Plant Soil (2024) 495:77–97
https://doi.org/10.1007/s11104-023-06094-4
RESEARCH ARTICLE
Revealing the molecular basis regulating the iron deficiency
response in quinoa seedlings by physio‑biochemical
and gene expression profiling analyses
Yao Zhao · Zhangyi Chen · Weimin Li · Fei Liu ·
Liangliang Sun · Min Wu · Ping Zhang ·
Leiping Hou · Meilan Li · Jin Xu
Received: 31 March 2023 / Accepted: 23 May 2023 / Published online: 16 June 2023
© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2023
Abstract
Background and aims Improving iron utilization
efficiency in crops is of great importance to food
security and sustainable agricultural development.
Quinoa (Chenopodium quinoa Willd.) is a high-iron
accumulation crop; however, the molecular mecha-
nisms underlying iron accumulation and iron defi-
ciency response in quinoa are unclear.
Methods We investigated the molecular basis of the
iron deficiency response in quinoa through physio-
biochemical and transcriptome analysis using the
methods of iron depletion and resupply.
Results Iron depletion inhibits photosynthesis but
does not cause obvious oxidative damage in quinoa.
Iron resupply generally induces the expression of
genes associated with sugar metabolism and primary
Responsible Editor: Jian Feng Ma.
Supplementary Information The online version
contains supplementary material available at https://​doi.​
org/​10.​1007/​s11104-​023-​06094-4.
Y. Zhao · Z. Chen · W. Li · F. Liu · L. Sun · M. Wu ·
P. Zhang · L. Hou (*) · M. Li (*) · J. Xu (*)
College of Horticulture, Shanxi Agricultural University,
Taigu 030801, China
e-mail: sxndhlp@126.com
M. Li
e-mail: mlli@sxau.edu.cn
J. Xu
e-mail: xujin@sxau.edu.cn
nitrogen metabolism in roots. The expression of
iron-responsive transcription factors, including FIT,
BTS and bHLH101, as well as the metal transporters
MTP and NRAMP, is induced under iron depletion
and continues to be expressed after 6 h of iron resup-
ply. Iron depletion increases the levels of cytokinin,
ABA, SA and ethylene precursor ACC but decreases
JA levels. The contents of cytokinin and ACC gradu-
ally recovered after 6 h of iron resupply, but the ABA
content did not change significantly; in contrast, the
JA content further decreased. Iron depletion markedly
induces DELLA gene expression. Iron depletion does
not change the auxin level in roots, but it inhibits the
auxin pathway.
Conclusion These results indicated that the iron
deficiency response is tightly modulated by phytohor-
mone signaling. This study elucidates the regulatory
network of the iron deficiency response and provides
a theoretical basis for further exploring the molecular
mechanism of high-iron accumulation in quinoa.
Keywords Iron depletion and resupply · Primary
metabolism · Metal transporters · Iron-responsive
transcription factors · Phytohormones
Introduction
Iron (Fe) is crucial for plant growth and development
(Balk and Schaedler 2014). As a cofactor for numer-
ous proteins and enzymes, Fe is required for many
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biochemical processes, including mitochondrial res-
piration, photosynthetic electron transport, oxidative
stress protection, the tricarboxylic acid cycle (TCA)
and multiple metabolic pathways (Balk and Schaedler
2014). Fe deficiency is a serious issue that threatens
crop yield and quality (Briat et al. 2015). Fe is also
a necessary micronutrient for humans. However, due
to the lack of Fe in their plant-based diets, more than
2 billion people worldwide suffer from Fe deficiency
(DeLoughery 2017). Fe deficiency leads to hair loss,
heart failure, pulmonary hypertension and iron-defi-
ciency anemia (IDA) (Lukito and Wahlqcist 2020).
Therefore, improving Fe absorption and utilization
efficiency in crops is of great importance to human
health and sustainable agricultural development.
There are two major strategies for Fe acquisi-
tion in higher plants. Under low-Fe conditions, non-
graminaceous plants employ a reduction strategy
­ e3+ is more soluble in soil as a result
(strategy I): F
of rhizosphere acidification through protons extruded
by ­H+-ATPases and the secretion of phenolic com-
pounds, such as coumarin (Kobayashi and Nishizawa
2012). ­Fe3+ is reduced to ­Fe2+ by ferric reductase
oxidase 2 (FRO2) (Robinson et al. 1999; Mukher-
jee et al. 2006) in Arabidopsis roots. Subsequently,
iron-regulated transporter 1 (IRT1), a major Fe trans-
porter, transports F­ e2+ into roots (Eide et al. 1996;
Vert et al. 2002). Graminaceous plants, in contrast,
use a chelation-based strategy (strategy II) (Morrissey
and Guerinot 2009). Mugineic acids (MAs), which
are synthesized from L-methionine through nicotian-
amine (NA) in graminaceous plants (Mori and Nishi-
zawa 1987), have high affinity for F ­ e3+ (Mori 1999).
3+
­Fe -MAs are taken up through yellow stripe-like
(YSL) transporters (Curie et al. 2001).
After being absorbed by roots, Fe first accumulates
at the base of the shoot, the discrimination center
(DC), and is then transported to the shoot (Suzuki
et al. 2006; Tsukamoto et al. 2009; Yokosho et al.
2009; Ishimaru et al. 2011). Arabidopsis citrate trans-
porter ferric reductase defective 3 (FRD3), which
localizes to the plasma membrane in root pericycle
cells surrounding xylem, is involved in Fe transport
in the xylem (Yokosho et al. 2009). OsYSL16 and
OsYSL18 are ­Fe3+-DMA transporters; the former is
present in the plasma membrane of cells surrounding
the xylem and is involved in Fe distribution through
vascular bundles, and the latter is involved in Fe trans-
location in reproductive organs and phloem in joints
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Plant Soil (2024) 495:77–97
(Kakei et al. 2012). OsYSL2 transports ­Fe2+-NA and
is responsible for phloem Fe transport, particularly
into grains (Ishimaru et al. 2010). Phenolic com-
pounds are involved in the utilization of apoplastic
Fe that precipitates in the stele or root surface. Phe-
nolic efflux zero 1 (PEZ1) and PEZ2 are involved in
phenolic efflux transport in plants (Ishimaru et al.
2011; Bashir et al. 2011). Fe is stored partially in the
vacuole and is mobilized when needed. Vacuolar iron
transporters (VITs), which encode tonoplast Fe trans-
porters in Arabidopsis, transport Fe into vacuoles
(Kim et al. 2006), and natural resistance-associated
macromolecule protein 3 (NRAMP3) and NRAMP4
are responsible for transporting ­Fe2+ and other diva-
lent metals from vacuoles to the cytoplasm (Lanquar
et al. 2010).
Fe homeostasis in plants is tightly regulated by
transcription factors (TFs) or transcriptional regu-
lators that regulate ferroportin gene expression
directly or indirectly (Kobayashi et al. 2013). Under
Fe deficiency, the dimers formed by bHLH IVc TFs
[including bHLH34, bHLH104, IAA-leucine resist-
ant 3 (ILR3)/bHLH105 and bHLH115] can directly
activate the expression of bHLH Ib genes (including
bHLH38/39/100/101), which can form heterodimers
with FER-like iron deficiency-induced transcrip-
tion factor (FIT) to regulate the expression of genes
related to ­ Fe3+ uptake, including IRT1 and fer-
ric reduction oxidase 2 (FRO2) (Yuan et al. 2008;
Wang et al. 2013). POPEYE (PYE) is a regulator
in the Fe deficiency response. Chip-on-chip experi-
ments revealed that PYE targets genes associated
with metal homeostasis, including nicotinic acid
synthase 4 (NAS4), FRO3, and zinc-induced facilita-
tor 1 (ZIF1) (Long et al. 2010). Arabidopsis brutus
(BTS) and its homologs brutus-like 1 (BTSL1) and
BTSL2 are potential Fe sensors that negatively regu-
late Fe homeostasis by directly targeting FIT in plants
(Kobayashi et al. 2013; Selote et al. 2015; Tissot et al.
2019). Recently, IRON MANs (IMAs) have been
identified as Fe sensors in plants. IMAs are inhibitors
of BTS under Fe-deficient conditions (Li et al. 2021).
Overexpression of IMA genes induces the expression
of bHLH Ib genes and upregulates the expression
of IRT1 and FRO2 genes in Arabidopsis (Hirayama
et al. 2018; Grillet et al. 2018).
Phytohormones play important roles in regulating
Fe deficiency-mediated Fe uptake processes (Grazi-
ano and Lamattina 2007; Séguéla et al. 2008; García
Plant Soil (2024) 495:77–97
et al. 2010; Romera et al. 2011). Auxin and ethylene
upregulate the expression of FIT to mediate the Fe
deficiency response in plants (Graziano and Lamat-
tina 2007; García et al. 2010; Romera et al. 2011).
In contrast, cytokinin (CK), abscisic acid (ABA) and
jasmonic acid (JA) inhibit the expression of these
genes (Séguéla et al. 2008).
Quinoa (Chenopodium quinoa Willd.) is one of
the major food crops in the Andes (Jacobsen 2003).
Due to its special nutritional qualities, such as high
protein and richness in trace elements, including Fe,
it is increasingly being cultivated and used as a new
functional food (Moscoso-Mujica et al. 2022). How-
ever, the molecular mechanism of high Fe utilization
efficiency in quinoa is still unclear. In this study, by
integrating physio-biochemical and transcriptomic
analyses, we investigated the Fe deficiency response
in quinoa seedlings using the method of Fe depletion
and resupply. The aim of this study was to explore the
molecular regulatory mechanisms underlying Fe defi-
ciency responses and Fe uptake in quinoa.
Materials and methods
Plant material and growth conditions
Seeds of quinoa (Chenopodium quinoa Willd.) culti-
var Yunli108 were kindly provided by Prof. Zhiquan
Cai (Foshan University). Quinoa seeds were steri-
lized with 2% (v/v) sodium hypochlorite solution for
15 min, rinsed with ­ddH2O 3–5 times, and then sown
on 1/2 MS medium for germination. Five-day-old
well-developed quinoa seedlings were transferred to
Hoagland solution for continued cultivation for 7 d.
Then, the seedlings were transferred to Fe-deficient
Hoagland solution (FeD, removal of Fe from the
nutrient solution) for 4 d. After 4 days of Fe deple-
tion, the seedlings were transferred to fresh Hoagland
solution with normal Fe for recovery growth for 3 h
(RE 3 h), 6 h (RE 6 h), 12 h (RE 12 h), 24 h (RE
24 h) and 3 d (RE 3d).
Phenotypic parameters and chlorophyll fluorescence
After treatment, the plant height and root length were
measured using ImageJ analysis software (v1.8.0). A
chlorophyll meter (SPAD-502 Plus, Konica-Minolta,
Japan) was used to measure the chlorophyll content
79
in the third leaf of the seedling. Chlorophyll fluo-
rescence was measured using a pulse amplitude
modulated fluorometer (Imaging-PAM M-series,
Walz, Germany). Leaves were placed in the dark
for 30 min and exposed to a weakly modulated light
source (0.5 μmol·m−2·s−1), and the initial fluores-
cence ­(F0) was determined. Then, the saturation pulse
(3000 μmol·m−2·s−1) emission was measured for
0.8 s to determine the maximum fluorescence (­Fm).
Far-red light (5 μmol·m−2·s−1) irradiation for 4 s was
used as the basic fluorescence ­(F0’), the steady-state
chlorophyll fluorescence ­(FS) under actinic illumina-
tion was measured, and the maximum light adaptive
fluorescence ­(Fm’) was measured using the second
saturation pulse. The nonphotochemical quench-
ing coefficient (qN & NPQ) and Y (NPQ) are cal-
culated according to the following formula (Kramer
et al. 2004): qN = ­(Fm-Fm′)/(Fm-Fo); NPQ = ­(Fm-Fm′)/
Fm′; Y (NPQ) = 1-Y (II) -1/[NPQ + 1 + qL ­(Fm/F0-1)],
where Y (II) = ­(Fm′- ­F0)/Fm′ and qL = [(Fm′- ­FS)*Fo′]/
[(Fm′- ­Fo)*FS].
Determination of malondialdehyde (MDA) contents
Approximately 0.4 g of sample was ground in a pre-
cooled mortar with 4 ml of precooled PBS solution
(100 mM pH = 7.8) in an ice bath. After centrifuga-
tion at 4 °C and 10,000 r/min for 20 min, the super-
natant was used to determine MDA contents using the
thiobarbituric acid (TBA) method (Zeng et al. 2018).
RNA sequencing and RT‒qPCR analysis
Total RNA was extracted from quinoa roots using
TRIzol (Takara, Beijing, China), and the purity and
concentration of RNA were detected using a Nan-
oDrop 2000 (Thermo, USA). The integrity of the
RNA was verified using an Agilent 2100 bioana-
lyzer (Agilent Technologies, Palo Alto, Calif.). The
sequencing libraries were constructed with a Next®
Ultra™ RNA Library Prep Kit (Illumina, NEB,
USA). RNA sequencing was performed on the Illu-
mina HiSeq X Ten platform at Beijing BMK Bio-
technology Co., Ltd. (Beijing, China). After remov-
ing the low-quality sequences (the number of bases
with quality Q ≤ 10 represented more than 50% of the
entire read), clean raw data were obtained. The reads
from all samples were aligned to the quinoa reference
genome (ASM168347v1) using HISAT2 software.
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Sequences were aligned to public databases using
BLAST software to obtain annotation information.
False discovery rate (FDR) < 0.05 and |log2(change
fold)|> 1 were used as filtering criteria for differen-
tially expressed genes (DEGs).
A coexpression network was constructed using
weighted gene coexpression network analysis
(WGCNA) (Langfelder and Horvath 2008). First, all
samples were clustered using the ‘WGCNA’ R pack-
age, filtering for outliers. The expression similarity of
genes was then assessed according to Pearson’s cor-
relation coefficient, resulting in a correlation matrix.
The correlation matrix is converted into a weighted
neighborhood matrix using a soft threshold func-
tion. The neighborhood matrix is again converted
to a topological overlap matrix (TOM), and soft
threshold power is introduced. Based on the above
analysis, hierarchical clustering was implemented to
identify modules, each containing at least 30 genes
(minimodule size = 30). Finally, key modules were
selected for Gene Ontology (GO) enrichment analy-
sis by association analysis between the modules and
the treatment groups, yielding pathways associated
with the response to Fe deficiency. Meanwhile, the
gene interaction network was drawn and visualized
according to the weighted value between the genes.
The RNA was reverse transcribed using a cDNA syn-
thesis kit (gDNA clean-up, novo protein, China), and
six genes were randomly selected for RT‒qPCR with
three independent biological and technical replicates.
Specific primers were designed using Primer Premier
5.0, and the sequences are shown in Supplemental
Table S1.
Determination of phytohormone contents
After grinding the root samples in liquid nitrogen,
approximately 0.1 g of powder was placed in an
Eppendorf tube with 1.5 ml of 50% ice-cold acetoni-
trile (ACN) (including isotopic internal standard).
After sonication in an ice bath for 3 min, the sam-
ples were placed in a rotary instrument overnight
at 4 °C and centrifuged at 13,000 rpm for 10 min at
4 °C. The supernatant was purified by an Oasis HLB
RP (1 cc/30 mg) column and washed with 30% (v/v)
ACN. Samples were further evaporated under a gen-
tle stream of gaseous nitrogen and redissolved in 30%
ACN for mass spectrometric analysis. The contents
of indole acetic acid (IAA), N6‐isopentenyladenine
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(iP), N6-(Delta2 isopentenyl) adenine riboside (iPR),
abscisic acid (ABA), 1-aminocyclopropane-1-car-
boxylic acid (ACC), salicylic acid (SA), SA 2-O-β-
glucoside (SAG), Jasmonic acid (JA) and JA-Ile were
determined using ultrahigh-performance liquid chro-
matography‒electrospray ionization tandem mass
spectrometry (UHPLC‒ESI‒MS/MS) as described
by Šimura et al. 2018. The mass spectrometry detec-
tion system was an AB SCIEX Qtrap5500 (AB
SCIEX in the United States). The obtained data were
sorted with MultiQuant (AB Science, USA) software
and supplemented by manual inspection. The mass of
hormone in the sample was calculated according to
the mass spectrum peak area. The concentrations of
ACC, SA, IAA, ABA and JA were calculated by the
internal standard method (Cohen et al. 1987; Shimizu
et al. 2015).
Determination of mineral elements
Seedlings were soaked in 1 mM ­Na2·EDTA for 30 min
and then rinsed with ­ddH2O 5 times. The roots and
leaves were dried in a high-temperature oven and
ground into powder (Zhang et al. 2019). Then, the
samples are decocted with concentrated ­HNO3. The
contents of Fe, elements (Zn), manganese (Mn),
magnesium (Mg) and potassium (K) were measured
using an inductively coupled plasma atomic emission
spectrometer (ICPAES, IRIS Advantage, iCAP 6300,
USA).
Statistics and analysis
All of the experiments were performed with three
biological replicates, and at least 18 seedlings were
used per biological replicate. Data were analyzed for
significance using Student’s t-test or Tukey’s test as
appropriate using SPSS software (P < 0.01).
Results
Fe deficiency represses quinoa seedling growth
Compared with the control, Fe depletion decreased
primary root (PR) length (Fig. 1A and C), root fresh
weight (FW) (Fig. 1F) and DW (Fig. 1G) by 42.9%,
39.25% and 32.52%, respectively. However, plant
height (Fig. 1 B) and leaf growth (Fig. 1D and E)
Plant Soil (2024) 495:77–97
Fig. 1  Fe deficiency represses quinoa growth. Twelve-day-old
quinoa seedlings were grown in Hoagland solution with (con-
trol) or without Fe for 4 d (FeD), and then transferred to nor-
mal Hoagland solution for recovery growth for 3 h (RE 3 h),
6 h (RE 6 h), 12 h (RE 12 h), 24 h (RE 24 h) or 3 d (RE 3d).
A, Representative images show the Fe deficiency phenotype
of quinoa seedlings. Bar = 7 cm. B-M, The plant height (B),
were not significantly inhibited at 4 d after Fe deple-
tion. These results indicated that Fe depletion has an
earlier inhibitory effect on the root system growth
than on the leaf growth of quinoa seedlings. After
12 h of Fe resupply, all the physiological param-
eters detected showed recovery to varying degrees
(Fig. 1B-G). The malondialdehyde (MDA) level is
an effective indicator of membrane lipid peroxida-
tion, which reflects the degree of oxidative damage
in plants (Hodges et al. 1999). Although Fe deple-
tion inhibited seedling growth, neither Fe depletion
81
primary root (PR) length (C), leaf fresh weight (D) and dry
weight (E), root fresh weight (F) and dry weight (G), MDA
contents in the leaves (H) and roots (I), SPAD (J), NPQ (K),
qN (L) and Y(NPQ) (M) were determined. Bars represent
the SEs, and different letters indicate significant differences
(P < 0.05)
nor resupply significantly affected MDA levels in
quinoa seedlings (Fig. 1H and I).
Compared with the control, Fe depletion decreased
SPAD by 24.58% (Fig. 1J). After 3 h, 6 h, 24 h and
3 d of Fe resupply, the SPAD decreased by 21.75%,
21.11%, 17.72% and 6.56%, respectively, compared
with the control, indicating that the chlorophyll
contents gradually recovered with the extension of
Fe resupply time. We then measured the changes
in chlorophyll fluorescence parameters. As shown
in Fig. 1K-M, compared with the control, NPQ
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decreased by 7.75% in Fe-depleted plants, and it
increased gradually after Fe resupply (increased by
22.47% after 12 h of Fe resupply) (Fig. 1K). Fe deple-
tion did not significantly affect qN and Y (NPQ) val-
ues. However, qN reached the highest value after 6 h
of Fe resupply (increased by 19.8% compared with
the control), and Y (NPQ) reached the highest value
after 12 h of Fe resupply (increased by 33.81% com-
pared with the control) (Fig. 1L and M).
Transcriptomic analysis
To elucidate the molecular regulatory mechanisms
underlying the Fe deficiency response, we performed
a global transcriptome analysis in quinoa roots. Fig-
ure 2A shows the experimental design and sampling
time points. The clean reads obtained from five treat-
ment groups (CT, FeD 4 d, RE 3 h, RE 6 h and RE
12 h) were 26,741,782, 26,637,586, 25,571,965,
24,051,150 and 25,653,602, respectively. The GC
content of all samples exceeded 43%, and the Q30 of
all samples exceeded 91%. These data show that the
Fig. 2  Transcriptome analysis. A, Experimental design and root sampling time points. B, Statistics of the differentially expressed
genes (DEGs). C, Heatmap of the expression patterns of DEGs. The scale represents the l­og10 (FPKM + 1)
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sequencing quality met the requirements of expres-
sion profile analysis (Table S2).
A total of 533 DEGs (296 upregulated and 237
downregulated) were identified in the FeD/CT com-
parison group, 1224 DEGs (730 upregulated and
494 downregulated) were identified in the RE 3 h/
CT comparison, 2025 DEGs (1196 upregulated and
829 downregulated) were identified in the RE 6 h/
CT comparison, 1620 DEGs (521 upregulated and
1099 downregulated) were identified in the RE 12 h/
CT comparison, 657 DEGs (441 upregulated genes
and 216 downregulated) were identified in the RE
3 h/FeD comparison, 1432 DEGs (1018 upregulated
and 414 downregulated) were identified in the RE
6 h/FeD comparison, and 821 DEGs (284 upregu-
lated and 537 downregulated) were identified in
the RE 12 h/FeD comparison (Fig. 2B and C). Six
genes were randomly selected for RT‒qPCR analy-
sis, which verified the accuracy of the transcriptome
sequencing data (Fig. S1A), and the correlation anal-
ysis also showed the reliability of the transcriptome
(Fig. S1B).
Plant Soil (2024) 495:77–97
Weighted gene coexpression network analysis
We then performed a weighted gene coexpression
network analysis (WGCNA) to reveal the regula-
tory network and explore hub genes in response to
Fe depletion and resupply in quinoa roots. A soft
threshold power of 14 was introduced in the network
topology to reveal the scale independence and aver-
age connectivity of the network (Fig. S2). Hierarchi-
cal clustering trees were constructed by weighted cor-
relation coefficients, and a total of 4340 genes from
the 15 samples were clustered and divided into six
modules (Fig. 3A). The correlation analysis between
the six modules and five treatment groups showed
that the blue module with the control group, brown
module with the ‘RE 3 h’ group and turquoise module
with the ‘RE 6 h’ group were significantly correlated
(Fig. 3B). Therefore, the three modules were chosen
to further explore the responses of these modules to
Fe deficiency by heatmap display of expression pro-
files of all coexpressed genes in these modules and
subsequent GO enrichment analysis.
The expression of genes in the blue module was
downregulated under Fe depletion and further down-
regulated in subsequent Fe resupply (Fig. 3C). GO
analysis revealed that genes in the blue module were
mainly enriched in the pathways of hydrogen perox-
ide catabolic process, xyloglucan metabolic process,
cell wall biogenesis and oxidative stress response
(Fig. 3D), indicating that genes involved in cell wall
modification are correlated with the module. Next, we
performed an intramodule gene connectivity analysis.
A gene interaction network was drawn for the top 10%
of the screening using Cytoscape_v3.8.2 (Shannon
et al. 2003) (Fig. 3E). Nine peroxidase genes involved
in hydrogen peroxide catabolic process, three xylo-
glucan endotransglucosylase/hydrolase (XTH) genes
involved in xyloglucan metabolic process, and eight
TFs including bHLH126 (gene16088), bHLH120
(gene39718), bHLH84 (gene35232), serine/threo-
nine-protein kinase-like protein CCR4 (gene11972
and gene55268), two-component response regula-
tor ORR9 (gene11404), zinc finger CCCH domain-
containing protein 29 (gene19434) and an unknown
newgene4130 were identified in the network. Other
key genes, including endoglucanase 5 (gene35085),
beta-glucosidase 24 (gene49548) and expansin-A8
(gene46229), were also identified in the network
(Fig. 3E). We also found that the expression of two
83
XTH genes was downregulated under Fe depletion,
while all seven identified differentially expressed
XTH genes were repressed during Fe resupply, espe-
cially after 6 h and 12 h of Fe resupply (Fig. S3;
Table S3).
The expression of genes in the brown module was
gradually upregulated at FeD and RE 3 h and gradu-
ally recovered from RE 6 h to RE 12 h (Fig. 3F). The
main GO pathways in the module include riboflavin
biosynthesis and metabolism, nicotianamine biosyn-
thesis, metal ion transmembrane transport, malate
transport and salicylic acid biosynthesis. Although
the role of secreted riboflavin in promoting Fe acqui-
sition is still unclear, it is speculated that it may
play the role of redox bridge, bind extracellular Fe
to promote Fe chelate reductase activity, or mediate
the dissolution of low soluble F ­ e3+ oxide by modi-
fying the extracellular Fe and changing the rhizos-
phere microbial community to promote Fe absorp-
tion (Cesco et al. 2010; Sisó-Terraza et al. 2016;
Gheshlaghi et al. 2021). Malic acid mediates metal
ion uptake and long-distance transport from roots to
shoots through chelation with metal ions in plants
(Wei et al. 2009). These results indicated that genes
involved in metal ion transmembrane transport were
enriched in the module. Subsequently, 22 hub genes
including eight TFs were screened from the mod-
ule, including riboflavin biosynthesis protein PYRD
(gene17628 and gene54967) and PYRR​ (gene43304),
bHLH55 (gene18505), bHLH19 (gene48000), aqua-
porin NIP5-1 (gene18777), WRKY65 (gene20159
and gene19025), E3 ubiquitin-protein ligase RIBA1
(gene2563), bifunctional riboflavin biosynthesis pro-
tein BAH1L1 (gene26491), ABC transporter C fam-
ily member 1 (ABCG1, gene33533), zinc transporter
29 (ZTP29, gene38651), detoxification 14 (DTX14,
gene40544), DTX43 (gene4328), metal tolerance
protein 10 (MTP10, gene56361), sensitive to proton
rhizotoxicity 2 (STOP2, gene34813), auxin response
factor 5 (ARF5, gene32058), transcription repressor
OFP8 (gene34205), zinc finger protein CONSTANS
(gene35730), serine/threonine-protein kinase WNK11
(gene43913), and transcription repressor OFP14
(gene45219) (Fig. 3H).
The expression of genes in the turquoise module
was upregulated after Fe resupply, reached its peak at
RE 6 h, and gradually recovered at RE12 h (Fig. 3I).
GO analysis revealed that genes in the module were
mainly enriched in the pathways of phytohormone
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Fig. 3  Weighted gene coexpression network analysis. A, A
hierarchical clustering tree was constructed by correlation
coefficients between genes. Different branches of the cluster-
ing tree represent different gene modules. B, Module-trait rela-
tionship. The depth of the color indicates the degree of corre-
lation. C, F, I, Heatmaps show the expression profiles of all
coexpressed genes in the blue (C), brown (F) and turquoise
(I) modules. The color scale indicates the expression level of
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genes, and the bars show the consistent expression patterns
in the module coexpressed genes. D, G, J, GO analysis of the
genes in the blue (D), brown (G) and turquoise (J) modules.
E, H, K, The predicted regulatory network for the blue (E),
brown (H) and turquoise (K) modules. The rhombuses repre-
sent the transcription factors, and the inner circles and rhom-
buses represent the hub genes
Plant Soil (2024) 495:77–97
responses and abscisic acid (ABA) pathway, amino
acid biosynthesis and metabolism, mitochondrial
pyruvate transport, oxidative stress response, lateral
root development and hydrogen peroxide catabolism,
indicating that genes in the module modulate phyto-
hormone signaling, root system development and pri-
mary metabolism processes (Fig. 3J). A total of 151
genes were analyzed for intramodule gene connec-
tivity, and 45 hub genes were identified in the mod-
ule, including 15 TFs, such as ethylene-responsive
transcription factor (ERF), homeobox-leucine zip-
per protein ATHB40, NAC29, bZIP53, agamous-like
MADS-box protein AGL80, cysteine-rich receptor-like
protein kinase 10 (CRK10) and WRKY75 (Fig. 3K).
Carbon metabolism and primary nitrogen metabolism
WGCNA showed that Fe deficiency affected primary
metabolism in quinoa roots. We thus investigated the
expression of genes involved in carbon metabolism
and primary nitrogen metabolism (Fig. 4; Table S4).
The carbon metabolic pathway mainly includes gly-
colysis, the pentose phosphate pathway and the tricar-
boxylic acid cycle (TCA cycle). Transcriptome analy-
sis indicated that the expression of most genes related
to carbohydrate synthesis and metabolism during Fe
resupply was upregulated in quinoa roots (Fig. 4).
In the glycolysis pathway, the expression of
hexokinase 2 (HK2), fructose-1,6-bisphosphatase
(FBP), fructose-bisphosphate aldolase (FBA), glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH) and
2,3-bisphosphoglycerate-independent phosphoglycer-
ate mutase-like (iPGAM) genes was upregulated after
Fe resupply, while the expression of HK3, ribulose
bisphosphate carboxylase (Rubisco) and phospho-
glycerate mutase-like (PGAM) genes was downregu-
lated after 6 h and 12 h of Fe resupply (Fig. 4). In
the pentose phosphate pathway, the expression of
transketolase (TK) and ribose-5-phosphate isomer-
ase (RPI) genes was downregulated after 3 h or 6 h of
Fe resupply; the expression of fructose-bisphosphate
aldolase (ALDO) and ribose-phosphate pyrophos-
phokinase (PRPS) genes was upregulated after Fe
resupply (Fig. 4). In the TCA pathway, the expres-
sion of PRPS, aconitate hydratase (ACON), malate
synthase (MS) and NADP-dependent malic enzyme
(NADP-ME) genes was upregulated under Fe deple-
tion and/or after 3 h and 6 h of Fe resupply, while the
expression of 2-oxoglutarate dehydrogenase (OGDH)
85
and malate dehydrogenase (MDH) genes was down-
regulated after 3 h and/or 12 h of Fe resupply (Fig. 4).
The direct carbon source of amino acid biosynthesis
mainly comes from glycolysis, the pentose phosphate
pathway and the TCA cycle, while nitrogen mainly
comes from primary nitrogen metabolism. In the pri-
mary nitrogen metabolism pathway, the expression
of nitrite reductase (NiR), high-affinity nitrate trans-
porter (NRT), glutamate dehydrogenase (GDH), and
cytochrome b5 domain-containing (CYB5D) genes
was upregulated after Fe resupply (Fig. 4).
Fe deficiency affects micronutrient element
accumulation in quinoa seedlings
WGCNA also revealed that Fe deficiency affected
the expression of genes associated with metal ion
transport and accumulation in quinoa roots. We thus
investigated the expression patterns of these DEGs.
A total of 62 DEGs involved in metal ion uptake,
transport, accumulation and detoxification were
identified, including 11 TFs/transcription regula-
tors (TRs), one MTP, two YSL, five oligopeptide
transporter (OPT), 11 VIT, three NRAMP, 13 ZRT/
IRT-like protein (ZIP), eight multidrug and toxic
compound extrusion (MATE) and eight heavy metal-
associated isoprenylated plant protein (HIPP) genes
(Fig. 5A; Table S5).
Fe depletion induced the expression of one
FIT, three BTS and three bHLH101 genes in
roots, while the expression of two FIT, two BTS
and two bHLH101 genes continued to be upregu-
lated after 3 h and 6 h of Fe resupply and grad-
ually returned to normal levels after 12 h of Fe
resupply, while the expression of two bHLH101
genes was downregulated (Fig. 5A).
MTP is a cation diffusion factor (CDF) transporter
(Haney et al. 2005; Montanini et al. 2007) that plays
an important role in the uptake, transport and detoxi-
fication of heavy metals in plants. We found that the
expression of MTP was upregulated under Fe defi-
ciency, and the expression of MTP continued to be
upregulated after 3 h and 6 h of Fe resupply, and then
its expression was downregulated after 12 h of Fe
resupply (Fig. 5A). The YSL protein family is iden-
tified based on sequence similarity with maize (Zea
mays) Yellow Stripe 1 (YS1) (Von et al. 1994). YSL
genes participate in metal absorption and long-dis-
tance transportation in plants (Ishimaru et al. 2010).
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Fig. 4  Effects of Fe depletion and resupply on the expression
of genes related to carbon metabolism and primary nitrogen
metabolism in the roots of quinoa seedlings. Asterisks in the
Fe deficiency induced the expression of one YSL
gene, which was upregulated under Fe deficiency and
after 3 h of Fe resupply. In contrast, another YSL gene
was downregulated under Fe depletion and after Fe
resupply in quinoa roots (Fig. 5A). OPTs are respon-
sible for transporting oligopeptides, glutathione
and glutathione chelates in plants (Lubkowitz et al.
1998). Some studies have demonstrated that OPTs
can also transport Fe or other metal ions (Vasconce-
los et al. 2008; Stacey et al. 2006). The expression
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Plant Soil (2024) 495:77–97
heatmaps represent the DEGs ­[log2 |fold change|≥ 1 or ≤ -1 and
false discovery rate (FDR) < 0.01]. The list of DEGs is shown
in Table S4
of two OPT genes was upregulated; however, almost
all OPT genes were downregulated after Fe resupply
in quinoa roots (Fig. 5A). VITs are involved in the
transport of Fe and Mn in vacuoles, regulate metal
homeostasis and improve the survival of plants under
excess metal ions (Sharma et al. 2020). We found
that Fe deficiency did not affect VIT gene expres-
sion, while 63.64% of VIT genes showed upregulated
expression after 3 h of Fe resupply, and 54.55% of
VIT genes were downregulated during Fe resupply
Plant Soil (2024) 495:77–97 87

Fig. 5  Fe deficiency affects

metal element accumula-
tion in quinoa seedlings.
A, Differentially expressed
genes involved in metal ion
uptake, transport and accu-
mulation in quinoa roots.
B, Contents of Fe, Zn and
Mn in the leaves and roots
of quinoa seedlings under
Fe deficiency and after 12 h
of Fe resupply. Values are
given as the means ± SDs
and different letters indicate
significant differences
(P < 0.05). The list of DEGs
is shown in Table S5

(Fig. 5A), suggesting that VIT genes are involved in
the maintenance of Fe homeostasis in quinoa roots.
NRAMP metal transporters are widely distributed in
plants and are mainly involved in the absorption and
transport of Fe, Mn, Cd and other metal ions (Xiao
et al. 2008; Peng et al. 2018). ZIP transporters partici-
pate in the transport of ­Zn2+, ­Ca2+, ­Fe2+ and ­Cd2+ in
plants (Chen et al. 2008). The expression of NRAMPs
was upregulated under Fe deficiency, continued to be
upregulated after 3 h of Fe resupply, and then down-
regulated after 6 h and 12 h of Fe resupply in quinoa
roots (Fig. 5A). However, the expression of most
ZIP genes was downregulated under Fe depletion
and during Fe supply (Fig. 5A). MATE transporters
are responsible for transporting diverse substrates,
including heavy metal ions (Zhou et al. 2014). A pre-
vious study demonstrated that MATE transporters are
also involved in maintaining Fe homeostasis in plants
(Durrett et al. 2007). Both Fe depletion and resupply
upregulated the expression of MATE genes in quinoa
roots (Fig. 5A). HIPPs are a family of metal chaper-
one proteins that reduce the toxicity of heavy metals
by combining with heavy metal ions, thus improving
heavy metal toxicity tolerance in cells (Zhao et al.
2013; Khan et al. 2019). The expression of HIPP
genes was downregulated after 6 h and 12 h of Fe
resupply in quinoa roots (Fig. 5A).
We then detected the contents of metal ions
in quinoa seedlings. Fe depletion reduced the Fe
content in quinoa leaves by 18.84%; after 12 h of
Fe resupply, the Fe content in leaves increased by
129.54% compared with the control (Fig. 5B).
Fe depletion reduced the Fe content in roots by
71.67%; after 12 h of Fe resupply, however, the Fe

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content in roots did not return to the normal level,
still 63.33% lower than the control (Fig. 5B). Fe
depletion increased the Zn content in quinoa roots;
after 12 h of Fe resupply, the Zn content returned
to the normal level (Fig. 5B). Fe depletion also did
not significantly affect Mn level in leaves; however,
it increased Mn content by 139.86% in roots. After
12 h of Fe resupply, the root Mn content gradually
returned to the normal level (Fig. 5B).
Changes in phytohormone levels and pathways
during Fe depletion and resupply.
Fig. 6  Phytohormone contents in quinoa roots under Fe deple-
tion and resupply. A, IAA. B, iPR. C, iP. D, ABA. E, ACC.
F, SA. G, SAG. H, JA. I, JA-Ile. The phytohormone contents
were determined in the roots of 12-day-old quinoa seedlings
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Phytohormones play an important role in modu-
lating plant growth and development and stress
responses. The above WGCNA revealed that Fe defi-
ciency altered phytohormone pathways. Therefore,
we investigated the changes in phytohormone con-
tents and the gene expression patterns in their bio-
synthesis and signaling pathways during Fe depletion
and resupply (Fig. 6 and 7; Table S6).
The expression of one tryptophan aminotrans-
ferase-related 1 (TAR1) gene was upregulated under
Fe depletion, and one YUCCA​ (YUC​) gene was
under Fe deficiency (FeD) for 4 d and after 6 h of Fe resup-
ply (RE 6 h). Values are given as the means ± SEs (n = 3), Stu-
dent’s t-test, *P < 0.05
Plant Soil (2024) 495:77–97
upregulated after 6 h of Fe resupply, while the expres-
sion of two YUC​ genes was downregulated during
Fe resupply. The expression of one TAR1 gene was
downregulated during Fe resupply, whereas glucosyl-
transferase (UGT​) gene expression was upregulated
after 3 h of Fe resupply (Fig. 7A). However, we found
that Fe depletion and resupply did not significantly
affect the IAA contents in quinoa roots (Fig. 6A).
We also found that the expression of one auxin influx
carrier auxin resistant 1 (AUX1) gene was down-
regulated during Fe depletion and/or resupply. In the
auxin signaling pathway, the expression of one auxin/
indole-3-acetic acid (AUX/IAA), one auxin response
factor (ARF) and two auxin-inactivating Gretchen
hagen 3 (GH3) genes was upregulated after 3 h or
6 h of Fe resupply, while the expression of almost
all small auxin upregulated RNA (SAUR​) genes was
downregulated in quinoa roots (Fig. 7A).
Fe depletion significantly increases the content of
N6‐isopentenyladenine (iP), a major form of cyto-
kinin in plants (Hirose et al. 2008), and its content
returns to normal levels after 6 h of Fe resupply
(Fig. 6C). The expression of one cytokinin trans-
hydroxylase CYP735A and one phosphate-isopente-
nyltransferase (IPT) gene was downregulated during
Fe resupply. In contrast, the expression of one glu-
cosyltransferase 73C (UGT73C) and three glucosyl-
transferase 85A (UGT85A) genes was upregulated
during Fe depletion and/or resupply (Fig. 7B). In the
cytokinin pathway, the expression of one AHP and
one B-type response regulator (B-ARR​) gene was
upregulated during Fe depletion and/or resupply,
while the expression of A-ARR​ genes was downregu-
lated during Fe resupply (Fig. 7B).
In the GA pathway, Fe deficiency upregulated
the expression of DELLA and gibberellin insensitive
dwarf 1 (GID1) genes, indicating that the GA signal
pathway was involved in the Fe-deficient response in
quinoa roots (Fig. 7C).
Fe depletion significantly induced an increase in
ABA content in roots, and the ABA content did not
recover after 6 h of Fe resupply (Fig. 6D). Fe deple-
tion downregulated the expression of two abscisic
acid-deficient 2 (ABA2) and two cytochrome P450
CYP707A genes, while Fe resupply upregulated the
expression of four zeaxanthin epoxidase (ZEP) genes
in quinoa roots (Fig. 7D).
Fe depletion significantly increased the content of
the ethylene precursor ACC in quinoa roots, which
89
gradually decreased after Fe resupply (Fig. 6E). The
expression of three aminocyclopropane-1-carboxylic
acid oxidase (ACO) genes was downregulated after
6 h and/or 12 h of Fe resupply, while the expres-
sion of the other three ACO genes was upregulated
(Fig. 7E). In the ethylene signaling pathway, the
expression of two ethylene receptor (ETR), two eIN3-
binding F-box 1 and 2 (EBF1/2) and four ethylene
response factor 1/2 (ERF1/2) genes was upregulated
after 6 h of Fe resupply (Fig. 7E).
Fe depletion significantly increased the contents
of SA and SA 2-O-β-glucoside (SAG), a derivative of
SA, while Fe resupply further increased their contents
(Fig. 6F). However, we did not find a DEG related to
SA biosynthesis in quinoa roots. In the SA pathway,
the expression of two TGACG-binding factor (TGA​
) and two pathogenesis-related genes1 (PR-1) genes
was upregulated under Fe depletion, while the expres-
sion of four TGA​ genes and almost all PR-1 genes
was also upregulated during Fe resupply (Fig. 7F),
indicating that Fe deficiency and resupply induced the
SA signal pathway in quinoa roots.
In contrast to SA, Fe depletion significantly
decreased the levels of JA and JA-Ile, and Fe resup-
ply further decreased their contents in roots (Fig. 6H
and I). The expression of two lipoxygenase (LOX)
genes was downregulated under Fe depletion. Mean-
while, the expression of four LOX, two allene oxide
synthase (AOS), one lecithin-cholesterol acyltrans-
ferase (LCAT​) and five defective in anther dehiscence
1 (DAD1) genes was downregulated during Fe resup-
ply. In contrast, the expression of one LOX and three
12-oxophytodienoate reductase (OPR) genes was
upregulated during Fe resupply (Fig. 7G). In the JA
pathway, the expression of two jasmonate zim (JAZ)
genes was downregulated, while one myelocyto-moto-
sis2 (MYC2) gene was upregulated under Fe deple-
tion. Meanwhile, the expression of four JAZ and five
MYC2 genes was upregulated, while the expression
of three MYC2 genes was downregulated during Fe
resupply (Fig. 7G).
Discussion
In this study, using physio-biochemical, transcrip-
tomic and bioinformatics methods, we analyzed the
physiological changes in quinoa seedlings under Fe
depletion and resupply, as well as the differentially
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◂Fig. 7  The expression patterns of genes involved in phyto-
hormone biosynthesis and signaling pathways in quinoa roots
under Fe depletion and resupply. A, auxin. B, cytokinin. C,
GA. D, ABA. E, ethylene. F, SA. G, JA. Asterisks in the heat-
maps represent the DEGs ­[log2 |fold change|≥ 1 or ≤ -1 and
false discovery rate (FDR) < 0.01]. The list of DEGs is shown
in Table S6
expressed genes and regulatory networks in roots. In
addition, we also revealed the molecular regulatory
mechanism of Fe deficiency on phytohormone path-
ways, primary carbon/nitrogen metabolism and metal
ion absorption and accumulation in quinoa roots.
These results provide a basis for further revealing the
molecular mechanism of Fe deficiency responses and
identifying and analyzing the key regulatory genes in
quinoa.
Physiological responses and reprogramming of
the core metabolic pathway in Fe‑deficient quinoa
seedlings
Fe is an essential trace element in chlorophyll biosyn-
thesis (Wang et al. 2018). One of the typical symp-
toms of Fe deficiency in plants is the reduction in
chlorophyll content and yellowing of leaves, which
leads to a reduction in photosynthetic efficiency.
Our results showed that the chlorophyll contents
decreased under Fe depletion in quinoa seedlings;
with the extension of Fe resupply time, the contents
of chlorophyll gradually increased (Fig. 1J). Nonpho-
tochemical quenching refers to the ability of plants to
convert excess light energy into heat, that is, the abil-
ity of light protection (Muller et al. 2001). qN is the
nonoptical chemical quenching coefficient, reflect-
ing the energy loss in the form of heat energy gener-
ated by the light energy absorbed by the PSII antenna
pigment but not transmitted by the photosynthetic
electrons (Horton et al. 1996). When excessive light
energy is absorbed by the PSII reaction center, if it
cannot be released in time, it will cause damage to the
photosynthetic system (Fleming et al. 2012). There-
fore, qN is a self-protection mechanism and has a pro-
tective effect on the photosynthetic system. Y (NPQ)
represents the energy dissipated into heat through the
regulatory light protection mechanism (Klughammer
and Schreiber 2008). In the process of Fe resupply,
the nonphotochemical quenching coefficients (qN and
NPQ) and Y (NPQ) gradually increased, suggesting
that the photoprotection ability gradually increased
91
during Fe resupply in quinoa seedlings (Fig. 1K-
M). In addition, we found that although Fe deple-
tion inhibited seedling growth, neither Fe depletion
nor resupply significantly affected MDA levels in
quinoa seedlings (Fig. 1H and I), indicating that Fe
deficiency did not cause serious oxidative damage to
quinoa seedlings.
Fe-containing enzymes commonly exist in plants,
including a series of reductases, such as nucleo-
tide reductase, nitrate reductase and nitrite reduc-
tase, hydrogenase, nitrogenase, peroxidase (includ-
ing cytochrome peroxidase), oxidase (including
cytochrome C oxidase), and hydratase, etc. (Pernil
and Schleiff 2019; Zhang and Zheng 2020). Fe-con-
taining enzymes are involved in the electron transfer
process and play a critical role in cell metabolism
(Richardson and Ponka 1997). Therefore, Fe defi-
ciency will result in metabolic pathway disorders in
plants. Our results showed that Fe deficiency altered
the primary metabolic pathways (Fig. 4), thereby
affecting plant growth and development.
The differential expression of metal transporters
and Fe deficiency‑responsive transcription factors
affected Fe absorption and accumulation in quinoa
Fe deficiency changes the gene expression patterns
of metal transporters and Fe deficiency-responsive
TFs, including 11 genes of three TF families and 35
metal transporter genes such as MTPs, YSLs, OPTs,
VITs, NRAMPs, ZIPs, MATEs and HIPPs (Fig. 5A).
MTPs are involved in the uptake and accumulation of
heavy metals, while YSLs mediate the long-distance
transportation of heavy metals in plants (Haney et al.
2005; Montanini et al. 2007; Gustin et al. 2011; Ishi-
maru et al. 2010). OsYSL2 is responsible for trans-
porting Fe(II)-NA, and OsYSL9 transports Fe(II)-NA
and Fe(III)-NA in rice (Ishimaru et al. 2010). OPTs
are responsible for transporting Fe or other metal ions
in plants (Vasconcelos et al. 2008; Stacey et al. 2006).
Rice OsOPT1/3/4 participates in the transportation
of Fe(II)-NA or Fe(III)-NA. Among them, OsOPT4
has the highest absorption efficiency for Fe(II)-NA
(Vasconcelos et al. 2008). AtOPT2 is induced by Fe
deficiency in Arabidopsis roots (Stacey et al. 2006).
VITs are vacuolar Fe transporters. Arabidopsis
AtVIT1 is responsible for transporting Fe to the vacu-
ole for storage, thus improving Fe toxicity tolerance
(Narayanan et al. 2015). In addition, NRAMPs and
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ZIPs are involved in the absorption and transport of
Fe, Mn, Cd, Zn, and other metal ions in plants (Xiao
et al. 2008; Chen et al. 2008). MATE transporters
are involved in maintaining Fe homeostasis in plants
(Durrett et al. 2007; Rogers et al. 2009). The Arabi-
dopsis MATE transporter AtFRD3 and rice OsFRD1
have citric acid efflux activity and can transport Fe
in plants (Rogers and Guerinot 2002; Yokosho et al.
2009). GmFRD3a/3b are induced by Fe deficiency
and can maintain Fe homeostasis in soybean (Rog-
ers et al. 2009). HIPPs metal chaperone proteins can
combine with heavy metal ions to alleviate heavy
metal toxicity (Zhao et al. 2013). Loss-of-function of
HIPP genes results in sensitivity to heavy metal tox-
icity in rice (Khan et al. 2019). Future research will
explore the molecular mechanism of these differen-
tially expressed metal transporter genes in mediating
Fe absorption in quinoa roots and Fe accumulation in
plants. Fe deficiency induces the expression of Fe-
responsive TF genes, and the expression of these TFs
is downregulated rapidly after Fe resupply in Arabi-
dopsis and rice (Kim et al. 2019; Liang et al. 2020).
In this study, we found that Fe depletion significantly
induced the expression of FIT, BTS and bHLH101
genes in quinoa roots (Fig. 5A). However, the expres-
sion of these genes continued to be upregulated after
3 and 6 h of Fe resupply and gradually returned to
normal levels until 12 h (Fig. 5A). High expression
of these TFs affected the expression of metal trans-
porter genes, such as MTP, NRAMP and MATE genes
(Fig. 5A), thus promoting the absorption and accu-
mulation of Fe and other metal ions at the early stage
of Fe resupply. Similar to the results of Valentinuzzi
et al. (2020), we found that after 12 h of Fe resupply,
the Fe content in leaves increased rapidly (three times
higher than that under Fe depletion), but the Fe con-
tent in roots did not increase significantly (Fig. 5B).
These results indicated that the Fe absorbed by the
root system was rapidly transported to the shoots after
Fe resupply. This may be one of the mechanisms of
high Fe accumulation in quinoa.
Phytohormones participate in regulating the Fe
deficiency response in quinoa roots
Fe depletion and resupply did not affect IAA con-
tents in quinoa roots (Fig. 6A), but they regulated
the auxin pathway (Fig. 7A). The expression of
AUX1 and SAUR​genes was downregulated under Fe
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depletion and resupply. In contrast, the expression
of one gene encoding Aux/IAA repressor protein
and two auxin-inactivating GH3 genes was upreg-
ulated after Fe resupply, indicating that the auxin
pathway is repressed by Fe depletion and resupply.
However, one ARF gene was induced after 3 h of Fe
resupply in quinoa roots (Fig. 7A). Qi et al. (2012)
found that OsARF12 positively regulates Fe content
by modulating the abundance of OsIRT1 in rice.
Future research will elucidate the function of this
ARF gene in the response to Fe deficiency and its
effect on Fe accumulation in quinoa roots.
Cytokinin induces the expression of ZIP1 and
ZIP5 genes in rice (Gao et al. 2019). A previous
study demonstrated that cytokinin negatively regu-
lates Fe uptake by repressing IRT1, FRO2 and FIT
in Arabidopsis roots (Séguéla et al. 2008). However,
we found that Fe deficiency increased the cytokinin
iP content in quinoa roots (Fig. 6C). Meanwhile, the
expression of AHP and B-ARR​ genes was upregu-
lated during Fe depletion and/or resupply; in con-
trast, the expression of A-ARR​ genes, the negative
regulators of cytokinin signaling by repressing
B-ARRs, was downregulated during Fe resupply
(Fig. 7B). These results indicated that Fe depletion
and resupply promoted cytokinin signaling in qui-
noa roots. However, the detailed mechanisms of the
cytokinin-mediated Fe deficiency response and Fe
accumulation in quinoa roots still need to be further
elucidated.
Wild et al. (2016) found that Fe deficiency inhib-
its root growth by inducing DELLA accumula-
tion in Arabidopsis roots. DELLA interacts with
bHLH38/39 and FIT to inhibit their transcriptional
activity. Meanwhile, DELLA exclusion from the epi-
dermal cells of the root differentiation zone releases
FIT/bHLH38/39 and therefore promotes Fe uptake at
the site (Wild et al. 2016). We found that Fe deple-
tion induced the expression of the GA receptor GID1
and the GA-signaling DELLA repressors in quinoa
roots (Fig. 7C), thereby modulating the Fe deficiency
response and Fe accumulation.
Fe depletion and resupply significantly increased
ABA contents in quinoa roots (Fig. 6D). Fan et al.
(2014) found that ABA increases Fe accumulation,
which is independent of IRT1 in Arabidopsis. We
found that the expression of SnRK2 and ABF genes in
the ABA signaling pathway was upregulated under Fe
depletion and/or resupply (Fig. 7D), indicating that
Plant Soil (2024) 495:77–97
the ABA pathway was induced, thus improving Fe
accumulation and Fe deficiency tolerance in quinoa.
Li and Li (2004) showed that Fe deficiency
increases ethylene levels in roots. Ethylene-respon-
sive ERF4 can interact with FIT, thereby regulating
the expression of FRO2 and IRT1 in response to Fe
deficiency in Arabidopsis (Liu et al. 2017; Balparda
et al. 2020). Consistent with this result, we found that
Fe depletion and resupply increased the contents of
the ethylene precursor ACC (Fig. 6E), and the expres-
sion of EBF1/2 and ERF1/2 in the ethylene pathway
was upregulated after 6 h of Fe resupply in quinoa
roots (Fig. 7E), indicating that Fe resupply triggered
the ethylene pathway in quinoa roots.
Exogenous SA increases the uptake of K, Mg and
Mn in maize (Gunes et al. 2007). SA also alleviates
Fe deficiency-induced oxidative stress and increases
chlorophyll contents under Fe deficiency (Kong
et al. 2014). In contrast, JA negatively regulates the
expression of FIT and bHLH38/39/100/101, thereby
inhibiting the expression of IRT1 and FRO2 under Fe
Fig. 8  A model of Fe deficiency response in quinoa roots. The red and blue arrows indicate an increase or decrease of the contents
of phytohormones or micronutrients after Fe depletion compared to the control
93
deficiency in Arabidopsis (Maurer et al. 2011; Cui
et al. 2018). Interestingly, we found that Fe depletion
and resupply increased SA levels but decreased JA
levels in quinoa roots (Fig. 6F and H). These results
indicated that plants modulated the equilibrium of SA
and JA in roots to affect Fe absorption and accumula-
tion and ultimately regulated Fe deficiency adaptation
in quinoa. Previous studies have shown that JA and
SA have antagonistic effects, and SA-induced gluta-
raldehyde is a potential regulatory component of SA/
JA antagonism (Ndamukong et al. 2007). However,
how these two plant hormones regulate the Fe defi-
ciency response in quinoa needs further clarification.
Conclusion
This study investigated the Fe deficiency response in
quinoa seedlings through physio-biochemical and
transcriptomic analyses, and revealed the molecular
regulatory mechanisms underlying the Fe deficiency
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response and Fe absorption in quinoa. We found that
(i) Fe depletion inhibits photosynthesis but does not
cause significant oxidative damage; (ii) Fe deple-
tion changes the gene expression patterns of 35 metal
transporters and 11 Fe deficiency-responsive TFs; (iii)
Fe resupply induces the expression of genes related to
sugar metabolism and primary nitrogen metabolism in
roots; and (iv) Fe depletion increased levels of cyto-
kinins, ABA, SA, and the ethylene precursor ACC but
decreased JA levels (Fig. 8). Meanwhile, we found that
the Fe absorbed by roots can be quickly transported to
the aboveground parts after Fe resupply. These may be
the cause of high Fe accumulation in quinoa. Future
research on the signal regulation processes of the Fe
deficiency response in quinoa will provide a theoretical
basis for breeding efficient Fe utilization crop varieties.
Author contributions Yao Zhao: Data curation, Writing—
original draft. Zhangyi Chen: Data curation, Investigation.
Weimin Li: Investigation, Formal analysis. Fei Liu: Conceptu-
alization, Methodology. Liangliang Sun: Investigation, Formal
analysis. Min Wu: Visualization. Ping Zhang: Investigation.
Leiping Hou: Supervision, Writing—original draft. Meilan
Li: Supervision, Writing—original draft. Jin Xu: Supervision,
Writing—original draft, Writing—review & editing.
Funding This research was supported by the China National
Natural Sciences Foundation (32070314), the Science and tech-
nology Innovation Fund Project of Shanxi Agricultural Univer-
sity (2020BQ24 and 2020QC13) and the Basic Research Pro-
gram of Shanxi Province (Free Exploration) (20210302124369
and 20210302124065).
Data availability All RNAseq datasets were deposited to the
NCBI Short Read Archive under the BioProject PRJNA771182.
Declarations
Competing interest The authors declare that they have no
conflict of interest.
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