Scopoletin 8-Hydroxylase-Mediated Fraxetin Production Is Crucial for Iron Mobilization^{<a href="#fn1" rid="fn1" class=" fn">1</a>,}^{<a href="#fn2" rid="fn2" class=" fn">[OPEN]</a>}

Abstract

As a key edaphic factor, soil pH has a strong impact on the availability of mineral nutrients and the distribution of species in natural plant communities. Iron (Fe) solubility decreases dramatically with increasing pH, excluding so-called calcifuge (“chalk-fleeing”) species from carbonate-rich, alkaline soils due to their inability to acquire sufficient Fe under such conditions. Calcicole behavior, i.e. the ability to thrive on alkaline soils, has been attributed to the efficiency of Fe acquisition of a cultivar or species, a trait that strongly contributes to the ability to survive and reproduce on such soils (Zohlen and Tyler, 2004).

Plants have been categorized into two phylogenetically distinct groups that have evolved mutually exclusive mechanisms to increase the solubility of Fe (Römheld and Marschner, 1986). In nongraminaceous species, such as Arabidopsis (Arabidopsis thaliana), Fe is mobilized by net proton extrusion into the rhizosphere mediated by the P-type ATPase AHA2 (Santi and Schmidt, 2009) and reduced through the root surface Fe chelate reductase FERRIC REDUCTION OXIDASE2 (FRO2; Robinson et al., 1999) before Fe ^{is taken up via the high-affinity Fe transporter IRON REGULATED TRANSPORTER1 (IRT1;} Eide et al., 1996; Vert et al., 2002). This mechanism has been referred to as strategy I (Römheld and Marschner, 1986; Brumbarova et al., 2015). Grasses (Poales) have adopted a system in which secretion of high-affinity Fe chelators of the mugineic acid family, generically referred to as phytosiderophores, precedes uptake of the Fe^{-phytosiderophore complex without prior reduction (strategy II;} Kobayashi and Nishizawa, 2012). However, recent studies suggest that secretion of Fe-chelating compounds is not unique to graminaceous species. In Arabidopsis, the scopoletin pathway is reprogrammed upon Fe deficiency to produce and secrete coumarins with Fe-mobilizing properties, which is of particular importance in alkaline soils in which the free ion activity of Fe is extremely low (Rodríguez-Celma et al., 2013; Schmid et al., 2014; Fourcroy et al., 2014; Schmidt et al., 2014). Expression profiling, genetic, and biochemical approaches implicated the 2-oxoglutarate and Fe(II)-dependent dioxygenase (2OGD) FERULOYL-COA 6'-HYDROXYLASE1 (F6′H1) and the ABC transporter PLEIOTROPIC DRUG RESISTANCE9 (PDR9) as key nodes in the biosynthesis and secretion of Fe-mobilizing coumarins, respectively (Kai et al., 2008; Yang et al., 2010; Lan et al., 2011; Rodríguez-Celma et al., 2013; Schmid et al., 2014; Fourcroy et al., 2014). F6'H1 mediates the ortho-hydroxylation of feruloyl CoA to generate scopoletin, which, together with its β-glucoside scopolin, constitutes the most abundant coumarin in Arabidopsis (Kai et al., 2008; Döll et al., 2018). Subsequent studies revealed that aglycones of coumarins containing a catechol moiety, such as esculetin and fraxetin, were the dominant active compounds in Arabidopsis (Schmidt et al., 2014; Strehmel et al., 2014; Sisó-Terraza et al., 2016a). However, the final step(s) of the biosynthetic pathway of these compounds has not yet been annotated.

The assimilation of Fe is under a surprisingly complex, multifaceted control, which is required to keep Fe within a tight cellular concentration. Similar to other key processes in Fe acquisition, the biosynthesis and secretion of coumarins is regulated by the basic helix-loop-helix (bHLH)-type transcription factor FIT (Ivanov et al., 2012; Colangelo and Guerinot, 2004; Schmidt et al., 2014). FIT dimerizes with the subgroup Ib bHLH proteins, bHLH38, bHLH39, bHLH100, and bHLH101, to regulate its target genes (Yuan et al., 2008; Sivitz et al., 2012; Wang et al., 2013). The transcription of FIT and its partners is responsive to Fe and directly activated by three other bHLH proteins, bHLH34, bHLH104, and bHLH105 (Li et al., 2016; Zhang et al., 2015). These three proteins also activate the expression of POPEYE, a negative regulator of the Fe deficiency response (Long et al., 2010), which regulates a subset of genes that do not overlap with the FIT regulatory network. The abundance of bHLH104 and bHLH105 is likely regulated by the Fe-binding hemerythrin domain-containing E3 ligase BRUTUS in a proteasomal-dependent manner (Long et al., 2010; Selote et al., 2015; Li et al., 2016; Zhang et al., 2015). Coregulation of all components of the reduction-based Fe uptake system suggests high cooperativity of the various processes. It is therefore reasonable to speculate that the final step(s) of the biosynthesis of Fe-mobilizing coumarins is part of the same regulon.

Iron deficiency-induced production of Fe-mobilizing compounds by nongraminaceous species and their subsequent release into the rhizosphere has been observed over several decades (Dakora and Phillips, 2002; Tsai and Schmidt, 2017). Since their discovery, the role of root exudates in Fe acquisition was subject to a long-standing debate. Both reductive release of Fe from Fe ^{chelates and chelation of Fe to provide the substrate for an unidentified ferric chelate reductase were suggested as functions of Fe deficiency-induced phenolic secretion (}Brown and Ambler, 1973; Hether et al., 1984; Römheld and Marschner, 1984). After the molecular identification of the Fe chelate reductase FRO2 in Arabidopsis (Robinson et al., 1999), the so-called “reductants” were considered as being of minor importance in Fe acquisition. Several aspects collectively led to a gross underestimation of the ecological significance of Fe-mobilizing compounds of strategy I species. First, in the laboratory, plants are generally grown at optimal, i.e. slightly acidic pH at which the Fe acquisition machinery works most efficiently. However, Fe chlorosis is most frequently observed at alkaline pH, conditions under which FRO2 activity is compromised (Susín et al., 1996). As a second factor, experiments are mostly conducted with highly soluble sources of Fe, such as Fe ^{complexed by aminopolycarboxylates, at concentrations that well exceed the} K_m of FRO2, a situation that is not likely to occur in situ. Under such conditions, the activity of FRO2 is presumably rate-limiting for Fe uptake, which makes the contribution of the secretion of Fe-scavenging compounds to Fe uptake insignificant.

The question as to the traits that allow calcicole plants to thrive on neutral or alkaline soil has puzzled plant biologists for more than two centuries (Link, 1789; Unger, 1836; Grime and Hodgson, 1968). Attempts to single out specific factors have been largely unsuccessful, an approach that is also hampered by the difficulties inherent to interspecies comparisons (De Silva, 1934; Lee, 1998; Schmidt and Fühner, 1998). The huge variety of the secreted substances and a lack of knowledge regarding the exact chemical nature and precise role of the secreted compounds have excluded targeted approaches and have rendered attempts to associate secreted secondary metabolites with the autecology of species difficult. In this study, we addressed the biosynthesis and ecological role of Fe-mobilizing coumarins secreted by Fe-deficient plants. We report that besides the ortho-hydroxylation of feruloyl CoA by F6’H1, the introduction of a second hydroxyl group in the ortho position, a reaction catalyzed by scopoletin 8-hydroxylase (S8H; At3g12900), is critical for producing the Fe-mobilizing coumarin fraxetin. Scopoletin hydroxylation at the C8 position by At3g12900 was shown previously (Siwinska et al., 2018). By comparing diverse Arabidopsis accessions differing in the ability to grow on alkaline media supplemented with an immobile Fe source, we further show that the abundance of fraxetin in the media determines the ability to acquire Fe at elevated pH.

Acknowledgments

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