of Fe availability.

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451 The advantage of a parallel iron uptake system for strategy I plants

From an ecological point of view, the existence of a chelation-based Fe(III) acquisition system
for Strategy I plants might be a strong advantage when soil conditions, such as high pH
(Römheld and Marschner, 1983; Terés et al., 2019), prevent Fe reduction by FRO2, necessary
for the uptake of this nutrient through the high affinity Fe(II) transporter IRT1 and to a lesser
extent NRAMP1. In contrast, the use of a proton coupled high affinity MA-Fe transport system
allows grasses to thrive in high pH soils. This is because the proton-coupled MA-Fe complex
transport can be achieved as long as the local pH gradient generated by proton extrusion is
maintained and the overall difference of the electrochemical potential remains negative. The
data presented herein support that Fraxetin-Fe complex uptake system is very similar to the one
of MA-Fe in grasses and may explain how non-grass species, including Arabidopsis, can thrive
when encountering carbonate-rich soils with low bioavailable Fe.

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468

We have found that fraxetin, unlike MA, has a limited capacity to chelate with non-ferric
metals. This is because fraxetin contains a catechol moiety that strongly coordinates Fe(III) due
to its high charge density, hard acid character, and preference for octahedral coordination. This
results in highly stable tris-catecholate complexes. In contrast, divalent metals like Cu(II),
Ni(II) and Mn(II), being softer and less charged, interact more weakly with catechol moieties.
This selectivity allows fraxetin to mobilize Fe(III) efficiently without promoting uptake of non-
469

target metals, which might confer an advantage to plants to maintain metal homeostasis under Fe-limiting conditions

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471

It was shown that fraxetin secretion of 22 different Arabidopsis accessions is highly correlated with plant Fe content and that accessions abundantly secreting fraxetin have the best growth parameters (Tsai et al., 2018). In addition, it was reported that local Arabidopsis populations adapted to alkaline soils (pH 7.5) presented a high fraxin content in roots and a high capacity for fraxetin secretion (Terés et al., 2019). On the contrary, root ferric reductase activity is similar between this population and the pH sensitive ones (Terés et al., 2019).

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477

The identification of a second Fe acquisition system is a major step towards the complete understanding of Fe uptake in non-grass plant species and might have important implications for improving cultural practices to prevent Fe deficiency. The selection of plants being able to secrete higher amounts of fraxetin might significantly reduce Fe induced chlorosis in the field without adding synthetic Fe chelates and thus allow the culture and yield improvement in soils with poor Fe availability. Previous studies have demonstrated that grass species can improve Fe nutrition of non-grass plants in intercropping systems (Brown et al., 1991; Hopkins et al., 1992; Kamal et al., 2000; Gunes et al., 2007; Xiong et al., 2013). Hence, it can be hypothesized that the transporter involved in Fe-coumarin complexes uptake might also be involved in the uptake of phytosiderophores. However, a previous study indicates that the reverse is improbable (Robe et al., 2021a).

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489

## Materials and methods

490

### Plant Material

491

*Arabidopsis thaliana* ecotype Columbia (Col-0) was used as wild type (WT). The *Arabidopsis thaliana* mutant lines used in this study are: *f6'h1-1* (Kai et al., 2008), *irt1-2* (Varotto et al., 2002), *fro2* (*frd1-1*) (Robinson et al., 1999), *bhlh121-2* (Gao et al., 2020a; Gao et al., 2020b),

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ysl1-2 (Le Jean et al., 2005), ysl2-1 (DiDonato et al., 2004), ysl3-1 and ysl1-2 ysl3-1 (Waters et al., 2006). The Fe-inefficient tomato (*Solanum lycopersicum*) mutant used in this study was T238fer (Brown et al., 1971).

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## Growth conditions

499

For *in vitro* culture, seeds were surface sterilized with a solution containing 12.5/37.5/50 (v/v/v) of bleach/water/ethanol for 5 minutes with strong agitation. Seeds were rinsed 3 times with 96% ethanol before drying. Seeds were then germinated on control Hoagland medium (50 µM Fe-EDTA at pH 5.5) solidified with 0.7% agar and grown under long day condition (16h light/8h dark).

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504

For all the experiments, control Hoagland medium contained 50 µM Fe-EDTA. Poorly available Fe Hoagland medium contained 25 µM FeCl₃, supplemented with 0.5 g l⁻¹ HEPES buffer and the pH adjusted to 7 with KOH. For agar plates, pH was adjusted after autoclave using a pH indicator paper (± 0.25 pH units).

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For coumarin uptake, *f6'h1* mutant plants were grown in hydroponic condition for 4 weeks in control Hoagland solution. Coumarins were added for 3 hours or 3 days at 50 µM in a new Hoagland solution containing poorly available Fe (25 µM FeCl₃ at pH 7). For orthovanadate and glibenclamide treatments, seedlings were transferred for 3 hours or 3 days in a Hoagland solution containing poorly available Fe, coumarins and 100 µM sodium orthovanadate or 150 µM glibenclamide (both from Sigma-Aldrich).

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514

For coumarin uptake kinetics and CCCP treatment, one-week-old seedlings were transferred to Hoagland agar plates containing poorly available Fe and 50 µM fraxetin.

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516

For *in vitro* mutant complementation, plants were directly germinated on poorly available Fe Hoagland plates (25 µM FeCl₃ at pH 7) containing the synthetic coumarins scopoletin, fraxetin or esculetin (all three from Sigma-Aldrich). Scopoletin was added at a concentration of 25 µM,

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518
519

esculetin at 50 µM and fraxetin at 100 µM. 100 µM concentration was not used for scopoletin or esculetin since detrimental effects on seedlings were observed.

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521

Tomato seeds were germinated for 1 week on wet tissue paper under long day condition. When cotyledons were fully developed, seedlings were transferred to a Hoagland solution containing 25 µM FeCl₃ at pH 7.5, supplemented or not with 100 µM fraxetin. Plants were then grown under short day condition (8 h light / 16h dark).

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526

## Coumarin imaging

527

Seedlings were grown for one week on control Hoagland agar plates (50 µM Fe-EDTA pH 5.5) prior to their transfer for 3 days on low Fe availability medium (25 µM FeCl₃, pH 7) supplemented with 25 µM of scopoletin, fraxetin or esculetin. The root cell walls were stained with 10 µg ml⁻¹ propidium iodide (PI) during 10 minutes. Coumarins were imaged with an LSM 780 multiphoton (i.e. two photon) microscope (Zeiss, Oberkochen, Germany) equipped with a Chameleon Ultra II laser (Coherent, Santa Clara, CA, USA) and a W Plan Apochromat 20x 1.0 objective (Talamond et al., 2015; Koyyappurath et al., 2015). Spectral imaging was performed using laser excitation at 720 nm and the 32-channel GaAsP spectral detector as described elsewhere (Robe et al., 2021a). The Linear Unmixing function in Zeiss Zen microscope software (version 2.10) was used to separate the signals of the auto-fluorescent compounds scopolin, fraxin, esculin and PI.

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539

## Chlorophyll Content

540

Chlorophyll from 2 to 5 mg of leaf tissue (fresh weight) were extracted in 1 ml of 100% acetone in the dark under agitation. The absorbance (A) at 661.8 and 644.8 nm was then measured.

541

542

Total chlorophyll content was assessed using the following equation: Chl a + Chl b = 7.05 x
543

$A_{661.6} + 18.09 \times A_{644.8}$ and was expressed as micrograms per gram fresh weight. Three seedlings were pooled for each biological replicate.

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545

546

## HPLC analysis of roots and leaves extracts

547

Coumarin extraction from roots, samples preparation for HPLC and coumarin quantification were performed as described previously (Robe et al., 2021a). HPLC-UV and HPLC-fluorescence analysis were performed using a 1220 Infinity II LC system (Agilent Technologies) coupled to a 1260 Infinity II fluorescence detector (Agilent Technologies). Separation was done on an analytical HPLC column (Kinetex 2.6 µm XB-C18 100 Å, 150 × 4.6 mm; Phenomenex), with a gradient mobile phase made with 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in acetonitrile (B) and a flow rate of 0.5 ml min⁻¹. The gradient program started at 8% B for 1 minute and increased linearly to 33% B in 11.5 minute and then to 50% B in 0.5 minute. This proportion was maintained for 3 minutes and returned linearly to initial conditions in 0.5 minute. The column was allowed to stabilize for 5 minutes at the initial conditions. Absorbance was monitored at λ = 338 nm. Fluorescence was monitored at λexc 365 and λem 460 nm.

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560

## LC-MS/MS analysis of soil grown roots

561

Wild type and *f6'h1* plants were co-cultivated for three weeks in soil supplemented with 0 to 0.6% (w/w) CaO. Plants were grown at a distance sufficient to prevent root mixture between genotypes. Soils with growing Arabidopsis plants were roughly washed with tap water. Roots of plants from both genotypes were then carefully rinsed with distilled water to remove all remaining soil particles. The absence of cross-contamination between the roots of both genotypes was verified by PCR genotyping (Figure S16). Then coumarins were extracted from roots as previously described for HPLC analysis (Robe et al., 2021a).

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567
568 LC-MS/MS analysis were performed using an Ultimate 3000 nano HPLC system (Thermo
569 Fisher Scientific Inc.) coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer
570 (Maxis Impact; Bruker Daltonics GmbH) as described previously (Robe et al., 2021a). The LC
571 conditions were those used for HPLC analysis with the exception of the flow rate, which was
572 reduced to 200 µl min⁻¹. Glycosylated coumarins usually lose their sugar moiety in the ESI
573 source. Therefore, after the verification of their identity by retention time, MS and MS/MS
574 profiles, fraxin, esculin, scopolin and sideretin glycosides were quantified by integrating the
575 MS Extracted Ion Chromatograms (EIC) peaks of their aglycone counterparts. As no standards
576 are commercially available for sideretin glycosides, calibration curves could not be performed
577 and thus, relative quantification was done for all the coumarins in this experiment. Results are
578 expressed as relative intensity peak areas (counts min⁻¹).

579

580 **Direct infusion mass spectrometry**

581 To study the *in vitro* interaction of Fe and other transition metals with coumarins, 50 µM
582 methanolic solutions of each ligand (fraxetin, scopoletin and esculetin) and 50 µM aqueous
583 solution of each metal salt (FeCl₃, MnCl₂, ZnCl₂, NiCl₂, AlCl₃ and CuCl₂) were freshly
584 prepared. ⁵⁷Fe³⁺ was obtained from ⁵⁷Fe₂O₃ (Eurisotop, Saint-Aubin, France) by overnight
585 solubilisation in 37% HCl followed by neutralisation with 1M ammonium bicarbonate.
586 Mixtures of metal:ligand ratios of 1:2 were made and diluted to 1/1000 in a solution of 50%
587 acetonitrile/50% 25 mM ammonium bicarbonate (adjusted to pH 8, 7, 6, 5 or 3 by addition of
588 formic acid). The obtained solutions were injected into the mass spectrometer via a syringe
589 pump at a rate of 200 µl h⁻¹. Source settings were the same as used for LC-MS analysis except
590 for mass range: 50 to 1000 m/z and ESI that was operated in negative mode.

591

592 **Capillary electrophoresis and Taylor dispersion analysis**
Capillary electrophoresis experiments were performed on an Agilent 7100 capillary electrophoresis instrument with a built-in diode array UV detector (Waldbronn), detection was performed at 214 nm. Fused silica capillaries of 50/375 µm I.D./O.D. were from Polymicro Technologies. Capillary dimensions were 33.5 cm long (25 cm to detection window). New capillaries were conditioned by performing the following washes at 1 bar: 1 M NaOH for 30 minutes and water for 15 min. Before each sample, the capillary was flushed with the buffer for 2 minutes. Samples were injected hydrodynamically on the inlet side of the capillary by applying a 50 mbar pressure for 3 seconds. Separations were achieved by applying a -15 kV voltage. Injections were repeated 3 times, between each repetition a 30 seconds flush with the buffer was done. Samples were prepared at a 1 µM concentration of fraxetin in a 50 mM ammonium bicarbonate (NH₄HCO₃) buffer at variable pH (5 to 8) adjusted with formic acid or in a 50 mM formic acid solution of pH 2.5.

The hydrodynamic radius of fraxetin was determined using Taylor dispersion Analysis (Chamieh and Cottet, 2014). Briefly, using the same capillary, instrument and buffers as for the capillary electrophoresis experiments, the samples were injected hydrodynamically by applying a 50 mbar pressure for 5 seconds and were mobilized with the buffer by applying a 100 mbar pressure. The elution profiles were treated as described elsewhere (Chamieh and Cottet, 2014) to calculate the diffusion coefficient and the hydrodynamic radius ($R_h$). In the case of Fe(III)-fraxetin complexes, the $R_h$ was estimated from the diffusion coefficient obtained using HydroPro software (Ortega et al., 2011) on structures drawn with Marvin Sketch (ChemAxon). The determination of the effective charge of fraxetin and Fe-fraxetin complexes was achieved by using the method based on the O'Brien-White-Ohshima model (OWO) (Ohshima, 2001; Ibrahim et al., 2013). Protocols and equations for the determination of the effective charge of fraxetin and Fe(III)-fraxetin complex and the pKa of Fraxetin are detailed in Methods S1 (Figure S17).
618

619 Fraxetin diffusion in liposomes

620 Giant unilamellar vesicles (GUVs) were prepared as previously shown (Velasco-Olmo et al.
621 2019). Dioleoyil-phosphatidylcholine (DOPC), dioleoyil-phosphatidylserine (DOPS) and
622 dioleoyl-phosphoethanolamine labeled with Atto 647N (Atto 647N DOPE) lipid stocks
623 dissolved in chloroform were mixed at 59.9:40:0.1 mol percentage, respectively, in a glass vial,
624 and dried under vacuum for 2-3 hours. The dried lipid film was then hydrated in a buffer
625 containing 25 mM HEPES at pH 7.4 to form multilamellar vesicles (MLVs) at a final
626 concentration of 1 g l⁻¹. Twenty µl of the MLVs solution was mixed with 2 µl of silica beads
627 (40 µm in diameter) (Microspheres-Nanospheres, USA), and deposited on parafilm. The
628 MLVs-silica beads mixture on parafilm was then completely dried on vacuum for 2 hours
629 leading to the formation of dried supported bilayers on the supported silica beads. The
630 supported silica beads were then collected using a glass capillary and placed on a hydration
631 chamber containing 1M trehalose at 60°C for 15 minutes) as the membrane pre-hydration step.
632 Pre-hydrated model membranes were then transferred to a 1.5 ml tube containing the working
633 buffer (25 mM HEPES, pH 7.4), and further hydrated for another 20 minutes. Lastly, the
634 mixture was gently stirred for 20-30 seconds in order to detach the hydrated model membranes
635 from the supporting silica beads, and the supernatant containing the detached GUVs was
636 transferred to a new tube containing fraxetin dissolved in the working buffer in a final
637 concentration of 1 mM. Upon 20 minutes of incubation, samples were imaged using a scanning
638 confocal microscope (inverted Nikon Eclipse Ti A1R microscope using a ×100, 1.3 NA oil
639 immersion objective (Nikon)).

640

641 Lipophilicity determination
642

To study hexane-water partition of iron chelates, 40 µM solutions of each ligand (fraxetin, deferiprone and hinokitiol; Grillo et al., 2017) and 13 µM $FeCl_3$ were prepared in 25 mM carbonate buffer at pH 8, mixed to an equal volume of hexane, shaken for 5 minutes and then centrifuged for 5 minutes to separate phases.

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644

645

646

647

## Metals measurements

648

649

650

651

652

653

About 2.5 mg of dry root or 7.5 mg of dry shoot tissue per sample were mixed with 750 µl of nitric acid (65% [v/v]) and 250 µl of hydrogen peroxide (30% [v/v]). After one night at room temperature, samples were mineralized at 85°C for 24 hours. Once mineralized, 4 ml of milliQ water was added to each sample. Metal contents present in the samples were then measured by microwave plasma atomic emission spectroscopy (MP-AES, Agilent Technologies). Around 100 seedlings were pooled for each biological replicate.

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655

## Western blot analysis

656

657

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659

Western blots were performed as described previously (Robe et al., 2021a). Immunodetection of ferritins was performed using a rabbit polyclonal antiserum raised against purified FER1 (Dellagi et al., 2005). Dilutions of antibodies applied were rabbit anti-ferritin 1:10000 (primary) and anti-rabbit HRP conjugated (Promega) 1:10000 (secondary).

660

661

## Accession Numbers

662

663

664

665

Sequence data from this article can be found in the GenBank/EMBL libraries under the following accession numbers: bHLH121 (AT3G19860), FRO2 (AT1G01580), F6'H1 (AT3G13610), IRT1 (AT4G19690), YSL1 (AT4G24120), YSL2 (AT5G24380) and YSL3 (AT5G53550).

666
667 Acknowledgements

668 We thank Sandrine Chay (IPSiM, SAME platform, Montpellier, France) for technical support
669 for plant Fe determination, Dr. Marie Lopez (IBMM, Montpellier, France) for help with
670 coumarin chemistry, and Dr Brigitte Touraine (IPSiM) for help with HPLC analysis of
671 coumarins. We thank Dr Catherine Curie (IPSiM) for kindly providing seeds for ysl mutants.

672 We also thank the imaging facility MRI, member of the national infrastructure France-
673 BioImaging supported by the French National Research Agency (ANR-10-INBS-04,
674 "Investments for the future"). Support to this work was provided to CD by the Agence Nationale
675 pour la Recherche (ANR-17-CE20-0008 and ANR-22-CE20-0006) and the National Research
676 Institute for Agriculture, Food and the Environment (INRAE, BAP Department). KR was
677 supported by a fellowship from the Agence Nationale pour la Recherche (ANR-17-CE20-0008)
678 and the BAP department of the INRAE, AR was supported by a fellowship from the Agence
679 Nationale pour la Recherche (ANR-22-CE20-0006) and the BAP department of the INRAE,
680 ML was supported by the China Scholarship Council, and SW by a Marie Skłodowska-Curie
681 Individual Fellowship in Horizon 2020 from the European Council (MSCA-IF-2020). AR
682 acknowledges funding from the Swiss National Fund for Research (#CRSII5_189996 and
683 #310030_200793) and the European Research Council Synergy (#951324-R2-TENSION). JE
684 acknowledges a long-term EMBO fellowship (EMBO ALTF 989-2022).

685

686 Author contributions

687 KR, JC, CD and EI conceived and designed the experiments. KR, MJJS, SW, PG, AR, ML, JC
688 and EI performed the experiments. KR, MJJS, SW, JE, PG, AR, ML, SH, JC, CD and EI
689 analyzed the data. SH and VS provided access to mass spectrometry facilities. KR, JC, CD and
690 EI wrote the paper with the help of all the authors.

691
692

693

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Figure legends

912
913 **Figure 1. Uptake of coumarins in the presence of iron.**

(A) Coumarin biosynthetic pathway. Enzymes are written in capital green letters. Catechol 914 groups are highlighted in yellow. Note that conversion of 6-hydroxycaffeoyl-CoA into esculetin 915 has only been demonstrated *in vitro*. Question marks indicate putative unknown enzymes. (B) 916 Scopolin, fraxin and sideretin glucoside content in the roots of three-week-old *Arabidopsis* 917 *thaliana* wild type (WT) and *f6'h1* mutant co-cultivated or not with the WT in soil 918 supplemented with different CaO concentrations (%, w/w). Coumarins quantification was 919 achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Coumarins were 920 identified by their retention time, MS and MS/MS profiles and quantified by integration of their 921 aglycone counterpart EIC (extracted-ion chromatograms) signals. EIC used for the 922 quantification of each coumarin were as follows: scopolin: *m/z* 193.04 +/- 0.02, sideretin 923 glycoside: *m/z* 225.03 +/- 0.02, fraxin: *m/z* 209.05 +/- 0.02. nd: non detected. Means within the 924 same genotype with the same letter are not significantly different according to one-way 925 ANOVA followed by post hoc Tukey test, *P* < 0.05 (n = 3 biological repeats of at least 20 926 seedlings). Bars represent means ± SD. (C) Three days kinetic analysis of fraxetin uptake in 927 *f6'h1* mutant grown in the presence of poorly available Fe (25 µM FeCl₃) and exogenous 928 fraxetin. Time course root samples were collected and fraxin was analysed by HPLC. Means 929 with the same letter are not significantly different according to one-way ANOVA followed by 930 post hoc Tukey test, *P* < 0.05 (n = 3 biological repeat of at least 50 seedlings each). Error bars 931 show means ± SD. Lines are used to connect the means. (D) Localisation of coumarins (i.e., 932 scopolin: purple, fraxin: green, esculin: dark yellow) in *f6'h1* mutant seedlings grown during 3 933 days in the presence of synthetic scopoletin, fraxetin or esculetin. Images were obtained by 934 spectral deconvolution and are representative of observations made in three independent 935 experiments where the roots of five to six seedlings were independently analysed. Bar = 25 µm. 936 e: endodermis, c: cortex, ep: epidermis. 937
938

939

**Figure 2. Identification of Fe-fraxetin and Fe-esculetin complexes.**

940

(A) Direct infusion ESI-QTOF MS analysis of a 2:1 (mol:mol) mixture of synthetic fraxetin and Fe(III) at pH 7. (B) Direct infusion ESI-QTOF MS analysis of 2:1 (mol:mol) mixture of synthetic esculetin and Fe(III) at pH 7. (A-C) ESI was operated in negative mode. Iron complexes were identified by their characteristic isotopic pattern and are highlighted in red.

941

942

943

944

Boxes show a zoom on the MS signal of the main coumarin species. MS intensity was measured in counts. (C) Electrophoretic mobility distributions for fraxetin (solid line) and a 2:1 (mol:mol) mixture of fraxetin and Fe(III) (dashed line) at pH 7 in a 50 mM Ammonium carbonate buffer.

945

946

947

The effective charge ($Z_{eff}$) was calculated using the model described in Oshima (2001). The structure of the main iron-containing complex is highlighted in red. The abundance of the protonated and deprotonated forms of fraxetin was calculated from its effective charge and is in agreement with fraxetin's $pK_a$.

948

949

950

951

952

**Figure 3. Effect of coumarins on *Arabidopsis thaliana* iron acquisition mutants.**

953

(A) Dose dependent chlorophyll content (top) and leaf phenotype (bottom) of *bhlh121* mutant supplemented with fraxetin. Fraxetin was added at different concentrations in Hoagland medium containing poorly available Fe (25 µM FeCl₃). (B) Chlorophyll content (top) and phenotype (bottom) of *bhlh121* mutant grown in the absence of Fe²⁺ ions. (C) Chlorophyll content (top) and phenotype (bottom) of *irt1* mutant grown in the absence of Fe²⁺ ions. (B-C)

954

955

956

957

958

The strong Fe²⁺ chelator ferrozine was added to the medium at 75µM. (A-C) Plants were grown for 1 week in control Hoagland agar plates and transferred for one week to new agar plates containing fraxetin and/or ferrozine. (D) Phenotype of several Fe acquisition mutants grown for 10 days on poorly available Fe Hoagland medium supplemented with scopoletin, esculetin or fraxetin. Plants were directly sown on coumarin containing medium. (E) Fresh weight content

959

960

961

962
of the shoots of 10 day old seedlings showed in (D). (F) Chlorophyll content of the shoots of 963
10 day old seedlings showed in (D). (A, B, C, E and F) Means within each genotype with the 964
same letter are not significantly different according to one-way ANOVA followed by post hoc 965
Tukey test, P < 0.05 (n = 3 to 4 biological repeats of at least 3 seedlings). Bars represent means 966
± SD. 967

968

969 **Figure 4. Characterization of the membrane diffusion capacity of fraxetin.**

(A) Fraxetin diffusion assay in liposome. 1 mM fraxetin was incubated during 1 hour with 970 liposomes. Left panel; red: liposome membrane, blue: fraxetin. Right panel; Fraxetin (blue) and 971 Atto 647N (lipid labelling, red) fluorescence plot profile intensity across the dashed line. (B) 972 Phase partitioning of deferiprone, fraxetin and hinokitiol Fe complexes into hexane (up) and 973 water (bottom). (C) Inhibition of coumarin uptake by orthovanadate (VO4³⁻) addition. Uptake 974 of coumarins by *f6'h1* roots grown in hydroponic Hoagland solution containing poorly available 975 Fe (+Fe; 25 µM FeCl3), supplemented or not with 50 µM VO4³⁻. Plants were kept for 3 hours 976 in the presence of coumarins and orthovanadate. Coumarins were analysed by HPLC. t-test 977 significant difference: \*P < 0.01 (n = 5 biological repeats). Bars represent means ± SD. 978

979

980 **Figure 5. Effect of fraxetin treatment on the accumulation of iron in Arabidopsis thaliana**
981 iron acquisition mutants.

(A) Shoots and (B) roots Fe and transition metals content of *irt1* and *bhlh121* mutants. *irt1* and 982 *bhlh121* mutants were germinated and grown for 10 days in control agar plates and then 983 transferred for another 10 days to agar plates containing poorly available Fe (25 µM FeCl3) 984 supplemented or not with 100 µM fraxetin and/or 50 µM orthovanadate (VO4³⁻). Means within 985 the same metal with the same letter are not significantly different according to one-way 986
ANOVA followed by post hoc Tukey test, P < 0.05 (n = 4 biological repeats of at least 20 seedlings each). Bars represent means ± SD. Fe: iron, Cu: copper, Zn: zinc and Mn: manganese.

989

Figure 6. Proposed model for Fe-coumarins uptake by *Arabidopsis thaliana* roots.

In calcareous or alkaline soils, FRO2/IRT1 high affinity Fe²⁺ transport system is almost inactive as FRO2 activity is strongly inhibited at high pH. As a response to this growth condition, Arabidopsis roots produce and secrete several coumarins such as scopoletin, esculetin, fraxetin and sideretin. Once secreted by PDR9, catechol coumarins solubilize Fe hydroxides by forming stable complexes with Fe(III) and also by reducing Fe(III) to Fe(II). Reduced Fe is then taken up by IRT1 and Fe(III)-coumarin complexes by a yet unknown transporter localized at the root epidermis plasma membrane. The main catechol coumarin involved in Fe(III) uptake is fraxetin and mostly act in a 3 fraxetin

999

1000

Supplemental information

1002

Supplemental items

1004

**Figure S1.** Direct infusion ESI-QTOF MS analysis of fraxetin and esculetin at pH 7 without any iron addition.

**Figure S2.** Analysis of isotopically labelled ⁵⁷Fe-fraxetin complexes.

**Figure S3.** Analysis of the stability of Fe-fraxetin complexes at different pH.

**Figure S4.** Determination of the pKa of fraxetin by capillary electrophoresis.

**Figure S5.** Proposed structures of Fe-fraxetin complexes.

**Figure S6.** Identification of non-ferric metals and iron complexes with fraxetin.
1012 **Figure S7.** Phenotypic effect of fraxetin application on *bhlh121* mutant.

1013 **Figure S8.** Fraxetin uptake in the presence of ferrozine.

1014 **Figure S9.** Coumarins accumulation in roots of iron acquisition mutants.

1015 **Figure S10.** Western Blot of ferritins accumulation in *irt1* mutant supplemented or not with coumarins.

1017 **Figure S11.** Effect of fraxetin on tomato *T3238fer* mutant.

1018 **Figure S12.** Inhibition of coumarin uptake by orthovanadate (VO₄³⁻) addition.

1019 **Figure S13.** Inhibition of fraxetin uptake by the ATP-dependent transport inhibitor glibenclamide and the proton uncoupler CCCP.

1021 **Figure S14.** Fraxetin uptake is not inhibited in *ysl1*, *ysl2*, *ysl3* and *ysl1 ysl3* mutants.

1022 **Figure S15.** Effect of fraxetin treatment on the accumulation of iron in *Arabidopsis thaliana* wild type plants.

1024 **Figure S16.** Genotyping of wild type (WT) and *f6'h1* mutant roots prior to coumarins LC-MS/MS analysis.

1026 **Figure S17.** Graphical determination of zeta potential (ζ).

1027 **Table S1.** Experimental and theoretical mass-to-charge ratios (m/z) and isotope abundance of 2:1 and 3:1 fraxetin-Fe complexes detected by direct infusion ESI-QTOF MS analysis.

1029 **Table S2.** Charge, hydrodynamic radius and electrophoretic mobility of Fe:Fraxetin complexes.

1031 **Table S3.** Natural isotope abundances of the analyzed metals.

1032

1033 Supplemental experimental procedures

1034

1035 **Methods S1** Detailed protocols for determination of the pKa of Fraxetin and the effective charge of fraxetin and Fe(III)-fraxetin complex.

1036
A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /></figure>

C

<figure><img src="image_5.png" /></figure>

D

<figure><img src="image_6.png" /></figure>
A

<figure><img src="image_7.png" /></figure>

B

<figure><img src="image_8.png" /></figure>

C

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>A</figcaption></figure>

bhlh121

<figure><img src="image_11.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_12.png" /></figure>

bhlh121

<figure><img src="image_13.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_14.png" /></figure>

irt1

<figure><img src="image_15.png" /><figcaption>D</figcaption></figure>

pH 7 + Scopoletin + Esculetin + Fraxetin

<figure><img src="image_16.png" /></figure>

bhlh121

<figure><img src="image_17.png" /></figure>

irt1

<figure><img src="image_18.png" /></figure>

fro2

<figure><img src="image_19.png" /></figure>

f6'h1

<figure><img src="image_20.png" /></figure>

WT

<figure><img src="image_21.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_22.png" /></figure>
A

Membrane

Fraxetin

Merged

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

B

<figure><img src="image_25.png" /></figure>

C

<figure><img src="image_26.png" /></figure>
<figure><img src="image_27.png" /><figcaption>bhlh121</figcaption></figure>

<figure><img src="image_28.png" /></figure>
<figure><img src="image_29.png" /></figure>