bioRxiv preprint doi: https://doi.org/10.1101/197806; this version posted October 4, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Scopoletin 8-hydroxylase: a novel enzyme involved in coumarin biosynthesis and iron- 2 deficiency responses in Arabidopsis 3 4 Running title: At3g12900 encodes a scopoletin 8-hydroxylase 5 6 Joanna Siwinska1, Kinga Wcisla1, Alexandre Olry2,3, Jeremy Grosjean2,3, Alain Hehn2,3, 7 Frederic Bourgaud2,3, Andrew A. Meharg4, Manus Carey4, Ewa Lojkowska1, Anna 8 Ihnatowicz1,* 9 10 1Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of 11 Gdansk, Abrahama 58, 80-307 Gdansk, Poland 12 2Université de Lorraine, UMR 1121 Laboratoire Agronomie et Environnement Nancy- 13 Colmar, 2 avenue de la forêt de Haye 54518 Vandœuvre-lès-Nancy, France; 3INRA, UMR 14 1121 Laboratoire Agronomie et Environnement Nancy-Colmar, 2 avenue de la forêt de Haye 15 54518 Vandœuvre-lès-Nancy, France; 16 4Institute for Global Food Security, Queen’s University Belfast, David Keir Building, Malone 17 Road, Belfast, UK; 18 19 [email protected] 20 [email protected] 21 [email protected] 22 [email protected] 23 [email protected] 24 [email protected] 25 [email protected] 26 [email protected] 27 [email protected] 28 *Correspondence: [email protected], +48 58 523 63 30 29 30 The date of submission: 02.10.2017 31 The number of figures: 9 (Fig. 2, 6, 9 in colour in print, in colour online) 32 The word count: 6 489 (from the start of the introduction to the end of the acknowledgements) 33 The number of supplementary data: 23 (Figs S1-S17, Table S1-S6) 1 bioRxiv preprint doi: https://doi.org/10.1101/197806; this version posted October 4, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 34 Highlight 35 A strongly iron-responsive gene of previously unknown function, At3g12900, encodes a 36 scopoletin 8-hydroxylase involved in coumarin biosynthesis and plays an important role in the 37 iron uptake strategy in Arabidopsis. 38 39 Abstract 40 Iron (Fe) deficiency represents a serious agricultural problem, particularly in alkaline soils. 41 Secretion of coumarins by Arabidopsis thaliana roots is induced under Fe-deficiency. An 42 essential enzyme for the biosynthesis of major Arabidopsis coumarins, scopoletin and its 43 derivatives, is Feruloyl-CoA 6’-Hydroxylase1 (F6′H1) that belongs to a large enzyme family 44 of the 2-oxoglutarate and Fe(II)-dependent dioxygenases. Another member of this family that 45 is a close homologue of F6’H1 and is encoded by a strongly Fe-responsive gene, At3g12900, 46 is functionally characterized in the presented work. We purified the At3g12900 protein 47 heterologously expressed in Escherichia coli and demonstrated that it is involved in the 48 conversion of scopoletin into fraxetin via hydroxylation at the C8-position. Consequently, it 49 was named scopoletin 8-hydroxylase (S8H). Its function in plant cells was confirmed by the 50 transient expression of S8H protein in Nicotiana benthamiana leaves followed by the 51 metabolite profiling and the biochemical and ionomic characterization of Arabidopsis s8h 52 knockout lines grown under various regimes of Fe availability. Our results indicate that S8H 53 is involved in coumarin biosynthesis as part of the Fe acquisition machinery. 54 55 Keywords: abiotic stress, Arabidopsis, enzyme activity, fraxetin, Fe- and 2OG-dependent 56 dioxygenase, plant–environment interactions, mineral nutrition 57 58 59 Introduction 60 Iron (Fe) is an essential micronutrient for all living organisms. In plants, chloroplast and 61 mitochondria have high Fe demand, and play key roles in the Fe homeostasis network in 62 photosynthetic cells (Nouet et al., 2011). Fe is abundant in soils, but its availability is often 63 limited due to soil conditions, which can be highly heterogeneous, such as pH and redox 64 presence of co-elements (Moosavi and Ronaghi, 2011; Marschner, 2012). Fe deficiency is a 65 serious agricultural problem particularly in alkaline and calcareous soils (Mengel, 1994). 66 These types of soils, which represent approximately 30 % of the world's cropland, are 2 bioRxiv preprint doi: https://doi.org/10.1101/197806; this version posted October 4, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 67 characterized by a higher pH that in combination with the presence of oxygen leads to Fe 68 precipitation in the form of insoluble ferric oxides (Fe2O3) (Morrissey and Guerinot, 2009). 69 70 Higher plants have developed two different types of strategies in response to Fe limitation. In 71 a reduction-based strategy (Strategy I), which occurs in all plant species except grasses, a 72 release of protons into the rhizosphere is enhanced (Marschner and Roemheld, 1994; Kim and 73 Guerinot, 2007) by Fe deficiency-induced proton-translocating adenosine triphosphatases, 74 such as AHA2 in Arabidopsis (Santi and Schmidt, 2009). As a result, at lower pH the ferric 75 oxide precipitates are being dissolved and the plasma membrane bound Ferric Chelate 76 Reductase 2 (FRO2) (Robinson et al., 1999) catalyse the reduction of ferric ions (Fe3+) into 77 more soluble and bioavailable to plants ferrous ions (Fe2+). Once reduced, Fe2+ are transported 78 into the root epidermal cells across the plasma membrane by the divalent metal transporter 79 Iron Regulated Transporter 1 (IRT1) (Vert et al., 2002; Varotto et al., 2002). A second 80 strategy, used by grasses, is based on the release of soluble mugineic acid family 81 phytosiderophores (PS) from the root epidermis and forming the complexes with Fe3+ (Takagi 82 et al., 1984; Marschner and Roemheld, 1994; Kim and Guerinot, 2007). The resulting Fe3+-PS 83 complexes are then transported into the root epidermis via a high-affinity uptake system 84 without the requirement of a reduction step (Curie et al., 2001; Kim and Guerinot, 2007). 85 86 The precise mechanisms underlying responses of Strategy I plants to low Fe availability in 87 calcareous soils are not clear. However, it is well documented that Fe deficiency enhanced 88 release of reductants/chelators (mainly phenolics) in many dicots (Marschner and Roemheld, 89 1994; Jin et al., 2007). Recently, it was shown that Fe deficiency induces the secretion of 90 secondary metabolites like scopoletin and its derivatives by Arabidopsis roots (Rodriguez- 91 Celma et al., 2013a; Fourcroy et al., 2014), and that Feruloyl-CoA 6'-Hydroxylase1 (F6'H1) is 92 required for the biosynthesis of the Fe(III)-chelating coumarin esculetin which is released into 93 the rhizosphere as part of the Strategy I-type Fe acquisition machinery (Schmid et al., 2014). 94 95 Scopoletin and, its corresponding glycoside, scopolin are the predominant coumarins in 96 Arabidopsis roots. But simultaneously, many other coumarin compounds such as skimmin, 97 esculetin, fraxetin and recently discovered coumarinolignans are present in the roots of this 98 model plant (Rohde et al., 2004; Bednarek et al., 2005; Kai et al., 2006; Kai et al., 2008; 99 Schmid et al., 2014; Schmidt et al., 2014; Ziegler et al., 2016; Siso-Terraza et al. 2016; 3 bioRxiv preprint doi: https://doi.org/10.1101/197806; this version posted October 4, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 100 Ziegler et al., 2017). In our previous work, we reported the presence of a significant natural 101 variation in scopoletin and scopolin accumulation between various Arabidopsis accessions 102 (Siwinska et al., 2014). It is an interesting issue due to the fact that Strategy I plants differ 103 considerably between plant species and genotypes in their tolerance to Fe deficiency. Among 104 coumarins significantly highly accumulated in response to Fe limited condition, scopoletin 105 and fraxetin together with their corresponding glycosides were detected (Fourcroy et al., 106 2014; Schmid et al., 2014; Schmidt et al., 2014). Up to now no enzymes involved in the last 107 step of fraxetin biosynthesis were identified. 108 109 The above findings point out that the biosynthesis of coumarins and their accumulation are 110 related to plant responses to Fe deficiency, but the exact mechanisms of action underlying 111 these processes have remained largely unknown. To better understand these mechanisms, we 112 selected and functionally characterize an enzyme of unknown biological role encoded by the 113 At3g12900 gene, which share significant homologies with the F6’H1 and F6’H2 described by 114 Kai et al. (2008) as involved in the synthesis of scopoletin, and which is pointed in the 115 literature as a strongly iron-responsive (Lan et al., 2011; Rodriguez-Celma et al., 2013b; Mai 116 et al., 2015; Mai and Bauer, 2016). This work lead us to identify the At3g12900 117 oxidoreductase as a scopoletin 8-hydroxylase (S8H) involved in the biosynthesis of fraxetin 118 that is associated with regulation of Fe homeostasis in Arabidopsis. 119 120 121 Materials and methods 122 Plant material 123 Arabidopsis thaliana (Arabidopsis) Col-0 accession was used as the wild-type.