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Iron acquisition strategies in land
plants: not so different after all
Summary
Due to its ability to accept and donate electrons, iron (Fe) is an
indispensable component of electron transport chains and a
cofactor in many vital enzymes. Except for waterlogged conditions,
under which the lack of oxygen prevents oxidation and precipitation
of iron as Fe3+ hydroxides, the availability of iron in soils is generally
far below the plant’s demand for optimal growth. Plants have
evolved two phylogenetically separated and elaborately regulated
strategies to mobilize iron from the soil, featuring mechanisms
which are thought to be mutually exclusive. However, recent
studies uncovered several shared components of the two strategies,
questioning the validity of the concept of mutual exclusivity. Here,
we use publicly available data obtained from the model species rice
(Oryza sativa) to unveil similarities and incongruities between co-
expression networks derived from transcriptomic profiling of iron-
deficient rice and Arabidopsis plants. This approach revealed
striking similarities in the topographies of the resulting co-expres-
sion networks with relatively minor deviations in the molecular
attributes of the comprised genes, which nonetheless lead to
different physiological functions. The analysis also discovered
several novel players that are possibly involved in the regulation
plant adaptation to iron deficiency.
A gene network controlling iron uptake and
homeostasis
Iron (Fe) participates in a multitude of redox reactions and is an
essential mineral nutrient for virtually all organisms. Although
highly abundant in the earth’s crust, the evolution of photosyn-
thesis dramatically reduced the activity of free iron in terrestrial and
aquatic environments, necessitating elaborate strategies in plants to
secure sufficient amounts of iron for growth and development. The
predominant nonsilicate iron compounds in aerated soils are Fe3+-
hydroxides, which are highly insoluble and are available to plants
only after prior mobilization. In principle, iron can be detached
from Fe3+-hydroxides by three processes; reduction, chelation and
protonation. Enzymes mediating these processes have evolved in
plants; their activity, however, can differ largely across species.
Grasses, for example, secrete iron-avid phytosiderophores (PS) of
the mugineic acid family into the rhizosphere, which form stable
complexes with Fe3+ and are taken up as such. In this strategy,
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reductive detachment of iron from its ligand occurs inside the cell
by an as yet unknown mechanism. All other cormophytes have
adopted a strategy in which iron is liberated from Fe3+-chelates
outside the cell by the action of a plasma membrane-bound surface
reductase (FRO). The released Fe2+ is taken up by a high-affinity
transporter (IRT), also localized on the plasma membrane (Eide
et al., 1996; Robinson et al., 1999). Iron uptake is preceded by
ATPase-driven proton extrusion, which decreases the pH of the
rhizosphere (Santi & Schmidt, 2009). The two strategies, referred
to as Strategy I (nongrasses) and Strategy II (grasses), are thought to
be mutually exclusive (R€omheld & Marschner, 1986). However,
recent data have shown that secretion of iron-mobilizing com-
pounds such as coumarins by roots of Strategy I species constitute a
critical component of this mechanism (reviewed in Tsai &
Schmidt, 2017). While secretion of phenolic compounds is part
of the original Strategy I concept (R€omheld & Marschner, 1986),
the importance of the secretion of iron-mobilizing compounds was
underestimated for decades due to the fact that mobilization of iron
by root exudates is particularly important under alkaline conditions
(Siso-Terraza et al., 2016; Rajniak et al., 2018; Tsai et al., 2018).
Moreover, peanut plants (Strategy I) were found to absorb Fe3+–PS
complexes generated by PS secreted from neighboring maize plants
(Xiong et al., 2013), and mugineic acid family PS were detected in
olives (Suzuki et al., 2016), suggesting that nongraminaceous
plants too can benefit from the PS system conceptually confined to
grasses. It should be mentioned that in contrast to PS, iron-
mobilizing compounds secreted by Strategy I plants such as flavins
or catechols possess reductive properties, thus providing an
additional source of electrons beside enzymatic reduction via
FRO2 or its orthologs in addition to their iron-chelating
properties. Also, rice and conceivably other Strategy II species
possess an IRT-dependent uptake system for iron (Ishimaru et al.,
2006), possibly as an adaptation to the presence of free Fe2+ under
anaerobic conditions. Moreover, several homologous signaling
compounds have been identified in the two model species rice and
Arabidopsis (Kobayashi & Nishizawa, 2012), indicating that the
iron regulon is, in large parts, evolutionarily conserved in land
plants.
To further explore putative homologies between the two iron
uptake strategies, and to uncover novel genes that have not yet been
associated with a particular component in either system, we
constructed a co-expression network of iron-regulated genes from
rice (Oryza sativa) and compared it to an already published
Arabidopsis network, representing Strategy II and Strategy I plants,
respectively. The basis of this network was a subset of iron-
responsive rice genes that showed signal changes greater than five-
fold in response to iron deficiency in a previous study (Zheng et al.,
2009), and co-expression calculated from publicly available
microarray hybridizations. In order to focus our analysis on the
genes most relevant to iron homeostasis and its regulation, we
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isolated a subnetwork of genes based on their known function in
iron homeostasis (Supporting Information Table S1), and of nodes
that were directly connected to at least two of these genes
(Table S2). We then predicted the Arabidopsis orthologs or the
closest homologs to each node of the network, subsequently
obtaining a rice gene network readily comparable to the Arabidop-
sis gene network from Rodrıguez-Celma et al. (2013) (Fig. 1).
SAM takes centre stage in the metabolism of iron-
deficient plants
Genes encoding enzymes involved in the methionine salvage
pathway are upregulated upon iron starvation in both Arabidopsis
and rice (Takahashi et al., 1999; Kobayashi et al., 2009; Lan et al.,
2011; Fig 1, 2). S-Adenosyl methionine (SAM) generated in this
pathway is required for the synthesis of the nonproteinogenic
amino acid nicotianamine (NA), an indispensable component for
the intracellular and intercellular distribution of iron in all species
(Anderegg & Ripperger, 1989; von Wiren et al., 1999; Schuler &
Bauer, 2011). The current network contains two genes encoding
NA synthase (NAS) genes, OsNAS2 and OsNAS1. The phyloge-
netic tree of Arabidopsis and rice NAS genes does not allow for
unambiguously determining which Arabidopsis gene is the closest
to the rice NAS genes because the proteins cluster by species
(Fig. S1). The only Arabidopsis NAS gene present in the iron-
responsive gene co-expression network is AtNAS4 (Rodrıguez-
Celma et al., 2013). In a reaction which appears to be confined to
grasses, NA is transaminated via NAAT to form the precursor of PS
(Kobayashi & Nishizawa, 2012). In Strategy I plants, NA either
remains inside the cell as a ligand for transition metals such as
copper (Cu), zinc (Zn), Fe and manganese (Mn), is exported into
the xylem or phloem together with a chelated metal, or secreted into
the rhizosphere to prevent the accumulation of excess zinc, as in the
case of the metal hyperaccumulator Arabidopsis halleri (Tsednee
et al., 2014). In Arabidopsis, SAM is required for the conversion of
trans-caffeoyl CoA to feruloyl CoA in the scopoletin pathway to
produce catechol-type coumarins such as fraxetin, which have been
shown to possess iron-mobilizing properties (Schmid et al., 2014;
Siso-Terraza et al., 2016; Tsai et al., 2018). This response is
analogous to the secretion of PS, but depends on a different
metabolic pathway with SAM as a converging point in the
biosynthesis of the two compounds (Fig. 2).
NA-related peptide transporters control the
distribution of iron
The pivotal role of NA in the transport of iron and other transition
metals is manifested by the multitude of metal–NA or NA-derived
substrates. YELLOW STRIPE-LIKE (YSL) is a family of metal–
NA transporters comprising eight members in Arabidopsis and 18
in rice (Curie et al., 2001; Koike et al., 2004). Assigning precise
functions to individual proteins in either species has turned out to
be a challenging task. The founding member of this protein family,
YELLOW STRIPE 1 (ZmYS1), imports Fe–PS complexes across
the root cell membrane (Von Wiren et al., 1994; Curie et al., 2001).
In rice, the uptake of Fe–PS complexes has been attributed to
OsYSL15 (Inoue et al., 2009). Another member of the YSL family,
OsYSL2, is upregulated in response to iron starvation and plays a
critical role in long-distance transport of iron and manganese
(Ishimaru et al., 2010). The most closely related homologs of

Fig. 1 Co-expression network of iron-responsive rice genes deduced from transcriptomic surveys and their predicted Arabidopsis homologs. The weight of the
edges is proportional to the Pearson correlation coefficient. Green nodes represent genes encoding enzymes involved in methionine salvage and nicotianamine
biosynthesis. Purple nodes represent genes encoding transporters of iron chelators and complexes. Gray nodes represent genes involved in oxidative stress
detoxification. Blue nodes represent genes encoding regulators of iron homeostasis. Red nodes represent genes with no obvious predicted function based on
BLAST search. White nodes represent the genes that do not belong to any of the earlier-mentioned categories.

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Fig. 2 S-Adenosyl methionine (SAM) plays a central role in iron uptake. SAM is a precursor for the ubiquitous iron chelator nicotianamine (NA) and is involved in
the biosynthesis of phytosiderophores of the mugineic acid family in rice (Strategy II; light-blue box) and the iron-mobilizing catechol-type coumarins such as
fraxetin in Arabidopsis (Strategy I; light-green box). Rice and Arabidopsis genes found in the network are represented in blue and green respectively, and genes
not found in the network are represented in gray. Solid lines represent chemical transformations and dashed lines represent transport steps. Genes were
matched based on sequence similarities but may control different physiological functions. SAM, S-adenosyl methionine; MTA, 5-methylthioadenosine; MTR,
5-methylthioribose; MTR-1-P, 5-methylthioribose-1-phosphate; MTRu-1-P, 5-methylthioribulose-1-phosphate; DHKMP, 1,2-dihydroxy-3-keto-5-
methylthiopentene; KMTB, 2-keto-4-methylthiobutyrate; Met, methionine; NA, nicotianamine; DMA, 20 -deoxymugineic acid.

OsYSL2 in Arabidopsis are AtYSL1, AtYSL2 and AtYSL3 (Curie
et al., 2009; Fig. S2). AtYSL1 and AtYSL3 are involved in iron
remobilization and seed loading (Le Jean et al., 2005; Waters et al.,
2006), the function of AtYSL2 is still unclear. Similar to OsYSL2,
AtYSL1, AtYSL2 and AtYSL3 are expressed in vascular tissues
(DiDonato et al., 2004; Koike et al., 2004; Le Jean et al., 2005;
Waters et al., 2006), but in contrast to OsYSL2, their expression is
not upregulated upon iron deficiency.
The main Fe3+-deoxymugineic acid importer OsYSL15, which
is the closest rice homolog to ZmYS1 (Le Jean et al., 2005; Inoue
et al., 2009), is predicted by InParanoid to be orthologous to an as
yet uncharacterized Arabidopsis OPT, At5g45450. However,
manual reciprocal BLAST searches and phylogeny-based predictions
suggest that AtYSL1, AtYSL2 or AtYSL3 are more closely related to
OsYSL15 (Curie et al., 2009; Fig. S2). The genes cluster by species
rather than by function, impairing the task to predict a functional
match.
The transporter mediating the secretion of PS in rice (OsTOM1;
Fig. 1) is a close homolog of the NA transporter AtZIF1, and two
similar proteins, AtZIFL1 and AtZIFL2 (Fig. S3). In Arabidopsis,
AtZIF1 but not AtZIF2 is co-expressed with iron-responsive genes
(Long et al., 2010), and is involved in the sequestration of zinc into
the vacuole (Haydon & Cobbett, 2007). Although the molecular
functions of OsTOM1 and AtZIF are analogous, their targeting to
different membranes lead to different physiological roles. Thus, it
appears that NA and DMA transporters have been recruited for
various, species-dependent functions to assure proper distribution
of transition metals.
YSL proteins belong to the oligopeptide transporters (OPTs), a
family encoding integral membrane proteins that is restricted to
plants, fungi, bacteria and archaebacteria (Yen et al., 2001;
Lubkowitz, 2011). While the function of YSLs appears to lie in
the distribution of metal chelates, OPTs form a separate clade with
less well-defined functions (Fig. S2). In Arabidopsis, mutants
defective in AtOPT3 expression accumulate high levels of iron in
leaves and show constitutively upregulated iron deficiency
responses in roots, indicative of a role of OPTs in long distance
iron signaling (Stacey et al., 2008). AtOPT3 was shown to mediate
iron loading into the phloem, and may regulate root iron deficiency
responses through the availability of iron in the phloem (Mendoza-
Cozatl et al., 2014; Zhai et al., 2014). The closest rice homolog to
AtOPT3 is OsOPT7 (Lubkowitz, 2011; Bashir et al., 2015),
sharing 89% identity. AtOPT3 and OsOPT7 are part of the iron-
responsive co-expression network in Arabidopsis and rice, respec-
tively (Fig. 1; Rodrıguez-Celma et al., 2013). Although iron
accumulation was less pronounced in the rice opt7-1 mutant than
in the Arabidopsis opt3-2 mutant, the two genes are likely
performing similar functions. It is conceivable that both proteins
are involved in phloem-mediated long-distance transport of iron.
A conserved cluster of proteins regulates iron uptake
and distribution
In Arabidopsis, the transcription factor AtFIT (AtbHLH29) forms
heterodimers with the subgroup Ib bHLH proteins AtbHLH38,
AtbHLH39, AtbHLH100 and AtbHLH101, and regulates the
major Strategy I-type iron acquisition genes (Fig. 3; Colangelo &
Guerinot, 2004; Yuan et al., 2008; Sivitz et al., 2012; Wang et al.,
2013). All three prediction methods (i.e. BLAST, InParanoid and
phylogeny) show that Arabidopsis subgroup Ib bHLH proteins are
closely related to OsIRO2 (Table S1; Fig. S4), a central regulator of
the iron deficiency responses in rice (Ogo et al., 2007). Overex-
pression of OsIRO2 in rice and AtbHLH39 in Arabidopsis resulted
in activation of species-specific iron deficiency responses, that is, PS

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secretion in rice and root Fe3+ reduction in Arabidopsis (Ogo et al.,
2007; Naranjo-Arcos et al., 2017), indicating that these proteins
are controlling the rate of iron uptake. Another bHLH protein,
OsIRO3, negatively regulates the iron deficiency responses in rice
(Zheng et al., 2010; Wang et al., 2013). OsIRO3 is most similar to
POPEYE (AtPYE), which plays a similar role in Arabidopsis (Long
et al., 2010; Selote et al., 2015).
The expression of OsIRO2/OsIRO3 is regulated by the subgroup
IVc bHLH transcription factor POSITIVE REGULATOR OF
IRON HOMEOSTASIS (OsPRI1; Zhang et al., 2017), the
turnover of which is controlled by the haemerythrin (HHE)
domain-containing proteins OsHRZ1. Similarly, in Arabidopsis
their homolog BRUTUS (AtBTS) promotes the degradation of the
subgroup IVc proteins bHLH115 and bHLH105 (Long et al.,
2010; Selote et al., 2015). All three HHE proteins possess
ubiquitination activity and regulate their down-stream targets in
a proteasomal-dependent manner (Kobayashi et al., 2013; Selote
et al., 2015; Zhang et al., 2015). Although several other elements of
iron signaling have been identified in both species which modulate,
fine-tune, or orchestrate the activity of iron homeostasis genes, the
function of the homologous groups OsHRZ-AtBTS, OsIRO2-
AtbHLH38/39/100/101, OsPRI1-AtbHLH105/115 and
OsIRO3-AtPYE appears to be conserved in both strategies (Fig. 3).
A recently discovered novel family of peptides named
IRONMAN (IMA) or FE-UPTAKE-INDUCING PEPTIDE
(FEP) positively regulates subgroup 1b bHLH genes in Arabidopsis
(Grillet et al., 2018; Hirayama et al., 2018). Overexpression of
IMAs causes iron and manganese accumulation, a function which is
dependent on a C-terminal amino acid motif that is conserved
among IMA peptides across species (Grillet et al., 2018). Genes
encoding IMA peptides in rice are highly induced by iron
deficiency and co-expressed with other iron homeostasis genes,
Fig. 3 Iron homeostasis genes in rice (blue) and Arabidopsis (green). Nodes
in contact with each other are homologous between the two species and,
conceivably, iron uptake strategies. Important regulators that were not
found in the network are represented in light blue for rice or light green for
Arabidopsis. Black lines and arrows correspond to regulatory pathways
common to both species and blue and green arrows correspond to pathways
only demonstrated in rice and Arabidopsis, respectively.
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suggesting that the function of IMA peptides is conserved. IMAs
likely represent the most upstream component of the iron signaling
cascade common to both iron acquisition strategies. IMAs in rice,
and generally in monocots, appear to be evolutionarily closer to
AtIMA3, mainly due to a longer aspartic acid repeat in the
conserved motif compared to other Arabidopsis IMA peptides
(Grillet et al., 2018; Fig. S5). All the monocot IMAs also harbor a
repeat of six aspartate residues while Amborella trichopoda IMAs
harbor a repeat of either seven to eight Asp. AtIMA3 has the longest
Asp stretch of all the eight Arabidopsis genes (6xAsp), suggesting
that it is also the closest to the ancestral gene. Interestingly, in
Arabidopsis IMA target genes include bHLH105 (ILR3)-regulated
genes (Tissot et al., 2019), suggesting that the molecular mecha-
nism of IMAs is related to the post-transcriptional regulation of
group IVc bHLH proteins.
ROS detoxification is a conserved component of the
iron deficiency response
Oxidative stress is not only caused by high iron levels but may also
result from an impaired efficiency of electron transport due to
suboptimal iron supply, a phenomenon that is poorly documented
(Walter et al., 2002). Four highly conserved chloroplast-localized
proteins, i.e. NEET, FERRITIN (FER), CONSERVED IN THE
GREEN LINEAGE AND DIATOMS 27 (AtCGLD27) and
NON-INTRINSIC ABC PROTEIN 1 (NAP1) participate in the
regulation of iron and reactive oxygen species (ROS) metabolism of
rice and Arabidopsis, and are conserved parts of the network (Fig. 1).
All four proteins play ancient roles in iron metabolism, in particular
in Fe-S/Fe transfer within cells (NEET; Nechushtai et al., 2012;
Tamir et al., 2015; Inupakutika et al., 2017); Fe-S cluster synthesis
(NAP1; Xu et al., 2005; Hu et al., 2017), xanthophyll metabolism
(CGLD27; Urzica et al., 2012), and storage/release of iron (FER;
Harrison & Arosio, 1996; Briat et al., 1995, 2010). Pleiotropic
phenotypes of neet, cgld27 and nap1 mutants in Arabidopsis imply
multiple roles of the proteins in cellular iron metabolism. Another
gene encoding a chloroplast localized protein, PGR5-LIKE A
(At5 g59400; AtPGR5LA), is tightly co-expressed with several
genes involved in ROS detoxification and iron distribution such as
FER1, FER4, NAP1, NAS3 and YSL1 in Arabidopsis co-expression
networks (Fig. S6). The name-giving proteins, PGR5 and PGR5-
LIKE A, are involved in tuning the ATP/NADPH ratios during
photosynthesis by regulating the relative activity of the two
photosystems (DalCorso et al., 2008; Hertle et al., 2013). PGR5
was shown to be essential for photoprotection (Suorsa, 2015). A
common function of the plastidial proteins listed earlier may lie in
the protection of cells from oxidative stress, a function that is of
particular importance in plastids.
IMAs and IRPs may play critical but yet unexplored
roles in iron homeostasis
In Arabidopsis, a subset of genes encoding proteins of unknown
function are highly induced by iron deficiency in both leaves and
roots. Based on their close co-expression with iron-responsive genes
with well-defined function in iron homeostasis, we referred to these
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genes as IRON-RESPONSIVE PROTEINs (IRPs; Rodrıguez-
Celma et al., 2013), indicating putative roles in the regulation of
iron uptake. Applying similar criteria to rice genes of unknown
function found in the co-expression network, we designated the so-
defined genes as OsIRPs (Table S3), denoted by red nodes in Fig. 1.
AtIRP1 and AtIRP2 have been later renamed to AtIMA1 and
AtIMA2, and share similarities with OsIMA1 and OsIMA2 in their
C-terminus (Grillet et al., 2018). OsIRP6 (Os03g52680) is
homologous to AtIRP5 and AtIRP6. OsIRP3, OsIRP4, OsIRP5
and OsIRP7 do not share sequence similarity with any of the other
Arabidopsis AtIRPs and may represent rice- or Strategy II-specific
proteins. While the function of these genes in iron metabolism
remains elusive, their expression lends support to speculate that
some if not all rice IRPs participate in iron signaling.
A complex recipe to satisfy hidden hunger
The co-expression network constructed from iron-responsive genes
in rice transcriptomes reveals a remarkably similar architecture to the
networks inferred for Arabidopsis (Schmidt & Buckhout, 2011;
Rodrıguez-Celma et al., 2013), highlighting a high congruency of
the iron deficiency response between the two species. The secretion
of iron-mobilizing compounds marks a key feature of both
strategies. In Strategy I plants, the presence of organic Fe3+
complexes is a prerequisite for root-mediated reduction. At alkaline
pH, Fe3+ reduction requires mobilization of iron by chelation via
root-borne coumarins before Fe2+ can be freed from the ligand and
taken up from the soil. Similarly, in Strategy II plants detachment of
iron from Fe3+ oxides via secretion of PS is critical for the acquisition
of iron from the soil. The major conceptual difference between the
two iron acquisition strategies appears to be related to the location of
reductive chelate splitting, which occurs either extracellularly or
intracellularly, an aspect which has received little attention. Our
analysis further suggests that key nodes such as NA metabolism,
ROS detoxification, iron sensing by HHE domain-containing
proteins, and iron signaling via bHLH transcription factors are
conserved in the two model organisms, and, presumably, among the
two iron acquisition strategies. Furthermore, the recruitment of
proteins to a novel physiological context by slightly modifying their
molecular function, for example OsNAAT in the synthesis of PS or
OsYSL15 in import of Fe–PS, provides a means to shape biological
processes in search for novel solutions. Assignment of a well-defined
molecular function to the proteins controlling and mediating iron
homeostasis will set the stage for a holistic understanding of the ‘iron
efficiency’ of a given species or cultivar. Identification of the traits
that allow plants to thrive on soils with immobile iron sources is a
prerequisite to prevent yield losses, improve the nutritional quality
of crops, and to combat iron deficiency-induced anemia in humans
by generating iron-efficient germplasm.
Materials and Methods
Network construction
A suite of 1349 genes that were highly iron-regulated either
positively or negatively in either leaves or roots (FC > 5; P < 0.05)
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Viewpoints Forum 15
were selected from the transcriptome published by Zheng et al.
(2009). The co-expression analysis was performed using rice
Affymettrix array data of c. 2700 hybridizations from
ARRAYEXPRESS (www.ebi.ac.uk/arrayexpress/). Pearson correlation
coefficients between probe set pairs were calculated using MACCU
software (http://maccu.sourceforge.net/), and the network was
computed with edges corresponding to a Pearson coefficient > 0.6,
resulting in the clustering of 1046 probe sets.
Arabidopsis loci assignment
Oligonucleotide sequences of the probes (e-value < 9.9e-6) were
mapped against the transcripts from the latest release of the Rice
Pseudomolecules and Genome Annotation Database (V7). We
then assigned Arabidopsis homologs to rice loci using the INPARA-
NOID software. To further strengthen the prediction, each rice gene
protein sequence was used in a BLAST search against the Arabidopsis
genome. The best Arabidopsis match was then used for a BLAST
search against the rice genome. If the result was consistent between
the two methods, the match was considered unambiguous. In the
case of ambiguous homolog assignments, we manually curated each
match using phylogenetic analysis (Figs S1–S5 & S7–S17) and, in
some cases attributed a single locus to multiple loci of the other
species.
Subnetwork selection
In order to focus the analysis on the genes most relevant to iron
homeostasis, we isolated a subnetwork of genes based on their
known function in iron homeostasis. The list of published genes
related to iron homeostasis is given in Table S1 and consists of iron
homeostasis genes listed in Kobayashi & Nishizawa (2012), genes
characterized after 2012, and uncharacterized putative iron
transporters from the ZIP, NRAMP and OPT-YSL families.
From the original 1046 gene network, we cropped the listed genes
as well as genes that were co-expressed with at least two of them,
resulting in the 55 genes subnetwork presented in Fig. 1.
Acknowledgements
The authors thank Wendar Lin (Bioinformatics Core Laboratory
IPMB) for bioinformatics support and Thomas J. Buckhout
(Humboldt University Berlin) for critical comments on the
manuscript. This work was supported by an Academia Sinica
Investigator Award to WS.
Author contributions
LG performed the analysis with the support of the Bioinformatics
Core Laboratory of the Institute of Plant and Microbial Biology.
WS and LG wrote the article.
ORCID
Louis Grillet https://orcid.org/0000-0002-2914-9023
Wolfgang Schmidt https://orcid.org/0000-0002-7850-6832
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Louis Grillet1 and Wolfgang Schmidt1,2,3*
1 Inupakutika MA, Sengupta S, Nechutsai R, Jennings PA, Onuchic JN, Azad RK,
Institute of Plant and Microbial Biology, Academia Sinica,
Taipei 11529, Taiwan;
2
Biotechnology Center, National Chung-Hsing University,
Taichung 40227, Taiwan;
3
Genome and Systems Biology Degree Program, College of Life
Science, National Taiwan University, Taipei 10617, Taiwan
(*Author for correspondence: tel +886 2 2782 7954;
email wosh@gate.sinica.edu.tw)
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Supporting Information
Additional Supporting Information may be found online in the
Supporting Information section at the end of the article.
Fig. S1 Phylogenetic tree of NAS proteins from rice and
Arabidopsis.
Fig. S2 Phylogenetic tree of OPT and YSL proteins from rice and
Arabidopsis.
Fig. S3 Phylogenetic tree of Major Facilitator Superfamily proteins
from rice and Arabidopsis.
Fig. S4 Phylogenetic tree of bHLH proteins from rice and
Arabidopsis.
Fig. S5 Phylogenetic tree of IMA peptides from Arabidopsis and a
few monocots.
Fig. S6 Network of genes co-expressed with Arabidopsis PGR5-like
A.
Fig. S7 Phylogenetic tree of ZIP proteins from rice and Arabidopsis.
Fig. S8 Phylogenetic tree of NRAMP proteins from rice and
Arabidopsis.
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18 Forum Viewpoints Phytologist

Fig. S9 Phylogenetic tree of FERRITIN proteins from rice and Fig. S17 Phylogenetic tree of aminotransferase proteins from rice
Arabidopsis. and Arabidopsis.

Fig. S10 Phylogenetic tree of HEMERYTHRIN proteins from rice

and Arabidopsis. Table S1 Rice iron homeostasis genes used to isolate the
subnetwork.
Fig. S11 Phylogenetic tree of Acyl-CoA binding proteins from rice
and Arabidopsis. Table S2 Rice iron homeostasis genes, putative transporters and
first degree neighbors that constitute the subnetwork, and their
Fig. S12 Phylogenetic tree of glycoside hydrolase proteins from rice Arabidopsis homologs.
and Arabidopsis.
Table S3 Genes encoding iron-responsive proteins (IRPs) in rice
Fig. S13 Phylogenetic tree of alcohol dehydrogenase proteins from with unknown function and low or absent homology to Arabidopsis
rice and Arabidopsis. genes.

Fig. S14 Phylogenetic tree of cysteine-rich secretory proteins from
rice and Arabidopsis.
Fig. S15 Phylogenetic tree of remorin proteins from rice and
Arabidopsis.
Fig. S16 Phylogenetic tree of LEUCINE RICH REPEAT-kinase
proteins from rice and Arabidopsis most closely related to
LOC_Os05 g44990.
Please note: Wiley Blackwell are not responsible for the content or
functionality of any Supporting Information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
Key words: co-expression networks, evolution, gene regulation, iron, iron
deficiency, reactive oxygen species.
Received, 7 May 2019; accepted, 11 June 2019.

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