Forum

Viewpoints

reductive detachment of iron from its ligand occurs inside the cell
Iron acquisition strategies in land by an as yet unknown mechanism. All other cormophytes have
adopted a strategy in which iron is liberated from Fe3+-chelates
plants: not so different after all 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
Summary
ATPase-driven proton extrusion, which decreases the pH of the
Due to its ability to accept and donate electrons, iron (Fe) is an rhizosphere (Santi & Schmidt, 2009). The two strategies, referred
indispensable component of electron transport chains and a to as Strategy I (nongrasses) and Strategy II (grasses), are thought to
cofactor in many vital enzymes. Except for waterlogged conditions, be mutually exclusive (R€omheld & Marschner, 1986). However,
under which the lack of oxygen prevents oxidation and precipitation recent data have shown that secretion of iron-mobilizing com-
of iron as Fe3+ hydroxides, the availability of iron in soils is generally pounds such as coumarins by roots of Strategy I species constitute a
far below the plant’s demand for optimal growth. Plants have critical component of this mechanism (reviewed in Tsai &
evolved two phylogenetically separated and elaborately regulated Schmidt, 2017). While secretion of phenolic compounds is part
strategies to mobilize iron from the soil, featuring mechanisms of the original Strategy I concept (R€omheld & Marschner, 1986),
which are thought to be mutually exclusive. However, recent the importance of the secretion of iron-mobilizing compounds was
studies uncovered several shared components of the two strategies, underestimated for decades due to the fact that mobilization of iron
questioning the validity of the concept of mutual exclusivity. Here, by root exudates is particularly important under alkaline conditions
we use publicly available data obtained from the model species rice (Siso-Terraza et al., 2016; Rajniak et al., 2018; Tsai et al., 2018).
(Oryza sativa) to unveil similarities and incongruities between co- Moreover, peanut plants (Strategy I) were found to absorb Fe3+–PS
expression networks derived from transcriptomic profiling of iron- complexes generated by PS secreted from neighboring maize plants
deficient rice and Arabidopsis plants. This approach revealed (Xiong et al., 2013), and mugineic acid family PS were detected in
striking similarities in the topographies of the resulting co-expres- olives (Suzuki et al., 2016), suggesting that nongraminaceous
sion networks with relatively minor deviations in the molecular plants too can benefit from the PS system conceptually confined to
attributes of the comprised genes, which nonetheless lead to grasses. It should be mentioned that in contrast to PS, iron-
different physiological functions. The analysis also discovered mobilizing compounds secreted by Strategy I plants such as flavins
several novel players that are possibly involved in the regulation or catechols possess reductive properties, thus providing an
plant adaptation to iron deficiency. 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
A gene network controlling iron uptake and
anaerobic conditions. Moreover, several homologous signaling
homeostasis
compounds have been identified in the two model species rice and
Iron (Fe) participates in a multitude of redox reactions and is an Arabidopsis (Kobayashi & Nishizawa, 2012), indicating that the
essential mineral nutrient for virtually all organisms. Although iron regulon is, in large parts, evolutionarily conserved in land
highly abundant in the earth’s crust, the evolution of photosyn- plants.
thesis dramatically reduced the activity of free iron in terrestrial and To further explore putative homologies between the two iron
aquatic environments, necessitating elaborate strategies in plants to uptake strategies, and to uncover novel genes that have not yet been
secure sufficient amounts of iron for growth and development. The associated with a particular component in either system, we
predominant nonsilicate iron compounds in aerated soils are Fe3+- constructed a co-expression network of iron-regulated genes from
hydroxides, which are highly insoluble and are available to plants rice (Oryza sativa) and compared it to an already published
only after prior mobilization. In principle, iron can be detached Arabidopsis network, representing Strategy II and Strategy I plants,
from Fe3+-hydroxides by three processes; reduction, chelation and respectively. The basis of this network was a subset of iron-
protonation. Enzymes mediating these processes have evolved in responsive rice genes that showed signal changes greater than five-
plants; their activity, however, can differ largely across species. fold in response to iron deficiency in a previous study (Zheng et al.,
Grasses, for example, secrete iron-avid phytosiderophores (PS) of 2009), and co-expression calculated from publicly available
the mugineic acid family into the rhizosphere, which form stable microarray hybridizations. In order to focus our analysis on the
complexes with Fe3+ and are taken up as such. In this strategy, genes most relevant to iron homeostasis and its regulation, we

Ó 2019 The Authors New Phytologist (2019) 224: 11–18 11

New Phytologist Ó 2019 New Phytologist Trust www.newphytologist.com
 New
12 Forum Viewpoints Phytologist

isolated a subnetwork of genes based on their known function in copper (Cu), zinc (Zn), Fe and manganese (Mn), is exported into
iron homeostasis (Supporting Information Table S1), and of nodes the xylem or phloem together with a chelated metal, or secreted into
that were directly connected to at least two of these genes the rhizosphere to prevent the accumulation of excess zinc, as in the
(Table S2). We then predicted the Arabidopsis orthologs or the case of the metal hyperaccumulator Arabidopsis halleri (Tsednee
closest homologs to each node of the network, subsequently et al., 2014). In Arabidopsis, SAM is required for the conversion of
obtaining a rice gene network readily comparable to the Arabidop- trans-caffeoyl CoA to feruloyl CoA in the scopoletin pathway to
sis gene network from Rodrıguez-Celma et al. (2013) (Fig. 1). 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
SAM takes centre stage in the metabolism of iron-
analogous to the secretion of PS, but depends on a different
deficient plants
metabolic pathway with SAM as a converging point in the
Genes encoding enzymes involved in the methionine salvage biosynthesis of the two compounds (Fig. 2).
pathway are upregulated upon iron starvation in both Arabidopsis
and rice (Takahashi et al., 1999; Kobayashi et al., 2009; Lan et al.,
NA-related peptide transporters control the
2011; Fig 1, 2). S-Adenosyl methionine (SAM) generated in this
distribution of iron
pathway is required for the synthesis of the nonproteinogenic
amino acid nicotianamine (NA), an indispensable component for The pivotal role of NA in the transport of iron and other transition
the intracellular and intercellular distribution of iron in all species metals is manifested by the multitude of metal–NA or NA-derived
(Anderegg & Ripperger, 1989; von Wiren et al., 1999; Schuler & substrates. YELLOW STRIPE-LIKE (YSL) is a family of metal–
Bauer, 2011). The current network contains two genes encoding NA transporters comprising eight members in Arabidopsis and 18
NA synthase (NAS) genes, OsNAS2 and OsNAS1. The phyloge- in rice (Curie et al., 2001; Koike et al., 2004). Assigning precise
netic tree of Arabidopsis and rice NAS genes does not allow for functions to individual proteins in either species has turned out to
unambiguously determining which Arabidopsis gene is the closest be a challenging task. The founding member of this protein family,
to the rice NAS genes because the proteins cluster by species YELLOW STRIPE 1 (ZmYS1), imports Fe–PS complexes across
(Fig. S1). The only Arabidopsis NAS gene present in the iron- the root cell membrane (Von Wiren et al., 1994; Curie et al., 2001).
responsive gene co-expression network is AtNAS4 (Rodrıguez- In rice, the uptake of Fe–PS complexes has been attributed to
Celma et al., 2013). In a reaction which appears to be confined to OsYSL15 (Inoue et al., 2009). Another member of the YSL family,
grasses, NA is transaminated via NAAT to form the precursor of PS OsYSL2, is upregulated in response to iron starvation and plays a
(Kobayashi & Nishizawa, 2012). In Strategy I plants, NA either critical role in long-distance transport of iron and manganese
remains inside the cell as a ligand for transition metals such as (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.

New Phytologist (2019) 224: 11–18 Ó 2019 The Authors

www.newphytologist.com New Phytologist Ó 2019 New Phytologist Trust
New
Phytologist Viewpoints Forum 13

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

Ó 2019 The Authors New Phytologist (2019) 224: 11–18

New Phytologist Ó 2019 New Phytologist Trust www.newphytologist.com
 New
14 Forum Viewpoints Phytologist

secretion in rice and root Fe3+ reduction in Arabidopsis (Ogo et al., suggesting that the function of IMA peptides is conserved. IMAs
2007; Naranjo-Arcos et al., 2017), indicating that these proteins likely represent the most upstream component of the iron signaling
are controlling the rate of iron uptake. Another bHLH protein, cascade common to both iron acquisition strategies. IMAs in rice,
OsIRO3, negatively regulates the iron deficiency responses in rice and generally in monocots, appear to be evolutionarily closer to
(Zheng et al., 2010; Wang et al., 2013). OsIRO3 is most similar to AtIMA3, mainly due to a longer aspartic acid repeat in the
POPEYE (AtPYE), which plays a similar role in Arabidopsis (Long conserved motif compared to other Arabidopsis IMA peptides
et al., 2010; Selote et al., 2015). (Grillet et al., 2018; Fig. S5). All the monocot IMAs also harbor a
The expression of OsIRO2/OsIRO3 is regulated by the subgroup repeat of six aspartate residues while Amborella trichopoda IMAs
IVc bHLH transcription factor POSITIVE REGULATOR OF harbor a repeat of either seven to eight Asp. AtIMA3 has the longest
IRON HOMEOSTASIS (OsPRI1; Zhang et al., 2017), the Asp stretch of all the eight Arabidopsis genes (6xAsp), suggesting
turnover of which is controlled by the haemerythrin (HHE) that it is also the closest to the ancestral gene. Interestingly, in
domain-containing proteins OsHRZ1. Similarly, in Arabidopsis Arabidopsis IMA target genes include bHLH105 (ILR3)-regulated
their homolog BRUTUS (AtBTS) promotes the degradation of the genes (Tissot et al., 2019), suggesting that the molecular mecha-
subgroup IVc proteins bHLH115 and bHLH105 (Long et al., nism of IMAs is related to the post-transcriptional regulation of
2010; Selote et al., 2015). All three HHE proteins possess group IVc bHLH proteins.
ubiquitination activity and regulate their down-stream targets in
a proteasomal-dependent manner (Kobayashi et al., 2013; Selote
ROS detoxification is a conserved component of the
et al., 2015; Zhang et al., 2015). Although several other elements of
iron deficiency response
iron signaling have been identified in both species which modulate,
fine-tune, or orchestrate the activity of iron homeostasis genes, the Oxidative stress is not only caused by high iron levels but may also
function of the homologous groups OsHRZ-AtBTS, OsIRO2- result from an impaired efficiency of electron transport due to
AtbHLH38/39/100/101, OsPRI1-AtbHLH105/115 and suboptimal iron supply, a phenomenon that is poorly documented
OsIRO3-AtPYE appears to be conserved in both strategies (Fig. 3). (Walter et al., 2002). Four highly conserved chloroplast-localized
A recently discovered novel family of peptides named proteins, i.e. NEET, FERRITIN (FER), CONSERVED IN THE
IRONMAN (IMA) or FE-UPTAKE-INDUCING PEPTIDE GREEN LINEAGE AND DIATOMS 27 (AtCGLD27) and
(FEP) positively regulates subgroup 1b bHLH genes in Arabidopsis NON-INTRINSIC ABC PROTEIN 1 (NAP1) participate in the
(Grillet et al., 2018; Hirayama et al., 2018). Overexpression of regulation of iron and reactive oxygen species (ROS) metabolism of
IMAs causes iron and manganese accumulation, a function which is rice and Arabidopsis, and are conserved parts of the network (Fig. 1).
dependent on a C-terminal amino acid motif that is conserved All four proteins play ancient roles in iron metabolism, in particular
among IMA peptides across species (Grillet et al., 2018). Genes in Fe-S/Fe transfer within cells (NEET; Nechushtai et al., 2012;
encoding IMA peptides in rice are highly induced by iron Tamir et al., 2015; Inupakutika et al., 2017); Fe-S cluster synthesis
deficiency and co-expressed with other iron homeostasis genes, (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

Fig. 3 Iron homeostasis genes in rice (blue) and Arabidopsis (green). Nodes
in contact with each other are homologous between the two species and, roles in iron homeostasis
conceivably, iron uptake strategies. Important regulators that were not
In Arabidopsis, a subset of genes encoding proteins of unknown
found in the network are represented in light blue for rice or light green for
Arabidopsis. Black lines and arrows correspond to regulatory pathways function are highly induced by iron deficiency in both leaves and
common to both species and blue and green arrows correspond to pathways roots. Based on their close co-expression with iron-responsive genes
only demonstrated in rice and Arabidopsis, respectively. with well-defined function in iron homeostasis, we referred to these

New Phytologist (2019) 224: 11–18 Ó 2019 The Authors

www.newphytologist.com New Phytologist Ó 2019 New Phytologist Trust
New
Phytologist Viewpoints Forum 15

genes as IRON-RESPONSIVE PROTEINs (IRPs; Rodrıguez- were selected from the transcriptome published by Zheng et al.
Celma et al., 2013), indicating putative roles in the regulation of (2009). The co-expression analysis was performed using rice
iron uptake. Applying similar criteria to rice genes of unknown Affymettrix array data of c. 2700 hybridizations from
function found in the co-expression network, we designated the so- ARRAYEXPRESS (www.ebi.ac.uk/arrayexpress/). Pearson correlation
defined genes as OsIRPs (Table S3), denoted by red nodes in Fig. 1. coefficients between probe set pairs were calculated using MACCU
AtIRP1 and AtIRP2 have been later renamed to AtIMA1 and software (http://maccu.sourceforge.net/), and the network was
AtIMA2, and share similarities with OsIMA1 and OsIMA2 in their computed with edges corresponding to a Pearson coefficient > 0.6,
C-terminus (Grillet et al., 2018). OsIRP6 (Os03g52680) is resulting in the clustering of 1046 probe sets.
homologous to AtIRP5 and AtIRP6. OsIRP3, OsIRP4, OsIRP5
and OsIRP7 do not share sequence similarity with any of the other
Arabidopsis loci assignment
Arabidopsis AtIRPs and may represent rice- or Strategy II-specific
proteins. While the function of these genes in iron metabolism Oligonucleotide sequences of the probes (e-value < 9.9e-6) were
remains elusive, their expression lends support to speculate that mapped against the transcripts from the latest release of the Rice
some if not all rice IRPs participate in iron signaling. 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
A complex recipe to satisfy hidden hunger
protein sequence was used in a BLAST search against the Arabidopsis
The co-expression network constructed from iron-responsive genes genome. The best Arabidopsis match was then used for a BLAST
in rice transcriptomes reveals a remarkably similar architecture to the search against the rice genome. If the result was consistent between
networks inferred for Arabidopsis (Schmidt & Buckhout, 2011; the two methods, the match was considered unambiguous. In the
Rodrıguez-Celma et al., 2013), highlighting a high congruency of case of ambiguous homolog assignments, we manually curated each
the iron deficiency response between the two species. The secretion match using phylogenetic analysis (Figs S1–S5 & S7–S17) and, in
of iron-mobilizing compounds marks a key feature of both some cases attributed a single locus to multiple loci of the other
strategies. In Strategy I plants, the presence of organic Fe3+ species.
complexes is a prerequisite for root-mediated reduction. At alkaline
pH, Fe3+ reduction requires mobilization of iron by chelation via
Subnetwork selection
root-borne coumarins before Fe2+ can be freed from the ligand and
taken up from the soil. Similarly, in Strategy II plants detachment of In order to focus the analysis on the genes most relevant to iron
iron from Fe3+ oxides via secretion of PS is critical for the acquisition homeostasis, we isolated a subnetwork of genes based on their
of iron from the soil. The major conceptual difference between the known function in iron homeostasis. The list of published genes
two iron acquisition strategies appears to be related to the location of related to iron homeostasis is given in Table S1 and consists of iron
reductive chelate splitting, which occurs either extracellularly or homeostasis genes listed in Kobayashi & Nishizawa (2012), genes
intracellularly, an aspect which has received little attention. Our characterized after 2012, and uncharacterized putative iron
analysis further suggests that key nodes such as NA metabolism, transporters from the ZIP, NRAMP and OPT-YSL families.
ROS detoxification, iron sensing by HHE domain-containing From the original 1046 gene network, we cropped the listed genes
proteins, and iron signaling via bHLH transcription factors are as well as genes that were co-expressed with at least two of them,
conserved in the two model organisms, and, presumably, among the resulting in the 55 genes subnetwork presented in Fig. 1.
two iron acquisition strategies. Furthermore, the recruitment of
proteins to a novel physiological context by slightly modifying their
Acknowledgements
molecular function, for example OsNAAT in the synthesis of PS or
OsYSL15 in import of Fe–PS, provides a means to shape biological The authors thank Wendar Lin (Bioinformatics Core Laboratory
processes in search for novel solutions. Assignment of a well-defined IPMB) for bioinformatics support and Thomas J. Buckhout
molecular function to the proteins controlling and mediating iron (Humboldt University Berlin) for critical comments on the
homeostasis will set the stage for a holistic understanding of the ‘iron manuscript. This work was supported by an Academia Sinica
efficiency’ of a given species or cultivar. Identification of the traits Investigator Award to WS.
that allow plants to thrive on soils with immobile iron sources is a
prerequisite to prevent yield losses, improve the nutritional quality
Author contributions
of crops, and to combat iron deficiency-induced anemia in humans
by generating iron-efficient germplasm. 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.
Materials and Methods

Network construction ORCID

A suite of 1349 genes that were highly iron-regulated either Louis Grillet https://orcid.org/0000-0002-2914-9023
positively or negatively in either leaves or roots (FC > 5; P < 0.05) Wolfgang Schmidt https://orcid.org/0000-0002-7850-6832

Ó 2019 The Authors New Phytologist (2019) 224: 11–18

New Phytologist Ó 2019 New Phytologist Trust www.newphytologist.com
 New
16 Forum Viewpoints Phytologist

Inoue H, Kobayashi T, Nozoye T, Takahashi M, Kakei Y, Suzuki K, Nakazono M,

Nakanishi H, Mori S, Nishizawa NK. 2009. Rice OsYSL15 is an iron-regulated
iron(III)-deoxymugineic acid transporter expressed in the roots and is essential for
Louis Grillet1 and Wolfgang Schmidt1,2,3* iron uptake in early growth of the seedlings. Journal of Biological Chemistry 284:
3470–3479.
1 Inupakutika MA, Sengupta S, Nechutsai R, Jennings PA, Onuchic JN, Azad RK,
Institute of Plant and Microbial Biology, Academia Sinica,
Padilla P, Mittler R. 2017. Phylogenetic analysis of eukaryotic NEET proteins
Taipei 11529, Taiwan; uncovers a link between a key gene duplication event and the evolution of
2
Biotechnology Center, National Chung-Hsing University, vertebrates. Scientific Reports 7: 42571.
Taichung 40227, Taiwan; Ishimaru Y, Masuda H, Bashir K, Inoue H, Tsukamoto T, Takahashi M,
3
Genome and Systems Biology Degree Program, College of Life Nakanishi H, Aoki N, Hirose T, Ohsugi R et al. 2010. Rice metal-nicotianamine
transporter, OsYSL2, is required for the long-distance transport of iron and
Science, National Taiwan University, Taipei 10617, Taiwan
manganese. The Plant Journal 62: 379–390.
(*Author for correspondence: tel +886 2 2782 7954; Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, Wada
email [email protected]) Y, Watanabe S, Matsuhashi S, Takahashi M et al. 2006. Rice plants take up iron
as an Fe3+-phytosiderophore and as Fe2+. The Plant Journal 45: 335–346.
References Kobayashi T, Nagasaka S, Senoura T, Itai RN, Nakanishi H, Nishizawa NK. 2013.
Iron-binding haemerythrin RING ubiquitin ligases regulate plant iron responses
Anderegg G, Ripperger H. 1989. The normalizing factor for the tomato mutant and accumulation. Nature Communications 4: 2792.
chloronerva. Correlation between metal-complex formation and biological Kobayashi T, Nakanishi Itai R, Ogo Y, Kakei Y, Nakanishi H, Takahashi M,
activity of nicotianamine analogs. Journal of the Chemical Society–Chemical Nishizawa NK. 2009. The rice transcription factor IDEF1 is essential for the early
Communications 10: 647–650. response to iron deficiency, and induces vegetative expression of late
Bashir K, Ishimaru Y, Itai RN, Senoura T, Takahashi M, An G, Oikawa T, Ueda embryogenesis abundant genes. The Plant Journal 60: 948–961.
M, Sato A, Uozumi N et al. 2015. Iron deficiency regulated OsOPT7 is essential Kobayashi T, Nishizawa NK. 2012. Iron uptake, translocation, and regulation in
for iron homeostasis in rice. Plant Molecular Biology 88: 165–176. higher plants. Annual Review of Plant Biology 63: 131–152.
Briat JF, Fobis-Loisy I, Grignon N, Lobreaux S, Pascal N, Savino G, Thoiron S, Koike S, Inoue H, Mizuno D, Takahashi M, Nakanishi H, Mori S, Nishizawa NK.
von Wiren N, Van Wuytswinkel O. 1995. Cellular and molecular aspects of iron 2004. OsYSL2 is a rice metal-nicotianamine transporter that is regulated by iron
metabolism in plants. Biology of the Cell 84: 69–81. and expressed in the phloem. The Plant Journal 39: 415–424.
Briat JF, Ravet K, Arnaud N, Duc C, Boucherez J, Touraine B, Cellier F, Gaymard Lan P, Li W, Wen TN, Shiau JY, Wu YC, Lin W, Schmidt W. 2011. iTRAQ
F. 2010. New insights into ferritin synthesis and function highlight a link between protein profile analysis of Arabidopsis roots reveals new aspects critical for iron
iron homeostasis and oxidative stress in plants. Annals of Botany 105: 811–822. homeostasis. Plant Physiology 155: 821–834.
Colangelo EP, Guerinot ML. 2004. The essential basic helix-loop-helix protein Le Jean M, Schikora A, Mari S, Briat JF, Curie C. 2005. A loss-of-function
FIT1 is required for the iron deficiency response. Plant Cell 16: 3400–3412. mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading. The
Curie C, Cassin G, Couch D, Divol F, Higuchi K, Le Jean M, Misson J, Schikora A, Plant Journal 44: 769–782.
Czernic P, Mari S. 2009. Metal Movement within the plant: contribution of Long TA, Tsukagoshi H, Busch W, Lahner B, Salt DE, Benfey PN. 2010. The
nicotianamine and yellow stripe1-like transporters. Annals of Botany 12: 465. bHLH transcription factor POPEYE regulates response to iron deficiency in
Curie C, Panaviene Z, Loulergue C, Dellaporta SL, Briat JF, Walker EL. 2001. Arabidopsis roots. Plant Cell 22: 2219–2236.
Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) Lubkowitz M. 2011. The oligopeptide transporters: a small gene family with a
uptake. Nature 409: 346–349. diverse group of substrates and functions? Molecular Plant 4: 407–415.
DalCorso G, Pesaresi P, Masiero S, Aseeva E, Nemann DS, Finazzi G, Joliot P, Mendoza-Cozatl DG, Xie QQ, Akmakjian GZ, Jobe TO, Patel A, Stacey MG,
Barbato R, Leister D. 2008. A complex containing PGRL1 and PGR5 is involved Song LH, Demoin DW, Jurisson SS, Stacey G et al. 2014. OPT3 is a component
in the switch between linear and cyclic electron flow in Arabidopsis. Cell 132: 273– of the iron-signaling network between leaves and roots and misregulation of
285. OPT3 leads to an over-accumulation of cadmium in seeds. Molecular Plant 7:
DiDonato RJ, Roberts LA, Sanderson T, Eisley RB, Walker EL. 2004. Arabidopsis 1455–1469.
Yellow Stripe-Like2 (YSL2): a metal-regulated gene encoding a plasma membrane Naranjo-Arcos MA, Maurer F, Meiser J, Pateyron S, Fink-Straube C, Bauer P.
transporter of nicotianamine-metal complexes. The Plant Journal 39: 403–414. 2017. Dissection of iron signaling and iron accumulation by overexpression of
Eide D, Broderius M, Fett J, Guerinot ML. 1996. A novel iron-regulated metal subgroup Ib bHLH039 protein. Scientific Reports 7: 10911.
transporter from plants identified by functional expression in yeast. Proceedings of Nechushtai R, Conlan AR, Harir Y, Song LH, Yogev O, Eisenberg-Domovich Y,
the National Academy of Sciences, USA 93: 5624–5628. Livnah O, Michaeli D, Rosen R, Ma V et al. 2012. Characterization of
Grillet L, Lan P, Li W, Mokkapati G, Schmidt W. 2018. IRON MAN is a Arabidopsis NEET reveals an ancient role for NEET proteins in iron metabolism.
ubiquitous family of peptides that control iron transport in plants. Nature Plants Plant Cell 24: 2139–2154.
4: 953–963. Ogo Y, Itail RN, Nakanishi H, Kobayashi T, Takahashi M, Mori S, Nishizawal
Harrison PM, Arosio P. 1996. Ferritins: molecular properties, iron storage function NK. 2007. The rice bHLH protein OslRO2 is an essential regulator of the genes
and cellular regulation. Biochimica et Biophysica Acta-Bioenergetics 1275: 161– involved in Fe uptake under Fe-deficient conditions. The Plant Journal 51: 366–
203. 377.
Haydon MJ, Cobbett CS. 2007. A novel major facilitator superfamily protein at the Rajniak J, Giehl RFH, Chang E, Murgia I, von Wiren N, Sattely ES. 2018.
tonoplast influences zinc tolerance and accumulation in Arabidopsis. Plant Biosynthesis of redox-active metabolites in response to iron deficiency in plants.
Physiology 143: 1705–1719. Nature Chemical Biology 14: 442–450.
Hertle AP, Blunder T, Wunder T, Pesaresi P, Pribil M, Armbruster U, Leister D. Robinson NJ, Procter CM, Connolly EL, Guerinot ML. 1999. A ferric-chelate
2013. PGRL1 is the elusive ferredoxin-plastoquinone reductase in photosynthetic reductase for iron uptake from soils. Nature 397: 694–697.
cyclic electron flow. Molecular Cell 49: 511–523. Rodrıguez-Celma J, Pan IC, Li WF, Lan P, Buckhout TJ, Schmidt W. 2013. The
Hirayama T, Lei GJ, Yamaji N, Nakagawa N, Ma JF. 2018. The putative peptide transcriptional response of Arabidopsis leaves to Fe deficiency. Frontiers in Plant
gene FEP1 regulates iron deficiency response in Arabidopsis. Plant and Cell Science 4: 276.
Physiology 59: 1739–1752. R€omheld V, Marschner H. 1986. Evidence for a specific uptake system for iron
Hu X, Kato Y, Sumida A, Tanaka A, Tanaka R. 2017. The SUFBC2 D complex is phytosiderophores in roots of grasses. Plant Physiology 80: 175–180.
required for the biogenesis of all major classes of plastid Fe-S proteins. The Plant Santi S, Schmidt W. 2009. Dissecting iron deficiency-induced proton extrusion in
Journal 90: 235–248. Arabidopsis roots. New Phytologist 183: 1072–1084.

New Phytologist (2019) 224: 11–18 Ó 2019 The Authors

www.newphytologist.com New Phytologist Ó 2019 New Phytologist Trust
New
Phytologist Viewpoints Forum 17

Schmid NB, Giehl RFH, Doll S, Mock HP, Strehmel N, Scheel D, Kong XL, Hider von Wiren N, Klair S, Bansal S, Briat J-F, Khodr H, Shioiri T, Leigh RA, Hider
RC, von Wiren N. 2014. Feruloyl-CoA 60 hydroxylase1-dependent coumarins RC. 1999. Nicotianamine chelates both FeIII and FeII. Implications for metal
mediate iron acquisition from alkaline substrates in Arabidopsis. Plant Physiology transport in plants. Plant Physiology 119: 1107–1114.
164: 160–172. Xiong H, Kakei Y, Kobayashi T, Guo X, Nakazono M, Takahashi H, Nakanishi H,
Schmidt W, Buckhout TJ. 2011. A hitchhiker’s guide to the Arabidopsis ferrome. Shen H, Zhang F, Nishizawa NK et al. 2013. Molecular evidence for
Plant Physiology and Biochemistry 49: 462–470. phytosiderophore-induced improvement of iron nutrition of peanut intercropped
Schuler M, Bauer P. 2011. Heavy metals need assistance: the contribution of with maize in calcareous soil. Plant, Cell & Environment 36: 1888–1902.
nicotianamine to metal circulation throughout the plant and the Arabidopsis NAS Xu XM, Adams S, Chua NH, Moller SG. 2005. AtNAP1 represents an atypical
gene family. Frontiers in Plant Science 2: 69. SufB protein in Arabidopsis plastids. Journal of Biological Chemistry 280: 6648–
Selote D, Samira R, Matthiadis A, Gillikin JW, Long TA. 2015. Iron-Binding E3 6654.
Ligase mediates iron response in plants by targeting basic helix-loop-helix Yen MR, Tseng YH, Saier MH. 2001. Maize Yellow Stripe1, an iron-
transcription factors. Plant Physiology 167: 273–286. phytosiderophore uptake transporter, is a member of the oligopeptide transporter
Sis 
o-Terraza P, Rios JJ, Abadia J, Abadia A, Alvarez-Fern andez A. 2016. Flavins (OPT) family. Microbiology 147: 2881–2883.
secreted by roots of iron-deficient Beta vulgaris enable mining of ferric oxide via Yuan YX, Wu HL, Wang N, Li J, Zhao WN, Du J, Wang DW, Ling HQ. 2008.
reductive mechanisms. New Phytologist 209: 733–745. FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene
Sivitz AB, Hermand V, Curie C, Vert G. 2012. Arabidopsis bHLH100 and expression for iron homeostasis in Arabidopsis. Cell Research 18: 385–397.
bHLH101 control iron homeostasis via a FIT-independent pathway. PLoS ONE Zhai ZY, Gayomba SR, Jung HI, Vimalakumari NK, Pi~ neros M, Craft E, Rutzke
7: e44843. MA, Danku J, Lahner B, Punshon T et al. 2014. OPT3 is a phloem-specific iron
Stacey MG, Patel A, McClain WE, Mathieu M, Remley M, Rogers EE, Gassmann transporter that is essential for systemic iron signaling and redistribution of iron
W, Blevins DG, Stacey G. 2008. The Arabidopsis AtOPT3 protein functions in and cadmium in Arabidopsis. Plant Cell 26: 2249–2264.
metal homeostasis and movement of iron to developing seeds. Plant Physiology Zhang H, Li Y, Yao X, Liang G, Yu D. 2017. POSITIVE REGULATOR OF IRON
146: 589–601. HOMEOSTASIS1, OsPRI1, facilitates iron homeostasis. Plant Physiology 175:
Suorsa M. 2015. Cyclic electron flow provides acclimatory plasticity for the 543–554.
photosynthetic machinery under various environmental conditions and Zhang J, Liu B, Li MS, Feng DR, Jin HL, Wang P, Liu J, Xiong F, Wang JF, Wang
developmental stages. Frontiers in Plant Science 6: 800. HB. 2015. The bHLH transcription factor bHLH104 interacts with IAA-
Suzuki M, Nozoye T, Nagasaka S, Nakanishi H, Nishizawa NK, Mori S. 2016. The LEUCINE RESISTANT3 and modulates iron homeostasis in Arabidopsis. Plant
detection of endogenous 20 -deoxymugineic acid in olives (Olea europaea L.) Cell 27: 787–805.
indicates the biosynthesis of mugineic acid family phytosiderophores in non- Zheng LQ, Huang FL, Narsai R, Wu JJ, Giraud E, He F, Cheng LJ, Wang F, Wu P,
graminaceous plants. Soil Science and Plant Nutrition 62: 481–488. Whelan J et al. 2009. Physiological and transcriptome analysis of iron and
Takahashi M, Yamaguchi H, Nakanishi H, Shioiri T, Nishizawa NK, Mori S. phosphorus interaction in rice seedlings. Plant Physiology 151: 262–274.
1999. Cloning two genes for nicotianamine aminotransferase, a critical Zheng LQ, Ying YH, Wang L, Wang F, Whelan J, Shou HX. 2010. Identification
enzyme in iron acquisition (strategy II) in graminaceous plants. Plant of a novel iron regulated basic helix-loop-helix protein involved in Fe homeostasis.
Physiology 121: 947–956. BMC Plant Biology 10: 166.
Tamir S, Paddock ML, Darash-Yahana-Baram M, Holt SH, Sohn YS, Agranat L,
Michaeli D, Stofleth JT, Lipper CH, Morcos F et al. 2015. Structure-function
analysis of NEET proteins uncovers their role as key regulators of iron and ROS
homeostasis in health and disease. Biochimica et Biophysica Acta-Molecular Cell
Supporting Information
Research 1853: 1294–1315. Additional Supporting Information may be found online in the
Tissot N, Robe K, Gao F, Grant-Grant S, Boucherez J, Bellegarde F, Maghiaoui A,
Supporting Information section at the end of the article.
Marcelin R, Izquierdo E, Benhamed M et al. 2019. Transcriptional integration
of the responses to iron availability in Arabidopsis by the bHLH factor ILR3. New
Phytologist 223: 1433–1446. Fig. S1 Phylogenetic tree of NAS proteins from rice and
Tsai HH, Rodrıguez-Celma J, Lan P, Wu YC, Velez-Berm udez IC, Schmidt W. Arabidopsis.
2018. Scopoletin 8-hydroxylase-mediated fraxetin production is crucial for iron
mobilization. Plant Physiology 177: 194–207.
Fig. S2 Phylogenetic tree of OPT and YSL proteins from rice and
Tsai H-H, Schmidt W. 2017. Mobilization of iron by plant-borne coumarins.
Trends in Plant Science 22: 538–548. Arabidopsis.
Tsednee M, Yang SC, Lee DC, Yeh KC. 2014. Root-secreted nicotianamine from
Arabidopsis halleri facilitates zinc hypertolerance by regulating zinc bioavailability. Fig. S3 Phylogenetic tree of Major Facilitator Superfamily proteins
Plant Physiology 166: 839–553. from rice and Arabidopsis.
Urzica EI, Casero D, Yamasaki H, Hsieh SI, Adler LN, Karpowicz SJ, Blaby-Haas
CE, Clarke SG, Loo JA, Pellegrini M et al. 2012. Systems and trans-system level
analysis identifies conserved iron deficiency responses in the plant lineage. Plant Fig. S4 Phylogenetic tree of bHLH proteins from rice and
Cell 24: 3921–3948. Arabidopsis.
Von Wiren N, Mori S, Marschner H, Romheld V. 1994. Iron inefficiency in maize
mutant ys1 (Zea mays L cv Yellow-Stripe) is caused by a defect in uptake of iron Fig. S5 Phylogenetic tree of IMA peptides from Arabidopsis and a
phytosiderophores. Plant Physiology 106: 71–77.
few monocots.
Walter PB, Knutson MD, Paler-Martinez A, Lee S, Xu Y, Viteri FE, Ames BN.
2002. Iron deficiency and iron excess damage mitochondria and mitochondrial
DNA in rats. Proceedings of the National Academy of Sciences, USA 99: 2264–2269. Fig. S6 Network of genes co-expressed with Arabidopsis PGR5-like
Wang N, Cui Y, Liu Y, Fan H, Du J, Huang Z, Yuan Y, Wu H, Ling HQ. 2013. A.
Requirement and functional redundancy of Ib subgroup bHLH proteins for iron
deficiency responses and uptake in Arabidopsis thaliana. Molecular Plant 6: 503–513.
Fig. S7 Phylogenetic tree of ZIP proteins from rice and Arabidopsis.
Waters BM, Chu HH, DiDonato RJ, Roberts LA, Eisley RB, Lahner B, Salt DE,
Walker EL. 2006. Mutations in Arabidopsis Yellow Stripe-Like1 and Yellow
Stripe-Like3 reveal their roles in metal ion homeostasis and loading of metal ions in Fig. S8 Phylogenetic tree of NRAMP proteins from rice and
seeds. Plant Physiology 141: 1446–1458. Arabidopsis.

Ó 2019 The Authors New Phytologist (2019) 224: 11–18

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

and Tansley insights.

26

www.newphytologist.com

np-centraloffi[email protected]
np-usaoffi[email protected]

www.newphytologist.com

New Phytologist (2019) 224: 11–18 Ó 2019 The Authors

www.newphytologist.com New Phytologist Ó 2019 New Phytologist Trust