Fei Gao
email: fei.gao@supagro.fr

Christian Dubos
email: christian.dubos@inrae.fr

Transcriptional integration of the plant responses to iron availability

Keywords: Arabidopsis thaliana, basic helix-loop-helix, bHLH, iron homeostasis, rice, transcription factor

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Iron (Fe) is an essential micronutrient for almost all living organisms. In humans, iron deficiency anemia is a major global health issue affecting about one billion people worldwide [START_REF] Camaschella | Iron-deficiency anemia[END_REF]. Therefore, increasing the iron content in plants, especially in crops (biofortification), would have enormous benefits for human health. To achieve this goal, a critical question is to uncover the mechanisms controlling iron homeostasis in plants. Like in humans, iron is an essential microelement for plant growth and development. It functions in various vital metabolic processes (e.g. photosynthesis, respiration, amino acid biosynthesis) by acting as cofactor for several metalloproteins or component of electron transport chains [START_REF] Hänsch | Physiological functions of mineral micronutrients (cu, Zn, Mn, Fe, Ni, Mo, B, cl)[END_REF][START_REF] Touraine | Iron-sulfur protein NFU2 is required for branched-chain amino acid synthesis in Arabidopsis roots[END_REF]. However, the excess of iron is deleterious to plants because of its capacity to interact with oxygen, generating reactive oxygen species (ROS) via the Fenton Reaction. Thus, the levels of iron in plant cells must be tightly regulated to avoid iron deficiency or iron excess, both of which severely affecting crops yield and the quality of their derived products [START_REF] Briat | Iron nutrition, biomass production, and plant product quality[END_REF].

Although iron is the fourth most-abundant element on earth, much of it is not readily available for plant use due to the poor solubility of its main (hydro)oxides, especially in neutral-to-alkaline soils [START_REF] Guerinot | Iron: nutritious, noxious, and not readily available[END_REF]Colombo et al., 2018). To adapt to low iron conditions and acquire iron from soil, higher plants have evolved two different strategies [START_REF] Marschner | Strategies of plants for acquisition of iron[END_REF] (Figure 1). Dicots and non-graminaceous monocots (nongrass species) employ the reduction strategy (named Strategy I). The graminaceous utilize the chelation strategy (named Strategy II). In strategy I, non-grass plants are able to acidify the rhizosphere to promote iron solubility through protons release and mobilize Fe 3+ through coumarins (i.e. fraxetin and sideretin) or riboflavins secretion [START_REF] Santi | Dissecting iron deficiency-induced proton extrusion in Arabidopsis roots[END_REF]Fourcroy et al., 2016;Robe et al., 2020). Following iron mobilization, Fe 3+ is reduced into Fe 2+ (a more soluble form of iron) that is subsequently transported into the root epidermal cells via high affinity iron transporters from the Zrt/Irt-like protein (ZIP) family.

In Arabidopsis thaliana, the release of protons into the rhizosphere is insured by the H + -ATPASE 2 (AHA2; [START_REF] Santi | Dissecting iron deficiency-induced proton extrusion in Arabidopsis roots[END_REF] whereas the reduction of iron and its translocation into the roots are ensured by FERRIC REDUCTION OXIDASE 2 (FRO2) and IRON-REGULATED TRANSPORTER1 (IRT1), respectively [START_REF] Brumbarova | Molecular mechanisms governing Arabidopsis iron uptake[END_REF]Connorton et al., 2017). AHA2, FRO2 and IRT1 associate into a complex at the surface of the Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraa556/6007775 by guest on 30 November 2020 A c c e p t e d M a n u s c r i p t root epidermal cells most likely allowing optimizing iron acquisition and therefore its uptake (Martín- [START_REF] Barranco | Dynamic control of the high-affinity iron uptake complex in root epidermal cells[END_REF]. Coumarins secretion is ensure by the PLEIOTROPIC DRUG RESISTANCE 9/ATP-BINDING CASSETTE G37 (PDR9/ABCG37) transporter (Fourcroy et al., 2014;Fourcroy et al., 2016). In strategy II, plants biosynthesize and secrete phytosiderophores of the mugineic acid family (MAs) into the rhizosphere to chelate Fe 3+ [START_REF] Kobayashi | Iron deficiency responses in rice roots[END_REF]. Fe 3+ -MAs complexes are then transported into root cells by transporters of the YELLOW STRIPE 1 (YS1) and YELLOW STRIPE 1-like (YSL) family [START_REF] Inoue | November 2020 A c c e p t e d M a n u s c r i p t uptake in early growth of the seedlings[END_REF][START_REF] Murata | A specific transporter for iron (III)-phytosiderophore in barley roots[END_REF]. Interestingly, it was recently shown that some plant species might use both strategies. For instance, when grown in waterlogged soil condition, rice (Oryza sativa), a graminaceous species, acquires Fe 2+ from the soil through the activity of two Fe 2+ transporters, OsIRT1 and OsIRT2 [START_REF] Ishimaru | Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+[END_REF]. The secretion of caffeic and protocatechuic acids via the PHENOLICS EFFLUX ZERO 2 (PEZ2) transporter participates to Fe 3+ mobilization and reduction into Fe 2+ [START_REF] Bashir | Rice phenolics efflux transporter 2 (PEZ2) plays an important role in solubilizing apoplasmic iron[END_REF]. If iron acquisition is the first step that participates to the maintenance of iron homeostasis in plants, it should be noted that it is not the sole mechanism involved in this process. Iron translocation, compartmentalization, assimilation and storage are also important processes required for the maintenance of iron homeostasis at the cellular and subcellular levels, throughout the whole plant body (Kobayashi and Nishizawa, 2012;Kobayashi et al., 2019).

Gene regulation is a crucial step for coping with iron fluctuations. For instance, FRO2 and IRT1 expression is induced when iron availability is low whereas the expression of the genes encoding the iron storage ferritin proteins (i.e. FER1, 3 and 4) is induced in response to iron excess [START_REF] Tissot | Transcriptional integration of the responses to iron availability in Arabidopsis by the bHLH factor ILR3[END_REF]. How plants control iron homeostasis, by regulating the expression of genes involved in the various facets of this complex mechanism, was a critical question for the past three decades. To address this question, several studies mostly based on forward and reverse genetic approaches were conducted, leading to the identification and characterization of several transcription factors (TFs). These studies, mostly conducted in Arabidopsis and rice, allowed establishing regulatory networks controlling iron homeostasis in which basic helix-loop-helix (bHLH) TFs play a preponderant role [START_REF] Tissot | Transcriptional integration of the responses to iron availability in Arabidopsis by the bHLH factor ILR3[END_REF]Gao et al., 2020a;[START_REF] Li | The Molecular Mechanisms Underlying Iron Deficiency Responses in Rice[END_REF] (Figure 2 and Table 1). bHLH proteins form one of the largest families of TFs [START_REF] Heim | The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity[END_REF] known to modulate several facets of plant growth and development, including cell differentiation, secondary metabolite biosynthesis, hormone signaling or the responses to environmental factors [START_REF] Carretero-Paulet | Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae[END_REF]. Indeed, A c c e p t e d M a n u s c r i p t additional TFs and protein regulating TFs activity were also identified [START_REF] Yan | A WRKY transcription factor regulates Fe translocation under Fe deficiency[END_REF][START_REF] Palmer | MYB10 and MYB72 are required for growth under iron-limiting conditions[END_REF]Rodríguez-Celma et al., 2019a).

Within this review, current knowledge on the control of iron homeostasis by TFs, especially from the bHLH family, will be presented and discussed with a special emphasis on the latest findings. The transcriptional and posttranslational regulation of the iron homeostasis regulatory networks will also be documented. Last, some perspectives on future research to be conducted in order to improve our understanding of this complex mechanism will be provided.

The transcriptional regulation of iron homeostasis in strategy I plants, a preponderant role for bHLH TFs

The model plant Arabidopsis has allowed the identification of several factors involved in the regulation of iron homeostasis, notably by studying its response to iron deficiency. Such studies highlighted that the regulation of iron homeostasis was essentially occurring at the transcriptional level and was involving several TFs, in particular those of the bHLH family. This topic has also been covered in recent reviews by [START_REF] Tissot | Transcriptional integration of the responses to iron availability in Arabidopsis by the bHLH factor ILR3[END_REF][START_REF] Kobayashi | Understanding the Complexity of Iron Sensing and Signaling Cascades in Plants[END_REF]Kobayashi et al., 2019;[START_REF] Wu | FIT-Binding Proteins and Their Functions in the Regulation of Fe Homeostasis[END_REF][START_REF] Schwarz | FIT, a regulatory hub for iron deficiency and stress signaling in roots, and FIT-dependent and -independent gene signatures[END_REF] To date, at least six bHLH TF subfamilies [START_REF] Heim | The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity[END_REF], encoding 17 different proteins, are known to participate to the maintenance of iron homeostasis in Arabidopsis. These bHLH TFs form an intricate regulatory network composed of two interconnected regulatory modules (Figure 3).

The first module relies on the activity of FIT/bHLH29 (FER-LIKE IRON DEFICIENCY INDUCED TRANSCRIPTION FACTOR), a clade IIIa bHLH TF [START_REF] Colangelo | The essential basic helix-loop-helix protein FIT1 is required for the iron deficiency response[END_REF][START_REF] Jakoby | FRU (BHLH029) is required for induction of iron mobilization genes in Arabidopsis thaliana[END_REF][START_REF] Yuan | AtbHLH29 of Arabidopsis thaliana is a functional ortholog of tomato FER involved in controlling iron acquisition in strategy I plants[END_REF]. FIT/bHLH29 is a direct regulator of IRT1 and FRO2 expression, highlighting its central role for the regulation of the iron uptake machinery (Wang et al., 2013b). FIT/bHLH29 activity relies on its interaction with the four members of the Ib bHLH clade (i.e. bHLH38, bHLH39, bHLH100 and bHLH101), forming heterodimer complexes displaying partial redundant activities [START_REF] Colangelo | The essential basic helix-loop-helix protein FIT1 is required for the iron deficiency response[END_REF][START_REF] Maurer | Responses of a triple mutant defective in three iron deficiency-induced basic helix-loop-helix genes of the subgroup Ib to iron deficiency and salicylic acid[END_REF][START_REF] Yuan | FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis[END_REF]Wang et al., 2013b) (Figure 3). Recently, the members of the IVa bHLH clade (i.e. bHLH18, bHLH19, bHLH20 and bHLH25) were identified as FIT/bHLH29 interacting proteins (Cui et al., 2018). These interactions were shown to A c c e p t e d M a n u s c r i p t promote the degradation of FIT/bHLH29 via the 26S proteasome pathway, in a jasmonic acid-dependent manner (Cui et al., 2018). It is noteworthy that the Ib bHLH and the IVa bHLH TFs antagonize the activity of each other in regulating FIT/bHLH29 protein accumulation to tightly regulate the iron uptake machinery in response to different environmental stimuli (Cui et al., 2018) (Figure 3).

The second module acts upstream from FIT/bHLH29. It involves the four members of the IVc bHLH clade, namely ILR3/bHLH105 (IAA-LEUCINE RESISTANT 3), IDT1/bHLH34 (IRON DEFICIENCY TOLERANT 1), bHLH104 and bHLH115. These four TFs play additive roles in the iron deficiency responses and their activity is thought to rely, at least in part, on their ability to form homo-or heterodimers [START_REF] Li | Two bHLH transcription factors, bHLH34 and bHLH104, regulate iron homeostasis in Arabidopsis thaliana[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] Liang | bHLH transcription factor bHLH115 regulates iron homeostasis in Arabidopsis thaliana[END_REF] (Figure 3). In response to iron deficiency, these TFs directly activate the expression of clade Ib bHLH and indirectly the one of FIT/bHLH29 [START_REF] Li | Two bHLH transcription factors, bHLH34 and bHLH104, regulate iron homeostasis in Arabidopsis thaliana[END_REF]Zhang et al., 2015;[START_REF] Liang | bHLH transcription factor bHLH115 regulates iron homeostasis in Arabidopsis thaliana[END_REF].

Clade IVb bHLH TFs (i.e. PYE/bHLH47, bHLH11 and URI/bHLH121), also participate to the regulation of iron homeostasis in Arabidopsis. PYE/bHLH47 is a negative regulator [START_REF] Long | The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots[END_REF], which contains an EAR motif at its C-terminal region (i.e. DLNxxP; [START_REF] Kagale | EAR motif-mediated transcriptional repression in plants: an underlying mechanism for epigenetic regulation of gene expression[END_REF] that directly represses the expression of genes participating to the maintenance of iron homeostasis. Interaction studies highlighted that PYE/bHLH47 could heterodimerize with ILR3/bHLH105 and bHLH115 [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] Tissot | Transcriptional integration of the responses to iron availability in Arabidopsis by the bHLH factor ILR3[END_REF]. However, the role of these interactions was still a matter of debate until recently. In a recent study, ILR3/bHLH105 was found to play a central role in the regulation of iron homeostasis where it acts as both transcriptional activator and repressor of the plant responses to iron deficiency and excess, respectively [START_REF] Kroh | Connecting the negatives and positives of plant iron homeostasis[END_REF][START_REF] Tissot | Transcriptional integration of the responses to iron availability in Arabidopsis by the bHLH factor ILR3[END_REF]. In this study, the authors showed that the repressive activity of ILR3/bHLH105 was conferred by its dimerization with PYE/bHLH47 (Figure 3). The authors also highlighted that ILR3-PYE heterodimers might repress the expression of PYE/bHLH47 when iron availability is not limiting via a negative feedback regulatory loop (Figure 3). bHLH11 is another transcriptional repressor also containing an EAR motif (i.e. LxLxL) in its C-terminal domain [START_REF] Tanabe | The basic helixloop-helix transcription factor, bHLH11 functions in the iron-uptake system in Arabidopsis thaliana[END_REF]. Overexpression studies suggest that bHLH11 inhibits the plant tolerance to iron deficiency and the expression of IRT1 and FRO2, most probably by indirectly repressing the expression of FIT/bHLH29 [START_REF] Tanabe | The basic helixloop-helix transcription factor, bHLH11 functions in the iron-uptake system in Arabidopsis thaliana[END_REF] (Figure 3). In contrast to PYE/bHLH47 and bHLH11, URI/ bHLH121 (UPSTREAM REGULATOR OF regulator of the plant responses to iron deficiency [START_REF] Lei | bHLH121 Functions as a Direct Link that Facilitates the Activation of FIT by bHLH IVc Transcription Factors for Maintaining Fe Homeostasis in Arabidopsis[END_REF][START_REF] Lockhart | Personal Trainer: bHLH121 Functions Upstream of a Transcriptional Network of Heavy Lifters Involved in Balancing Iron Levels[END_REF]Gao et al., 2020a;[START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF]. URI/bHLH121 can form heterodimers with clade IVc bHLH TFs [START_REF] Lei | bHLH121 Functions as a Direct Link that Facilitates the Activation of FIT by bHLH IVc Transcription Factors for Maintaining Fe Homeostasis in Arabidopsis[END_REF]Gao et al., 2020a;[START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF] (Figure 3). These interactions participate to the relocation of URI/bHLH121 from the cytosol into the nucleus [START_REF] Lei | bHLH121 Functions as a Direct Link that Facilitates the Activation of FIT by bHLH IVc Transcription Factors for Maintaining Fe Homeostasis in Arabidopsis[END_REF]. It also suggests that the transcriptional activation of clade IVc bHLH target genes requires, at least in part, URI/bHLH121 [START_REF] Lei | bHLH121 Functions as a Direct Link that Facilitates the Activation of FIT by bHLH IVc Transcription Factors for Maintaining Fe Homeostasis in Arabidopsis[END_REF]Gao et al., 2020a;[START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF].

In support of this assertion, it was demonstrated that several genes directly targeted by URI/bHLH121 are identical to that of clade IVc bHLH TFs (Figure 3). The expression of FIT/bHLH29 also relies on URI/bHLH121 activity, most probably via an indirect mechanism [START_REF] Lei | bHLH121 Functions as a Direct Link that Facilitates the Activation of FIT by bHLH IVc Transcription Factors for Maintaining Fe Homeostasis in Arabidopsis[END_REF]Gao et al., 2020a;[START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF]. It is noteworthy that under iron deficiency condition, URI/bHLH121 accumulates in its phosphorylated form (Figure 3) that increases its binding capacity to the promoter region of its target genes such as the clade Ib bHLH TFs [START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF]. Interestingly, both URI/bHLH121 transcript and protein accumulate constitutively regardless of iron status (Gao et al., 2020a;[START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF]. In contrast, URI/bHLH121 cellular localization in roots differs depending on iron availability (Gao et al., 2020a). When iron is not limiting, URI/bHLH121 mainly localizes in the stele and the endodermis, whereas under iron deficiency condition, URI/bHLH121 is primarily observed in the cortex and the epidermis cells, where it promotes iron uptake (Gao et al., 2020a). The thorough characterization of URI/bHLH121 indicates that it plays a key role in the control of plant iron homeostasis mainly because it directly or indirectly regulates the expression of most of the known genes involved in this regulatory network (Gao et al., 2020a;[START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF] (Figure 3). Interestingly, it was recently reported that URI/bHLH121 directly activates FER1, FER3 and FER4 expression when iron availability is not in excess, indicating that URI/bHLH121 positively regulates the transient storage of iron in addition to the iron deficiency response (Gao et al., 2020b).

Functional homologs of most of the above-described Arabidopsis bHLH TFs have been characterized in several dicots (Figure 2 As stated above, plants have developed two different strategies to take up iron from the soil, distinguishing the non-graminaceous (Strategy I, reduction strategy) and graminaceous (Strategy II, chelation strategy) species (Figure 1). However, the in depth study of the responses to iron deficiency in rice has highlighted that the regulation of iron homeostasis in strategy II plants involves the activity of several bHLH TFs belonging to the same clades than those identified in strategy I plant species (Figures 2 and4, Table 1; [START_REF] Kobayashi | Understanding the Complexity of Iron Sensing and Signaling Cascades in Plants[END_REF]Kobayashi et al., 2019).

OsFIT/OsbHLH156 was recently identified as a positive regulator of the iron deficiency responses [START_REF] Liang | Oryza sativa Fer-like Fe deficiency-induced transcription factor (OsFIT/OsbHLH156) interacts with OsIRO2 to regulate iron homeostasis[END_REF][START_REF] Wang | A transcription factor OsbHLH156 regulates Strategy II iron acquisition through localising IRO2 to the nucleus in rice[END_REF]. Loss-of-function of OsFIT/OsbHLH156 resulted in strong iron deficiency symptoms under upland condition, whereas no iron deficiency symptoms were observed when plants were grown in waterlogged soil (Wang et al., 2020). These results imply that the strategy II iron uptake system was impaired in the Osfit mutant, an hypothesis that is supported by the disruption of the expression of strategy II iron uptake related genes. It includes genes encoding enzyme involved in MAs biosynthesis (e.g. NAS1 and NAS2, NICOTIANAMINE SYNTHASE 1 and 2) or Fe 3+ -MA transport (e.g.

OsYSL15, YELLOW STRIPE-LIKE 15) (Figure 1) [START_REF] Liang | Oryza sativa Fer-like Fe deficiency-induced transcription factor (OsFIT/OsbHLH156) interacts with OsIRO2 to regulate iron homeostasis[END_REF][START_REF] Wang | A transcription factor OsbHLH156 regulates Strategy II iron acquisition through localising IRO2 to the nucleus in rice[END_REF].

Interestingly, it was demonstrated that OsFIT/OsbHLH156 regulates the expression of OsIRT1 and therefore participates also to the regulation of strategy I iron uptake mechanism [START_REF] Liang | Oryza sativa Fer-like Fe deficiency-induced transcription factor (OsFIT/OsbHLH156) interacts with OsIRO2 to regulate iron homeostasis[END_REF] (Figure 1). OsFIT/OsbHLH156 interacts with OsIRO2/OsbHLH56, a clade Ib bHLH, and promotes its nuclear accumulation (Figure 4) [START_REF] Liang | Oryza sativa Fer-like Fe deficiency-induced transcription factor (OsFIT/OsbHLH156) interacts with OsIRO2 to regulate iron homeostasis[END_REF][START_REF] Wang | A transcription factor OsbHLH156 regulates Strategy II iron acquisition through localising IRO2 to the nucleus in rice[END_REF]. OsIRO2/OsbHLH56 was the first characterized bHLH TF involved in the control of iron homeostasis in rice, where it acts as a positive regulator of the iron deficiency response [START_REF] Ogo | Isolation and characterization of IRO2, a novel ironregulated bHLH transcription factor in graminaceous plants[END_REF][START_REF] Ogo | The rice bHLH protein OsIRO2 is an essential regulator of the genes involved in Fe uptake under Fe-deficient conditions[END_REF]. Expression analysis indicated that the regulation of OsIRT1 expression by OsFIT/OsbHLH156 might be different to that of Arabidopsis or that it might involve other Ib bHLH TFs than OsIRO2/OsbHLH56. Three out of the four clade IVc bHLH TFs (i.e. OsPRI1/OsbHLH060, OsPRI2/bHLH058, OsPRI3/OsbHLH059; POSITIVE REGULATOR OF IRON HOMEOSTASIS 1, 2 and 3) have been identified in rice as playing a positive role in the iron deficiency responses [START_REF] Zhang | Positive regulator of iron homeostasis1, OsPRI1, facilitates iron homeostasis[END_REF]Kobayashi et al., 2019;Zhang et al., 2020b). The characterization of loss-offunction mutants suggested that these three TFs directly regulate the expression of OsIRO2/OsbHLH56, and indirectly the expression of OsNAS1, OsNAS2, and OsYSL15 via OsIRO2/OsbHLH56 (Figure 4) (Zhang et al., 2020b;[START_REF] Zhang | Positive regulator of iron homeostasis1, OsPRI1, facilitates iron homeostasis[END_REF]. Among the potential direct targets of OsPRI1/OsbHLH060, OsPRI2/bHLH058 and OsPRI3/OsbHLH059, there is also OsIRO3/OsbHLH63 (Figure 4) (Zhang et al., 2020b;[START_REF] Zhang | Positive regulator of iron homeostasis1, OsPRI1, facilitates iron homeostasis[END_REF]. OsIRO3/OsbHLH63 is a member of the IVb clade and the functional homolog of PYE/bHLH47 (Zheng et al., 2010). Like PYE/bHLH47, OsIRO3/OsbHLH63 functions as a negative regulator of the iron deficiency responses (Zheng et al., 2010). It is likely that OsIRO3/OsbHLH63 activity might antagonize OsIRO2/OsbHLH56 to tightly regulate iron uptake and avoid iron overload [START_REF] Zhang | The Arabidopsis mediator complex subunit16 positively regulates salicylate-mediated systemic acquired resistance and jasmonate/ethylene-induced defense pathways[END_REF]Zhang et al., 2020b;[START_REF] Zhang | Positive regulator of iron homeostasis1, OsPRI1, facilitates iron homeostasis[END_REF]Zheng et al., 2010). To date, there is no information on the role of OsbHLH057, the fourth member of the rice clade IVc bHLH. Indeed, based on the information gathered in Arabidopsis, it is likely that OsbHLH057 participates to the control of iron homeostasis in rice. Whether OsbHLH057 exerts a minor role in specific cell types or in specific environmental conditions remains to be determined.

OsbHLH133 functions as a negative regulator of iron translocation from roots to shoots (Wang et al., 2013a). It should be noted that OsbHLH133 is the only one member of the clade VIIIc reported to date as involved in the regulation of iron homeostasis in plants. The other members of this clade in Arabidopsis play a positive role in root hair development but have never been associated with the maintenance of iron homeostasis [START_REF] Bruex | A gene regulatory network for root epidermis cell differentiation in Arabidopsis[END_REF].

Whether these bHLHs have a similar and conserved role, as that of OsbHLH133 in the regulation of iron homeostasis in strategy II plants, is to be investigated.

The transcriptional regulation of iron homeostasis is not restricted to the activity of bHLH TFs

TFs from other families are involved in regulatory networks acting for iron homeostasis in both strategy I and strategy II plants (Figures 3 and4, Table 2).

Several R2R3-MYB TFs from different plant species have been shown to be involved in the regulation of iron deficiency responses. In Arabidopsis, MYB10 and MYB72 are two iron deficiency inducible TFs required for proper iron uptake and whose expression is partially dependent on FIT/bHLH29 and URI/bHLH121 activities (Figure 3) (Gao et al., 2020a;[START_REF] Palmer | MYB10 and MYB72 are required for growth under iron-limiting conditions[END_REF][START_REF] Zamioudis | β-Glucosidase BGLU42 is a MYB72dependent key regulator of rhizobacteria-induced systemic resistance and modulates iron deficiency responses in Arabidopsis roots[END_REF]. Furthermore, MYB72 has been identified as a A c c e p t e d M a n u s c r i p t transcriptional activator of genes associated with the biosynthesis and secretion of ironmobilizing coumarins, highlighting its role in strategy I iron uptake system [START_REF] Stringlis | MYB72-dependent coumarin exudation shapes root microbiome assembly to promote plant health[END_REF][START_REF] Zamioudis | β-Glucosidase BGLU42 is a MYB72dependent key regulator of rhizobacteria-induced systemic resistance and modulates iron deficiency responses in Arabidopsis roots[END_REF] (Figure 1). Recently, it was shown that the Arabidopsis MYB28 and MYB29 are at the interface of the plant sensitivity to ammonium stress and the modulation of iron homeostasis [START_REF] Coleto | Arabidopsis thaliana transcription factors MYB28 AND MYB29 shape ammonium stress responses by regulating fe homeostasis[END_REF]. Notably, the ammonium-dependent decrease of MYB28 and MYB29 expression (or the loss-of-function of both genes) leads to defects in iron translocation from roots to shoots and to the induction of the expression of FIT/bHLH29, clade Ib bHLHs, MYB72 as well as IMA1/FEP3 and IMA3/FEP1 in roots.

MdMYB58, a close homolog of MYB72, was recently characterized as a positive regulator of iron uptake and translocation in apple [START_REF] Wang | MdMYB58 modulates Fe homeostasis by directly binding to the MdMATE43 promoter in plants[END_REF]. Further investigation showed that MdMYB58 transcriptional activity is inhibited by its heterodimerization with MdSAT1/MdbHLH18, a IVa clade bHLH TF [START_REF] Wang | MdMYB58 modulates Fe homeostasis by directly binding to the MdMATE43 promoter in plants[END_REF]. In contrast to the abovementioned MYB TFs, MxMYB1 may function as a negative regulator of iron uptake and storage [START_REF] Shen | An MYB transcription factor from Malus xiaojinensis has a potential role in iron nutrition[END_REF].

In Arabidopsis, WRKY46 plays a role in iron translocation between root and shoot by directly regulating the expression of VITL1 (VACUOLAR IRON TRANSPORTER-LIKE 1), a potential iron transporter involved in iron sequestration into the vacuoles [START_REF] Yan | A WRKY transcription factor regulates Fe translocation under Fe deficiency[END_REF][START_REF] Gollhofer | Vacuolar-irontransporter1-like proteins mediate iron homeostasis in Arabidopsis[END_REF]. HAP5A/NF-YC1 is also involved in iron translocation between root and shoot by regulating the expression of NAS1 (Zhu et al., 2020). ERF4 and ERF72 are two Arabidopsis TFs belonging to AP2/ERF family that have been reported as potential negative regulators of iron deficiency responses by repressing the expression of genes involved in iron uptake such as IRT1 (Liu et al., 2017a;Liu et al., 2017b). Similarly, MbERF4 and MbERF72

from Malus baccata as well as MxERF4 from Malus xiaojinensis act as negative regulators of the iron deficiency responses in these two apple species [START_REF] Liu | An ethylene response factor (MxERF4) functions as a repressor of Fe acquisition in Malus xiaojinensis[END_REF]Zhang et al., 2020a). In contrast, ERF95 was recently proposed to promote iron storage in Arabidopsis seeds [START_REF] Sun | Ethylene promotes seed iron storage during Arabidopsis seed maturation via ERF95 transcription factor[END_REF]. However, how iron distribution is controlled in seeds is not clearly established even if it was recently proposed that B3 TFs, which are involved in the regulation of embryo development and seed maturation, might be good candidates [START_REF] Roschzttardtz | Transcriptional Regulation of Iron Distribution in Seeds: A Perspective[END_REF].

IDEF1 and IDEF2 (IRON DEFICIENCY-RESPONSIVE ELEMENT FACTOR 1 and 2) are two rice TFs belonging to two different families, the ABI3/VP1 and NAC, respectively (Figure 4) [START_REF] Kobayashi | The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants[END_REF][START_REF] Ogo | A novel NAC transcription factor, IDEF2, that recognizes the iron deficiency-responsive element 2 regulates the genes involved in iron homeostasis in plants[END_REF]. These two TFs participate to the regulation of iron homeostasis in rice. IDEF1 is required for the coordinated activation of A c c e p t e d M a n u s c r i p t genes related to iron uptake and translocation, including OsIRT1, OsNAS1, OsNAS2 and OsYSL15 [START_REF] Kobayashi | The rice transcription factor IDEF1 is essential for the early response to iron deficiency, and induces vegetative expression of late embryogenesis abundant genes[END_REF][START_REF] Kobayashi | The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants[END_REF]. In addition, IDEF1 positively regulates the expression of OsIRO2, indicating that IDEF1 functions upstream of OsIRO2 in the iron deficiency regulatory network (Figure 4) [START_REF] Kobayashi | The rice transcription factor IDEF1 is essential for the early response to iron deficiency, and induces vegetative expression of late embryogenesis abundant genes[END_REF][START_REF] Kobayashi | The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants[END_REF]. Interestingly, it was shown that IDEF1 could bind to iron and zinc atoms and that this capacity was necessary for its activity (Kobayashi et al., 2012). It was therefore proposed that IDEF1 could sense the cellular metal ion balance caused by changes in iron availability, suggesting that IDEF1 could be a cellular iron sensor allowing the tight regulation of the iron deficiency responses (Kobayashi et al., 2012). Similarly to IDEF1, IDEF2 plays a positive role in the plant response to iron deficiency [START_REF] Ogo | A novel NAC transcription factor, IDEF2, that recognizes the iron deficiency-responsive element 2 regulates the genes involved in iron homeostasis in plants[END_REF].

A family of peptides named IMA/FEP (IRONMAN/FE-UPTAKE-INDUCING PEPTIDE)

has been reported to play a positive role in iron deficiency responses in Arabidopsis, by regulating a set of deficiency-inducible genes including Ib bHLH TFs, a function that seems to be conserved across plant species [START_REF] Grillet | IRON MAN is a ubiquitous family of peptides that control iron transport in plants[END_REF][START_REF] Hirayama | The putative peptide gene FEP1 regulates iron deficiency response in Arabidopsis[END_REF]. Two recent studies showed that URI/bHLH121 is a direct positive regulator of IMA1/FEP3 and IMA2/FEP2 expression (Gao et al., 2020a;[START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF]. These results indicate that IMAs/FEPs are implicated in the bHLH-dependent regulatory network regulating iron homeostasis. However, the precise regulatory mechanisms by which IMAs/FEPs act are still unknown.

Post-translational regulation of the iron homeostasis regulatory networks

Protein-protein interactions as well as post-translational modifications (i.e. ubiquitination, sumoylation, phosphorylation) can significantly affect the regulatory activities of TFs. Such mechanisms were shown to play an important role in the maintenance of iron homeostasis in plants. This topic has also been covered in recent reviews by [START_REF] Kobayashi | Understanding the Complexity of Iron Sensing and Signaling Cascades in Plants[END_REF]Rodríguez-Celma et al., 2019a;[START_REF] Schwarz | FIT, a regulatory hub for iron deficiency and stress signaling in roots, and FIT-dependent and -independent gene signatures[END_REF][START_REF] Spielmann | The many facets of protein ubiquitination and degradation in plant root iron deficiency responses[END_REF][START_REF] Wu | FIT-Binding Proteins and Their Functions in the Regulation of Fe Homeostasis[END_REF].

As described earlier, the transcriptional activity of bHLH TFs involved in the control of iron homeostasis is extensively dependent on in vivo protein-protein interactions, in the form of homo-or heterodimers (Figures 3 and4, Tables 1 and2). For instance, FIT/bHLH29 heterodimerization with clade Ib bHLH TFs is required for its transcriptional activity and A c c e p t e d M a n u s c r i p t stability, whereas its interaction with clade IVa members promotes its degradation via the 26S proteasome pathway (Cui et al., 2018). bHLH39 nuclear localization also depends on its interaction with FIT/bHLH29 since in the cells lacking FIT/bHLH29, bHLH39 localizes predominantly in the cytoplasm [START_REF] Trofimov | Mobility and localization of the iron deficiency-induced transcription factor bHLH039 change in the presence of FIT[END_REF]. Similarly, OsFIT/OsbHLH156 facilitates the nuclear localization of OsIRO2/OsbHLH156, the functional homolog of bHLH39 in rice, suggesting that this post-translational regulatory mechanism is conserved within the plant kingdom [START_REF] Liang | Oryza sativa Fer-like Fe deficiency-induced transcription factor (OsFIT/OsbHLH156) interacts with OsIRO2 to regulate iron homeostasis[END_REF][START_REF] Wang | A transcription factor OsbHLH156 regulates Strategy II iron acquisition through localising IRO2 to the nucleus in rice[END_REF]. FIT/bHLH29 activity and/or stability are also modulated by its interaction with several additional protein partners that do not belong to the bHLH family of TFs (Figures 3, Tables 1) (reviewed in [START_REF] Kobayashi | Understanding the Complexity of Iron Sensing and Signaling Cascades in Plants[END_REF][START_REF] Schwarz | FIT, a regulatory hub for iron deficiency and stress signaling in roots, and FIT-dependent and -independent gene signatures[END_REF][START_REF] Spielmann | The many facets of protein ubiquitination and degradation in plant root iron deficiency responses[END_REF][START_REF] Wu | FIT-Binding Proteins and Their Functions in the Regulation of Fe Homeostasis[END_REF].

Several ubiquitin E3 ligases have been identified and characterized as negative regulators of iron uptake to avoid potential iron overload by targeting bHLH TFs for their degradation (for details, see [START_REF] Spielmann | The many facets of protein ubiquitination and degradation in plant root iron deficiency responses[END_REF]Rodríguez-Celma et al., 2019a) (Figures 3 and4).

BTS (BRUTUS), whose expression is induced by iron deficiency in roots, is proposed to be a critical iron sensing E3 ubiquitin ligase in Arabidopsis [START_REF] Long | The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots[END_REF]. BTS interacts with ILR3/bHLH105 and bHLH115 to facilitate their degradation via the 26S proteasome pathway, allowing the fine tuning of the expression of downstream iron deficiency response genes (Figure 3) [START_REF] Selote | Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors[END_REF][START_REF] Long | The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots[END_REF]. Similarly, OsHRZ1 and OsHRZ2

(HAEMERYTHRIN MOTIF-CONTAINING REALLY INTERESTING NEW GENE (RING) AND ZINC-FINGER PROTEIN 1 and 2), two rice ubiquitin E3 ligases displaying strong sequence similarities with BTS, have been reported as potential iron sensors playing a negative role in iron acquisition under iron sufficient conditions [START_REF] Kobayashi | Iron-binding haemerythrin RING ubiquitin ligases regulate plant iron responses and accumulation[END_REF]. It was shown that OsHRZ1 could interact with OsPRI1/OsbHLH60, OsPRI2/OsbHLH58 and OsPRI3/OsbHLH59 and mediate their degradation via the 26S proteasome (Figure 4) (Zhang et al., 2020b;[START_REF] Zhang | Positive regulator of iron homeostasis1, OsPRI1, facilitates iron homeostasis[END_REF]. However, different results were reported in another study only validating the sole interactions between OsHRZ1 and OsHRZ2 with OsPRI1/OsbHLH60 and OsPRI2/OsbHLH58 (Kobayashi et al., 2019). Another in vitro ubiquitination study showed that neither OsPRI1/OsbHLH60, nor OsPRI2/OsbHLH58 or OsPRI3/OsbHLH59 were ubiquitinated by OsHRZ1 or by OsHRZ2 (Kobayashi et al., 2019).

These discrepancies may be due to the different methodologies and materials used in these studies. Whether these interactions participate to the ubiquitination and degradation of OsPRI1/OsbHLH60, OsPRI2/OsbHLH58 and OsPRI3/OsbHLH59 remains to be further demonstrated and confirmed. IDEF1, whose activity is necessary for HRZs expression, is also A c c e p t e d M a n u s c r i p t degraded via the 26S proteasome pathway through a yet uncharacterized mechanism [START_REF] Zhang | The Bowman-Birk Trypsin Inhibitor IBP1 Interacts with and Prevents Degradation of IDEF1 in Rice[END_REF]. However, IDEF1 degradation is inhibited by its interaction with the IBP1.1 (IDEF1-BINDING PROTEINS 1.1) Bowman-Birk trypsin inhibitor protein (Figure 4) [START_REF] Zhang | The Bowman-Birk Trypsin Inhibitor IBP1 Interacts with and Prevents Degradation of IDEF1 in Rice[END_REF]. BTSL1 and BTSL2, two Arabidopsis homologs of BTS, function redundantly as negative regulators of the iron deficiency response [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]. In addition, BTSL1 and BTSL2 could directly target FIT/bHLH29 and promote its ubiquitination and subsequent degradation via the 26S proteasome pathway, thus negatively regulating the expression of iron uptake related genes (Figure 3) (Rodríguez-Celma et al., 2019b;Rodríguez-Celma et al., 2019a). In apples, MdBT1 and MdBT2, two BTB-TAZ proteins, interact with MdbHLH104. MdBT proteins interact as well with MdCUL3

(CULLIN-RING UBIQUITIN LIGASE 3) to form MdBT MdCUL3 complexes required for MdbHLH104 ubiquitination and degradation via the 26S proteasome pathway (Zhao et al., 2016a). In contrast, MdSIZ1, a SIZ/PIAS-type SUMO E3 ligase, directly sumoylates MdbHLH104, especially under iron deficiency conditions, to enhance its stability (Zhou et al., 2019). Recently, it was shown that the mutation of an alanine into valine within IDT1/bHLH34 enhances both its stability and nuclear localization [START_REF] Sharma | The dual benefit of a dominant mutation in Arabidopsis IRON DEFICIENCY TOLERANT1 for iron biofortification and heavy metal phytoremediation[END_REF].

Since this amino acid is conserved among clade IVc bHLH TFs in both monocots and dicots [START_REF] Sharma | The dual benefit of a dominant mutation in Arabidopsis IRON DEFICIENCY TOLERANT1 for iron biofortification and heavy metal phytoremediation[END_REF], one might speculate that this amino acid plays an important role in regulating the stability of clade IVc bHLH and thus their degradation via the 26S proteasome.

Phosphorylation plays also an important role in determining TF activity in the iron homeostasis networks (Figure 3). In Arabidopsis, FIT/bHLH29 phosphorylation by the calcium-dependent protein kinase CIPK11 positively regulates its activity by favoring its nuclear accumulation and dimerization with bHLH39 [START_REF] Gratz | CIPK11-dependent phosphorylation modulates FIT activity to promote Arabidopsis iron acquisition in response to calcium signaling[END_REF]. CIPK11 is induced and activated via a CBL1/9-mediated Ca 2+

sensing pathway under iron deficiency conditions. CIPK21 could also interact with FIT/bHLH29, indicating that this protein kinase might have a potential role in the regulation of the FIT/bHLH29-dependent iron deficiency responses [START_REF] Gratz | CIPK11-dependent phosphorylation modulates FIT activity to promote Arabidopsis iron acquisition in response to calcium signaling[END_REF]. Recently, the phosphorylation of URI/bHLH121 was also reported as a key mechanism that regulates its activity [START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF]. It was shown that the phosphorylated form of URI/bHLH121 only accumulates in response to iron deficiency, whereas the turnover of phosphorylated URI/bHLH121 is dependent on BTS activity [START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF]. Under iron deficiency, URI/bHLH121 showed enhanced heterodimerization capability with IVc bHLH TFs and increased binding ability to the promoter of its target genes, indicating that the et al., 2019). Nevertheless, since URI/bHLH121 can activate the expression of FER1, FER3

and FER4 in the stele when iron availability is not limiting (Gao et al., 2020b), a growth condition where the phosphorylated form of bHLH121 is degraded [START_REF] Kim | The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI[END_REF], it is likely that URI/bHLH121 is transcriptionally active independently of its phosphorylation state. Interestingly, as stated earlier, the pattern of accumulation of URI/bHLH121 within the root cells is controlled by an iron-dependent mechanism. URI/bHLH121 is preferentially accumulated in the epidermis and the cortex when plants are grown under iron deficiency and in the stele when grown under iron sufficiency. From these observations, one might hypothesize that URI/bHLH121 cellular distribution, rather than its transcriptional activity, is regulated by its phosphorylation state. However, the precise regulatory mechanism leading to the phosphorylation of URI/bHLH121 remains to be characterized.

Conclusions and future prospects

In the past two decades, remarkable progresses have been made in decrypting the molecular mechanisms that regulate iron homeostasis in both strategy I and strategy II plants (Figure 1).

They highlighted that iron homeostasis in plants is regulated at the transcriptional level and involves several bHLH TFs that function in intricate regulatory networks (Figures 2 to 4).

The function of most of these bHLH TFs (i.e. clades Ib, IIIa, IVb and IVc) is conserved among grass and non-grass species (Figure 2), in contrast to the downstream target genes of iron acquisition machinery, which are distinctive in strategy I and strategy II plants (Figure 1). How these bHLH functional homologs have evolved to target different genes is an intriguing question. bHLH from clade IVa are involved in the regulation of iron homeostasis in Arabidopsis but whether or not this clade of bHLH TFs has a similar role in other plant species needs to be investigated. Additional investigations will also be necessary to determine strategy II, and between annual and perennial species, is still on its own a question that deserves to be addressed.

How plants sense iron status and switch on or repress the downstream regulatory network to regulate iron uptake, translocation, storage or assimilation has remained elusive. To date, haemerythrin E3-ubiquitin ligases are the main candidates (Rodríguez-Celma et al., 2019a).

These E3-ubiquitin ligases are induced in response to iron deficiency, participate to the degradation of key bHLH TFs (i.e. clade IVc and FIT/bHLH29) and are destabilized upon iron binding at their haemerythrin motifs. IDEF1, the rice iron binding TF mentioned earlier,

was also proposed as a potential iron sensor (Kobayashi et al., 2012;[START_REF] Kobayashi | The rice transcription factor IDEF1 is essential for the early response to iron deficiency, and induces vegetative expression of late embryogenesis abundant genes[END_REF][START_REF] Kobayashi | The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants[END_REF]. The characterization of IDEF1 functional homologs in non-grass species would be an additional element in support of this later hypothesis. Another important question concerns the regulation of the most upstream bHLH TFs. For instance, the main challenges would be to determine how the expression of clade IVc bHLH is controlled and to identify which protein kinase modulates URI/bHLH121 activity.

Recently, epigenetic regulation has emerged as playing important role in the control of iron homeostasis, by regulating DNA accessibility to promoters, and thus the expression of both TFs and iron uptake genes. This is for instance the case in Arabidopsis with the irondependent deposition of repressive marks on H3 histone (i.e. H3K27m3) at the promoter loci of FIT/bHLH29, IRT1 and FRO2 [START_REF] Park | PRC2-mediated H3K27me3 contributes to transcriptional regulation of FIT-dependent iron deficiency response[END_REF]. This additional layer of regulation in the transcriptional networks controlling iron homeostasis raises questions on the importance and the significance of such regulatory mechanism in this process.

Within this review, it is possible to grasp the extent of the complexity of the regulatory networks that regulate iron homeostasis in plants. Nevertheless, another level of complexity still to be investigated concerns the apparent redundancies existing between several TFs, the localization of their target genes, and thus the physiological functions that are controlled.

This will be achieved by the in depth study of the tissue and cellular localization of the Unfortunately, such data are only available for a few of these proteins [START_REF] Long | The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots[END_REF]Gao et al., 2020a;[START_REF] Samira | The bHLH transcription factor ILR3 modulates multiple stress responses in Arabidopsis[END_REF].

In conclusion, much work still lies ahead to fully comprehend the transcriptional regulatory network that regulate iron homeostasis in plants, which might offer novel opportunities for improving plant growth and health and for generating iron-fortified crops.

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  A c c e p t e d M a n u s c r i p t bHLH TFs involved in the regulation of iron homeostasis in strategy II plants

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  s c r i p t different TFs as well as the proteins involved in their regulation. Such knowledge is necessary to fully decrypt and understand the dynamics of this regulatory process.

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  as well as phosphorylation and sumoylation, are crucial posttranslational modifications for the regulation of key bHLH TFs activities involved in the iron homeostasis networks, notably clade IVc bHLH FIT/bHLH29 and URI/bHLH121. Whether such modifications participate to the regulation of other TF activities within this network is still to be assessed. Determining the degree of conservation of this regulatory network between plants from strategy I and

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	homeostasis. Other regulatory proteins are also conserved between plants from both
	strategies. It is for instance the case for MYB and ERF TFs (Table 2) or for haemerythrin E3-
	ubiquitin ligases. Interestingly, BTB-TAZ-CUL3 ubiquitin ligase complexes participate to

the degradation of clade IVc bHLH in apples. To date, such mechanism has only been observed in apple, raising the question of the conservation of this regulatory mechanism in other plants species, from non-grass to grass and from perennials to annuals. Ubiquitination,

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Acknowledgements

This work was supported by grants from the "Agence Nationale de la Recherche (ANR)" to C.D. Support was provided by the China Scholarship Council to F.G. We thank Florence Vignols for help in preparing this article.

Author Contribution

The tree was constructed by using the neighbor-joining method with the MEGA software (v10.0.5) using full-length bHLH amino acid sequences. The bootstrap analysis was carried out with 1000 replicates. Sequences were aligned prior to the construction of the phylogenetic tree using the ClustalX software (v2.0.11). The different bHLH clades are designated as previously reported [START_REF] Heim | The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity[END_REF]


N Védrenne

A Montmerle-Bonnefois

C B Lim

C Petit

J.-F Sauvage

S Meimon

P Perrault

F Mendez

B Fleury

J Montri

J.-M Conan

V Michau

Z Sodnik

C Voland

First experimental demonstration of adaptive optics pre-compensation for GEO feeder links in a relevant environment

Keywords: adaptive optics, anisoplanatism, ground station for optical feeder links. I

The future of very high throughput optical links between the ground and GEO satellites depends on the ability to overcome turbulence channel disruptions caused by the optical propagation through the atmosphere. Pre-compensation by adaptive optics has been identified as a game changer, as it could theoretically provide for the uplink the additional margin necessary to secure the link budget. Several experimental activities have been reported to investigate the practical increase that can be expected from AO pre-compensation, demonstrating very promising results. Due to the proximity of the line of sight from ground and the strong variability of turbulence conditions these results would benefit from a confirmation on a more representative line of sight. The line of sight between ESA's Optical Ground Station and the top of Mount Teide provides an unprecedented opportunity to provide this confirmation in a relevant environment. We report here the results of the first experimental demonstration of uplink pre-compensation performed on this line of sight in April 2019. The increase of average optical power is demonstrated and compared to expected performance. The influence of anisoplanatism is demonstrated and quantified;

consequences on the future of GEO feeder links are inferred.

INTRODUCTION

Link budget of uplink GEO feeder links is compromised by atmospheric turbulence influence. Adaptive optics (AO) has been identified as an enabling technology to mitigate channel impairments. Several experiments have been recently reported that demonstrated the technology feasibility for pre-compensation. These demonstrations took place either on a short path [START_REF] Brady | Experimental validation of phase-only precompensation over 494 m free-space propagation[END_REF] or on a 10 km long horizontal path [START_REF] Saathof | Pre-correction adaptive optics performance for a 10 km laser link[END_REF]. In both cases the representativeness of the line of sight is limited. Short distance implies limited turbulence strength; horizontal path induces strong perturbations that will affect wavefront sensing performance and the relevancy of precompensation efficiency. As a consequence, adaptive optics compensation still has to be demonstrated in representative turbulent environment. Although analytical models are available for quantifying the effect of point ahead angle (PAA) anisoplanatism on ground-space optical links (see [START_REF] Conan | Adaptive optics for ground -GEO-satellites optical links: from system design to experimental demonstration[END_REF] and references therein), an experimental assessment of its impact on the link budget remains of great interest.

The FEEDEr LInk adaptive Optics (FEEDELIO) project aimed at demonstrating the feasibility of AO pre-compensation in a relevant environment and to assess expected performance for variable turbulent conditions in the presence of anisoplanatism.

II. FEEDELIO EXPERIMENT

A. Overview of the experimental setup

The FEEDELIO experiment took place in Tenerife during the two first weeks of April 2019 on a 13.2 km 5° elevation slant path between ESA's optical ground station (OGS) and top of mount Teide [START_REF] Montmerle-Bonnefois | Adaptive optics precompensation of a GEO feeder link: the FEEDELIO experiment[END_REF].

The hardware block diagram of the experiment setup, already presented in [START_REF] Védrenne | Adaptive optics pre-compensation for GEO feeder links: Towards an experimental demonstration[END_REF] [START_REF] Bonnefois | Adaptive optics pre-compensation for GEO feeder links: the FEEDELIO experiment[END_REF] is recalled Figure 1.

To provide the adaptive optics reference beam and the detection of the uplink pre-compensated beam a satellite terminal breadboard (STB) emulator was attached to the concrete wall of the cable car accommodation building.

To provide the uplink pre-compensated beam a ground terminal breadboard (GTB) was attached on the south pillar of ESA's OGS.

B. Satellite terminal breadboard

The STB was providing the 1550 nm reference beacon for wavefront sensing and the uplink detection. Uplink detection was performed through two multimode fibers coupled 1.7 mm diameter collimators sending the light on a 20 kHz low noise PIN detectors installed in two separate modules: one on axis module also providing the reference beacon, and one off axis movable module. In order to investigate the influence of point ahead angle, the off axis module was fixed on a translating platform remotely controlled hence providing the possibility to investigate a range from 3µrad up to 100µrad angular excursion.

C. Ground terminal breadboard

The GTB was composed of a 350 mm commercial telescope and of an optical breadboard carrying the adaptive optics system, the downlink measurement path and the uplink 1625 nm source. In order to cope with the evolution of turbulent conditions during the experiment the acquisition protocol was designed to address alternatively on and off axis module within a limited duration thanks to the use of a dedicated fast steering mirror on the uplink path. A 10s time period has been used here. In this timeframe turbulence conditions (Fried parameter and scintillation index) has proven to be stable enough with respect to AO error budget.

D. Adaptive optics

The adaptive optics system has been built from commercial off the shelf components: the deformable mirror was an Alpao 95-17 (fast upgrade), the wavefront sensor camera was a Raptor Owl HS, and the real-time computer was provided by Shakti upon ONERA's requirements. The wavefront sensor counted 8x8 subapertures and was used at a 1.5 kHz frequency providing a 2,3 frame delay. The adaptive optics system has been to provide a 2.2 dB (0,6 attenuation) average coupling loss (wrt diffraction limit) for r 0 = 11 cm. Thanks to the large stroke of the deformable mirror no dedicated tip/tilt mirror has been used. A motorization of the deformable mirror mount was only used to cope with refraction index evolution encountered during daytime. More details on the AO loop performance for downlink are available in [START_REF] Lim | Single-mode fiber coupling with adaptive optics for free-space optical communication under strong scintillation[END_REF]. We focus here on the uplink results.

III. EXPERIMENT RESULTS

A. Data acquisitions

Due to very poor weather conditions during the first week of the experiment (access to cable car closed), STB could only be installed at the very beginning of the second week of experiment. Experiment calibration and adjustments took 3 days, leaving four days for data acquisitions. We focus here on the last day of acquisitions (14/04/19). During one set of acquisitions uplink power measured on both on-axis and offaxis modules has been recorded simultaneously with a synchronized protocol. AO loop telemetry (wavefront slopes, voltages sent to the deformable mirror and intensity per subaperture) has been recorded jointly.

B. Turbulence parameters estimation

Wavefront sensor measurements are exploited to estimate optical turbulence conditions. A Fried parameter (r 0 ) estimate is obtained from a fit of the Zernike's spatial variance with Noll's power law [START_REF] Noll | Zernike polynomials and atmospheric turbulence[END_REF], downlink power fluctuations are computed from Shack-Hartmann wavefront sensor (SHWFS) intensity measurements. By exploiting the spatial correlation of slopes and intensity measurements, assuming small perturbations as described in [START_REF] Védrenne | Cn² profile measurement from Shack-Hartmann data: experimental validation and exploitation[END_REF], a C n ² profile estimate can be computed for every 5s time slot. This C n ² profile can be used to compute analytically another estimate of r 0 according to [START_REF] Fried | Statistics of a Geometric Representation of Wavefront Distortion[END_REF] and of power fluctuations normalized variance in a given square subaperture σ² i according to aperture averaged scintillation theory [START_REF] Fried | Aperture Averaging of Scintillation[END_REF] [START_REF] Churnside | Aperture averaging of optical scintillations in the turbulent atmosphere[END_REF]. Turbulence profile estimation also enables the computation of an isoplanatic angle estimate [START_REF] Fried | Anisoplanatism in adaptive optics[END_REF]. All these integrated parameters estimations are performed while taking into account the sphericity of the incoming beam from the STB module. The results of this turbulent profile estimation are presented in Figure 2. The top of the figure presents r 0 estimation from SHWFS data (obtained from Noll's fit) in black and the result of r 0 estimation from the estimated C n 2 as a function of time. The center of the figure presents σ² i measured by the SHWFS in black and its estimation from the C n ² profile in blue. Apart from the cases where the smooth perturbation approximation is obviously not satisfied (outside the Rytov regime, corresponding to log-amplitude variance above 0.3 ie σ² i > 1.2) the turbulence profile estimate provides a reliable evaluation of r 0 and σ² i hence a good confidence in the estimation of the isoplanatic angle θ 0 . In the following, the study is restricted to turbulence cases within the smooth perturbation approximation. Considering the elevations expected from future GEO-feeder links, above 30° in European area, this hypothesis shall be satisfied.

As illustrated here a wide variability of anisoplanatism conditions has been encountered, with isoplanatic angle θ 0 values ranging from 25µrad down to less than 5 µrad.

C. Anisoplanatism influence and turbulence conditions variability

We illustrate here for two cases the influence of anisoplanatism on the correction efficiency obtained during the experiment. We plot on Figure 3 the average coupling efficiency < I > of the flux in the receiver with respect to the geometrical losses (diffraction limited beam corresponds to < I > = 0.74 due to the uplink central obscuration). Two cases corresponding to different perturbation strength are illustrated: a favorable case, obtained by nighttime (22:56 p.m.), with r 0 = 10 cm, θ 0 = 10 µrad; a more perturbed case, obtained by daytime, around 10:15 a.m., with r 0 = 5 cm, θ 0 = 6 µrad.

To cope with potential turbulence non-stationarity both onaxis and off-axis measurements are performed alternatively with a 10s time period. We investigate four off-axis positions, each of them corresponding also to an on-axis measurement. We plotted the average of the four on-axis measurements (crosses) performed during the acquisition of the off-axis measurements and theoretical model results (round symbols) for 3 correction cases : red for a correction of all the degrees of freedom enabled by the deformable mirror, blue for a tip/tilt only correction, and without correction in black. Off axis measurements as a function of the point ahead angle are also reported: continuous lines correspond to measurements, dashed lines to theoretical performance models while taking into account the estimated turbulent profile. Anisoplanatism influence appears significantly different for both cases: by daytime, angular decorrelation of the perturbations induces a steep decrease of < I > as a function of the PAA. By nighttime the correction degradation is much more progressive.

For both cases the theoretical model (see [START_REF] Conan | Adaptive optics for ground -GEO-satellites optical links: from system design to experimental demonstration[END_REF] and references therein), not described here, adequately reproduces the measurement for on-axis and off axis cases.

D. Transposition to a GEO-feeder link case.

We analyze here the contributors to the error budget affecting off axis performance. We first study the on-axis measurements and compare them to theoretical evaluation to assess the on-axis error budget. Then we consider the offaxis terms for a PAA value representative of GEO-feeder link anisoplanatism conditions and compare it to theory.

In the presence of anisoplanatism (hence for off-axis measurement) the theoretical average power penalty term attributed to the correction of atmospheric effects can be written in a first approximation as:

< I > aniso ≈ ρ 0 exp[-σ fitting ² -σ scintillation ² -σ aniso ²(PAA)] (1)
In Equation ( 1), ρ 0 is the diffraction limited coupling efficiency for a 23% obscuration antenna (ρ 0 = 0.74 according to [START_REF] Klein | Optical Antenna Gain. 1: Transmitting Antennas[END_REF]), σ fitting ² stands for the fitting error term computed as follows [START_REF] Conan | Etude de la correction partielle en optique adaptative[END_REF]: σ fitting ² = 0.458 (n r +1) -5/3 (D/r 0 ) 5/3 (2) σ scintillation ² designates the variance of log amplitude fluctuations affecting coupling efficiency, it is approximated here, assuming the small perturbation approximation and neglecting the influence of aperture averaging at the scale of a subaperture, by σ i ²/4;while σ aniso ²(PAA) designates the anisoplanatism error contribution at PAA, and can be computed according to [START_REF] Chassat | Propagation optique a travers la turbulence atmospherique : etude modale de l'anisoplanetisme et application a l'optique adaptative[END_REF]. The ultimate limit to AO performance is fixed by the fitting error term. Its contribution is plotted in Figure 4 in dashed black lines. The on-axis measurements are plotted with black crosses. In order to limit the dispersion of the data plotted, measured values have been averaged over 1 cm bins in r 0 . Error bars correspond to the standard deviation of the values averaged on a 1 cm r 0 slot (when several measurements are available). Note that measurements have been normalized in units consistent with the theoretical average coupling efficiency defined by Eq. ( 1). Clearly on-axis measurements are smaller than fitting only performance. Adding the scintillation term of Eq. ( 1) however leads to a good fit of on-axis data. The choice of the FEEDELIO off-axis angle giving a relevant GEO-Feeder PAA emulation is a delicate question. Two options are discussed in [START_REF] Védrenne | Adaptive optics pre-compensation for GEO feeder links: Towards an experimental demonstration[END_REF]. The most relevant, that we will call further the modal approach is to consider an off-axis angle leading to a performance degradation, compared to on-axis, identical to the one expected for similar conditions in the case of GEOfeeder link. This implies a fine modeling of the anisoplanatism error accounting for the number of corrected modes and the turbulence profile. This is out of the scope of this paper. Another possibility is to consider a more straightforward but somewhat unprecise approach based on the conservation of the ratio between PAA and isoplanatic patch θ 0 . The PAA for a GEO-feeder link is 18 µrad. According to recently published statistics in Cerro-Paranal [START_REF] Osborn | Atmospheric turbulence forecasting with a general circulation model for Cerro Paranal[END_REF] median isoplanatic patch for such an observatory-like location is θ 0 =10 µrad. Without any exhaustive documentation of this quantity around the globe, considering such a value as representative might be questionable. Still, based on these elements one would want to conserve the ratio PAA/θ 0 =1.8 is. As exposed in [START_REF] Védrenne | Adaptive optics pre-compensation for GEO feeder links: Towards an experimental demonstration[END_REF], this simple approach could however lead to a (very) pessimistic estimate of anisoplanatism compared to the modal approach. Hence the trends identified in this case must be considered cautiously. Based on the analysis given in [START_REF] Védrenne | Adaptive optics pre-compensation for GEO feeder links: Towards an experimental demonstration[END_REF], a reasonable range from the equivalent PAA could be between PAA equivalent = θ 0 and PAA equivalent = 1.8 θ 0 . Results obtained from the experiment in these two cases together with their theoretical evaluation based on the estimated profile are plotted Figure 4 as a function of the encountered r 0 . They can be compared to on-axis tip/tilt only correction measurements (black crosses). These data points are measured on-axis but we remind that tip/tilt correction at PAA is barely affected by anisoplanatism [START_REF] Védrenne | Adaptive optics pre-compensation for GEO feeder links: Towards an experimental demonstration[END_REF]. This is confirmed by Figure 3: tip/tilt corrected results being constant in the range PAA = 0 and PAA= 1.8 θ 0 . The analysis of the experimental results demonstrate the interest for high order pre-compensation in the presence of anisoplanatism, as PAA affected measurements are systematically above tip/tilt only correction. Besides, theoretical performance evaluation from anisoplanatism models accurately fit the off-axis measurements. Assuming the relevant equivalent PAA is between PAA equivalent = θ 0 and PAA equivalent = 1.8 θ 0, the gain wrt tip/tilt only correction shall be between 1 dB and 2 dB depending on the turbulence profile.

E. Discussion

The analysis provided here has only been based on average power losses encountered on a single day of acquisition. In order to consolidate the data analysis, deeper investigations are therefore necessary: evaluation of power statistics (mean and fluctuations) in a wide range of turbulence conditions, precise evaluation of the equivalent PAA based on the modal approach. The temporal signature of power losses could also be studied and compared to the GEO-Feeder case. With respect to average power penalty, for the turbulence conditions encountered here, results obtained by precompensation led to a few dB gain compared to tip/tilt only correction. This may seem small but one has to remember that AO not only increases the average detected flux but also drastically reduces flux fluctuations and budget link gains can be shown to be mainly driven from this latter effect. Based on numerical modeling, higher order correction indeed gives few dB gains on average flux, compared to tiptilt, but gain is much more substantial (often more than 10 dB) in link availability thresholds Erreur ! Source du renvoi introuvable.. PAA anisoplanatism is still a strong limiting factor especially when going to large aperture (few 10 cm) emitters [START_REF] Conan | Adaptive optics for ground -GEO-satellites optical links: from system design to experimental demonstration[END_REF]. To truly benefit from the Tx full antenna gain one needs to find ways to reduce this anisoplanatism effect. Several strategies can be considered to circumvent PAA influence. Among others the design of appropriate control laws or the use of specific configurations of laser guide stars are already investigated. The most drastic solution is probably the concept suggested by Tyson in 1996 [START_REF] Tyson | Adaptive optics and ground-to-space laser communications[END_REF], ie the use of a free flyer support satellite to provide the beacon in the direction of the PAA.

CONCLUSION

We reported here the results of the first experimental demonstration of pre-compensation for GEO -feeder links in a relevant environment. The experiment took place in Tenerife April 2019 on a 13.2 km slant path line of sight between ESA's OGS and top of Mount Teide in Tenerife. Due to ground proximity, strong turbulence perturbations conditions were encountered. Restricting the analysis to smooth perturbations case, more relevant for GEO-feeder links, the feasibility of a pre-compensation by adaptive optics has been confirmed. Performance models to estimate the average power penalty fit well with measurements. The influence of PAA has been quantified experimentally and compared successfully to anisoplanatism error evaluation. The question of the off-axis angle equivalent to GEO-feeder PAA is discussed. Depending on the assumptions taken for this equivalence, gains in average power brought by correction beyond tip-tilt vary but are systematic. Besides, link budget has to account both for average power and statistical distributions. A systematic analysis of the collected data statistics is therefore now required. Particular interest shall also be granted to temporal signature of the power fluctuations. In any case, anisoplanatism highly restricts the potential gain of AO with significant size telescopes (few tens of cm). Appropriate strategies must be investigated to circumvent anisoplanatism limitation. Among others, the use of a free flyer satellite to provide the beacon for wavefront sensing in the direction of the PAA is conceptually the most promising one.

Figure 1 -

 1 Figure 1 -Hardware block diagram of the FEEDELIO experiment.

Figure 2 -

 2 Figure 2 -Top: Fried parameter estimated from Shack-Hartmann measurement (black) and from turbulence profile estimation (blue). Center: flux fluctuations variance per subaperture from Shack-Hartmann measurements (black) and estimated from turbulence profile (blue). Bottom: isoplanatic angle estimated from turbulence profile.

Figure 3 -

 3 Figure 3 -Evolution of the average coupling wrt diffraction limit as a function of the PAA, top for a small turbulence case (r 0 = 10 cm, θ 0 = 10 µrad), bottom for the stronger turbulence case (r 0 = 5 cm, θ 0 = 6 µrad). Continuous line: off-axis experiment, dashed line: offaxis theory. Crosses: on axis measurements; rounds: on axis theory. Red: full correction; blue: tip/tilt only; black: no correction.

Figure 4 -

 4 Figure 4 -Average coupling on the STB side as a function of r 0 . Black "+": measurements with all possibly corrected degrees of freedom, dashed lines: theoretical evaluation with fitting error term only; red dashed lines: theoretical evaluation including fitting and scintillation influence. Black crosses: on-axis tip/tilt correction only measurements; blue crosses: average coupling for PAA = 1.8 θ 0 (measures), dashed blue line: average coupling for PAA = 1.8 θ 0 (experiment).

ACKNOWLEDGMENT

This work has been performed in the framework of ESA's contract N°4000120300/17/NL/PS. FEEDELIO experiment could not have been possible without the very helpful contributions of the technical staff from Instituto Astrofísica de Canarias, Teide's National Park and Teide's cable car station. The author warmly thanks them for their fruitful help and collaboration.