Journal of Experimental Botany, Vol. 70, No. 10 pp. 2873–2886, 2019
doi:10.1093/jxb/erz089 Advance Access Publication 2 March 2019
© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
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AhFRDL1-mediated citrate secretion contributes to
adaptation to iron deﬁciency and aluminum stress in peanuts
Wei Qiu1, Nanqi Wang1, Jing Dai1, Tianqi Wang1, LeonV. Kochian2, Jiping Liu2 and Yuanmei Zuo1,*
1 Key Laboratory of Plant–Soil Interactions, MOE, College of Resources and Environmental Sciences, China Agricultural University,
Beijing 100193, China
2 Robert W.Holley Center, US Department of Agriculture-Agricultural Research Service, Ithaca, NY 14853, USA
* Correspondence: email@example.com
Received 30 November 2018; Editorial decision 18 February 2019; Accepted 17 March 2019
Editor: Karl-Josef Dietz, Bielefeld University, Germany
Although citrate transporters are involved in iron (Fe) translocation and aluminum (Al) tolerance in plants, to date none
of them have been shown to confer both biological functions in plant species that utilize Fe-absorption Strategy I.In
this study, we demonstrated that AhFRDL1, a citrate transporter gene from peanut (Arachis hypogaea) that is induced
by both Fe-deﬁciency and Al-stress, participates in both root-to-shoot Fe translocation and Al tolerance. Expression
of AhFRDL1 induced by Fe deﬁciency was located in the root stele, but under Al-stress expression was observed
across the entire root-tip cross-section. Overexpression of AhFRDL1 restored efﬁcient Fe translocation in Atfrd3
mutants and Al resistance in AtMATE-knockout mutants. Knocking down AhFRDL1 in the roots resulted in reduced
xylem citrate and reduced concentrations of active Fe in young leaves. Furthermore, AhFRDL1-knockdown lines had
reduced root citrate exudation and were more sensitive to Al toxicity. Compared to an Al-sensitive variety, enhanced
AhFRDL1 expression in an Fe-efﬁcient variety contributed to higher levels of Al tolerance and Fe translocation by
promoting citrate secretion. These results indicate that AhFRDL1 plays a signiﬁcant role in Fe translocation and Al
tolerance in Fe-efﬁcient peanut varieties under different soil-stress conditions. Given its dual biological functions,
AhFRDL1 may serve as a useful genetic marker for breeding for high Fe efﬁciency and Al tolerance.
Keywords: AhFRDL1, Al tolerance, citrate transporter, dual function, Fe translocation, high Fe-efﬁcient, MATE, peanut (Arachis
Aluminum (Al) and iron (Fe) are, respectively, the rst and
second most abundant metal elements in the Earth’s crust,
and they have contrasting eects on plant growth: Al is toxic
to growth in acidic soils, whereas Fe is an essential micro-
nutrient for growth and development (Guo et al., 2014;
Kochian et al., 2015; Curie and Mari, 2017). Plants have
therefore evolved various strategies to acquire Fe and to
cope with Al stress.
Al toxicity mainly targets the root apex, impairing root
growth and function (Ma etal., 2004; Yang etal., 2008, 2011;
Horst et al., 2010; Zhu et al., 2012, 2014). To cope with Al
toxicity, plants have evolved two resistance mechanisms: an
exclusion mechanism, which involves root exudation of
organic acids (OAs) to the rhizosphere to prevent the entry
of toxic Al3+ ions into the root cells; and an internal toler-
ance mechanism, in which Al is sequestrated into vacuoles
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2874 | Qiu etal.
in root cells and/or translocated from the roots and stored in
vacuoles of shoot cells (Liu etal., 2014; Kochian etal., 2015;
Wang etal., 2017).
In contrast, as an essential element, Fe is involved in funda-
mental biological processes, such as chlorophyll biosynthesis
and photosynthesis. Consequently, when Fe supply is limited,
plants frequently exhibit chlorosis in young leaves and stunted
growth (Jeong and Connolly, 2009). Generally, plants acquire
Fe from the soil through two mechanisms. In Strategy Ispe-
cies, which include non-graminaceous monocots and dicots,
plants secrete protons (H+) from the roots to acidify the soil
and synthesize ferric reductase to convert ferric Fe3+ to fer-
rous Fe2+ for root absorption. In Strategy II species, which
include graminaceous monocots, roots directly secrete che-
lates into the rhizosphere and take up the chelated Fe3+ from
the soil (Römheld and Marschner, 1986). Most of the genes
involved in these uptake processes have been identied and
characterized (Abadía et al., 2011; Bashir et al., 2016; Curie
and Mari, 2017). For long-distance root-to-shoot transport,
formation of an Fe–citrate complex in the xylem is required
for ecient translocation, which is dependent on citrate eux
into the xylem in both Strategy I and II species (Morrissey
and Guerinot, 2009). This eux is mainly facilitated by cit-
rate transporters localized in the root vasculature, which are
encoded by members of the multidrug and toxic compound
extrusion (MATE) gene family (Rogers and Guerinot, 2002;
Green and Rogers, 2004; Durrett etal., 2007).
MATE members constitute a large gene family that is widely
present in bacteria, fungi, plants, and mammals (Omote et al.,
2006). For instance, there are 56 MATE members in Arabidopsis
(Li etal., 2002; Liu et al., 2009). However, only a few mem-
bers of the MATE family have been functionally characterized,
and these include a group of citrate transporters. For example,
AtFRD3, a citrate transporter expressed in Arabidopsis root
pericycle cells, facilitates root-to-shoot Fe translocation via cit-
rate secretion into the xylem, where translocation-ecient Fe–
citrate complexes are formed (Durrett etal., 2007; Morrissey
and Guerinot, 2009). Functional homologs of FRD3 involved
in facilitating root-to-shoot Fe translocation have been iden-
tied in several crop species, including soybean (Glycine max),
rice (Oryza sativa), and rye (Secale cereale) (Rogers etal., 2009;
Yokosho etal., 2009, 2010).
A group of MATE-family citrate transporters also facilitates
Al-activated citrate exudation from roots into the rhizosphere,
a critical step in the Al exclusion mechanism. For instance, the
Al-resistance genes HvAACT1, SbMATE, AtMATE, ScFRDL2,
ZmMATE1, OsFRDL4, and TaMATE1B encode plasma-
membrane-localized citrate transporters that mediate exud-
ation from the root epidermis/cortex to the rhizosphere upon
Al stress in barley (Hordeum vulgare), sorghum (Sorghum bicolor),
Arabidopsis, rye, maize (Zea mays), rice, and wheat (Triticum aes-
tivum) (Furukawa etal., 2007; Magalhaes etal., 2007; Liu etal.,
2009; Yokosho etal., 2009, 2011; Maron etal., 2010; Tovkach
Among the MATE citrate transporters that mediate root-to-
shoot Fe translocation in Strategy II plant species, HvAACT1
also contributes to Al resistance in barley (Fujii et al., 2012).
HvAACT1 is expressed in the root stele, mediating citrate
release from root pericycle cells to the xylem and facilitating
root-to-shoot Fe translocation (Fujii etal., 2012). However, in
Al-tolerant barley accessions, a 1-kb insertion in the upstream
region of HvAACT1 alters its expression site from the stele to
the epidermis and cortex in the root tips, leading to Al-induced
root citrate exudation into the rhizosphere and enhanced
adaptation to acidic soils (Fujii et al., 2012). The dierential
physiological functions of HvAACT1 in Al resistance and Fe
nutrition are due to dierences in expression among tissues but
not due to dierent functions. Citrate transporters expressed in
the root stele facilitate root-to-shoot Fe translocation (Durrett
etal., 2007; Rogers etal., 2009; Yokosho etal., 2009), whereas
those expressed in epidermal/cortical cells are involved in Al
resistance (Liu etal., 2009; Yokosho etal., 2009, 2011; Tovkach
etal., 2013). However, among the Strategy Iplant species, it is
unclear whether MATE citrate transporters can be involved in
both Fe nutrition and Al resistance.
Peanut (Arachis hypogaea) is a Strategy I plant that ranks
fourth among major oil crops in terms of production and is
grown on more than 27 million ha worldwide, with a total
production of almost 43 million tons in 2016; 60% of the pro-
duction area is located in Asia (http://faostat. fao.org/) where
peanut is widely cultivated in both alkaline calcareous and acid
soils (Zuo and Zhang, 2008). Iron deciency in alkaline cal-
careous soils severely limits yield. Intercropping of peanuts and
maize in such soils improves Fe nutrition in the peanut plants
(Zuo and Zhang, 2008; Guo et al., 2014). In this intercrop-
ping system, phytosiderophore deoxymugineic acid (DMA)
secreted from the maize roots signicantly facilitates absorp-
tion of Fe3+-DMA by nearby peanut roots (Xiong etal., 2013).
However, the mechanism underlying root-to-shoot Fe trans-
location in peanuts is unclear.
Here, we report the identication and characterization of
AhFRDL1, a member of the MATE family, in Strategy Ipea-
nuts. It functions as a citrate transporter in Fe nutrition and Al
resistance, thus contributing to peanut ecological adaptation
under Fe-decient and Al-stress conditions.
Materials and methods
Plant material and growth conditions
After germination for 7 d, four peanut seedlings (Arachis hypogaea L.cv.
Luhua14, LH14) were transferred to a 3.5-l pot containing a complete
nutrient solution [2mM Ca(NO3)2, 0.5mM KH2PO4, 0.75mM K2SO4,
0.1mM KCl, 0.65mM MgSO4, 1μM MnSO4, 0.1μM ZnSO4, 0.1μM
CuSO4, 0.005μM (NH4)6Mo7O24, 1μM H3BO3, and 80μM Fe-EDTA;
pH 5.8–6.0] and grown for 7 d with continuous aeration. Four replicate
pots were used per treatment, and the nutrient solution was renewed
every 2 d. The seedlings were then transferred to a solution with or
without 80μM Fe-EDTA and grown under a 16/8h light/dark cycle
(300 μmol m–2 s–1) at 28/22 °C with a relative humidity of 70–75%.
Xylem sap samples were collected at 1, 4, 7, and 10 d.Root samples were
collected, frozen immediately in liquid nitrogen, and stored at –80°C
until RNA extraction.
For the Al treatment, after ger mination for 7 d of, ve seedlings were
transferred to pots containing 45ml of 0.5mM CaCl2 (pH 4.5) solu-
tion overnight and subsequently to fresh solutions of 0.5mM CaCl2
(pH 4.5) containing 0, 40, or 80μM AlCl3. The growth conditions were
as described above. Root length was measured at 0, 6, 12, 24, 36, and
48h; at each time point, 0–1cm of the root tips was collected for RNA
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AhFRDL1 and adaptation to Fe deﬁciency and Al stress | 2875
extraction, and the culture solution was decanted to measure organic acid
T-DNA insertion knockout (KO) mutants of AtMATE (SALK_081671)
and Atfrd3 (SALK_122235) were obtained from the Arabidopsis Biological
Resource Center (https://abrc.osu.edu) (Liu et al., 2009). Wild-type
(WT) and Atfrd3 mutant seeds were surface-sterilized and cold-treated
(4°C) for 3 d, grown on Murashige–Skoog (MS) medium for 2 weeks,
and then transferred to Rockwool soaked in 0.25×MS medium without
sucrose. Seedlings were grown under a 16/8h light/dark cycle (100μmol
m–2 s–1) at 21–22°C and watered once weekly with 0.25×MS medium.
After 7 weeks, the shoots were separated from the roots at the hypocotyl,
and xylem sap was collected as described by Durrett etal. (2007). Young
leaves (the top three fully emerged leaves) were collected for assessment
of HCl-extractable Fe (‘active Fe’), according to the procedure of Guo
etal. (2014). The HCl-extractable Fe concentration was determined by
inductively coupled plasma atomic emission spectrometry (ICP) using an
Optima 3300DV instrument (Perkin Elmer). Chlorophyll was extracted
from young leaves using 1ml of methanol, and total chlorophyll levels
were quantied according to the method of Durrett etal. (2007).
Seeds of the Arabidopsis WT and the AtMATE-KO mutant were sur-
face-sterilized, cold-treated (4°C) for 3 d, and sown onto a plastic mesh
oating on a hydroponic solution without or with 400μM AlCl3 (Liu
etal., 2009; Wang etal., 2017). After 7 d, root exudate was collected and
root length was measured.
To investigate the expression pattern of AhFRDL1, seedlings of the
peanut varieties Luhua14 (LH14, low Fe-eciency), Luhua11 (LH11,
high Fe-eciency), and Dabaisha (DBS, Al-sensitive) were subject to Fe
deciency (0μM) or Al toxicity (0, 40, 80, or 120μM AlCl3) for 7 d or
24h, respectively. Growth conditions were as described above for peanut.
After 7 d of Fe deciency, the chlorophyll contents of a young leaf (the
rst fully emerged leaf) or an old leaf (the fourth fully emerged leaf) were
determined using a SPAD-502 chlorophyll meter (Konica-Minolta), and
the active iron contents were measured as described above. After Al treat-
ment for 24 h, the lengths of the main roots of the peanut seedlings
were measured, and the roots were stained with 0.1% eriochrome cya-
nine R for 2h to assay Al toxicity. The culture solution was collected for
determination of citrate secretion, and root apexes (0–1cm) were cut,
frozen immediately in liquid nitrogen, and stored at –80°C until RNA
Collection and analysis of xylemsap
Three-week-old peanut seedlings were cut at 2cm above the root–shoot
junction, placed in 1.5-ml tubes, and covered by cotton balls. Xylem sap
was allowed to exude from the cut ends into the cotton balls for 2h and
was subsequently sucked out of the cotton using a disposable syringe.
Fe concentrations in the xylem sap were measured using the batho-
phenanthrolinedisulfonic acid (BPDS)-Fe2+ method with 0.1 M BPDS
and 0.5 M ascorbate to reduce ferric Fe3+. The absorbance of the
assay solutions at 533 nm was determined using a spectrophotometer
(NanoDrop). Standard curves were generated using 10, 50, and 100μM
ascorbate-reduced FeCl3. Citrate concentrations in the xylem sap were
measured using HPLC, as described by Yokosho etal. (2011).
RNA extraction and quantitative real-time PCRassays
Total RNA was extracted from the peanut root samples and reverse-
transcribed according to the method of Liu et al. (2009). Quantitative
real-time PCR (qRT-PCR) was perfor med using SYBR Green PCR
Master Mix (Applied Biosystems). The qRT-PCR primers were designed
at the 3´-untranslated region (3´-UTR) where the specic sites were
contained. The RT-PCR fragments were sequenced before the primers
were used for qRT-PCR for quantication of AhFRDL1 expression. The
qRT-PCR program comprised an initial denaturation step at 95 °C for
10min, followed by 40 cycles at 95°C for 15s and at 60°C for 1min,
and a nal dissociation step at 95°C for 15s, 60°C for 1min, and 95°C
for 15 s. The data were analysed using a 7500 Fast Real-Time PCR
System (Applied Biosystems). The expression levels of the genes were
normalized to those of the peanut ubiquitin gene using the △△CT method
(Xiong etal., 2013). All primers are listed in Supplementary Table S1 at
Cloning and sequence analysis of AhFRDL1cDNA
A fragment of AhFRDL1 was amplied using degenerate primers de-
signed from the conserved sequences of OsFRDL1 and AtFRD3 with
Primer Premier 5.0 (Biosoft; Supplementary Table S1). According to
the sequence of the AhFRDL1 fragment, 5′ and 3′ gene-specic pri-
mers were designed. The 5′- and 3′-regions of the AhFRDL1 mRNA
sequence were amplied using a SMART Rapid Amplication of cDNA
Ends (RACE) cDNA Amplication Kit (Clontech) and a 3′ RACE
System (Invitrogen), respectively. The PCR fragments were subcloned
into the pMD20-T vector (TaKaRa) and sequenced. After homologous
recombination, the full ORF of the AhFRDL1 cDNA was amplied
using KOD DNA polymerase (Toyobo). Sequence comparisons were
conducted using DNAMAN software (Lynnon Biosoft). Aphylogenetic
tree was constructed using the MEGA ver. 7.0 software.
Citrate transport assays in Xenopus oocytes
The AhFRDL1 ORF sequence was amplied, and subcloned into a
Xenopus laevis oocyte expression vector, pGEMKN.cRNA preparation,
micro-injection into oocytes, and a citrate transport activity assay were
performed as described previously by Yokosho etal. (2009). For electro-
physiological studies, oocytes injected with AhFRDL1 cRNA or water
were incubated in Modied Barth’s Saline (MBS) solution at 18°C. After
incubation for 1 d, 25ml of 200mM sodium citrate was injected into the
oocytes, which were then incubated for 0.5–2h. The net current across
the oocyte membrane was measured immediately after adding 0μM or
100μM Al to the solution (Furukawa etal., 2007). Current was recorded
using a two-electrode voltage clamp system with an amplier (MEZ-
7200 and CEZ-1200; Nihon Kohden) at membrane voltages of –20 to
–120 mV. The primers are shown in Supplementary Table S1.
Detection of root organic acid exudates
Surface-sterilized seeds (~2–3 mg) of the Arabidopsis WT and
AtMATE-KO were germinated in Magenta™ boxes containing sterile
hydroponic growth solution (pH 4.2) for 10 d.The seedlings were then
transferred to 20ml of lter-sterilized exudation solution (pH 4.2) with
or without 400μM AlCl3 for 24h (see Hoekenga etal., 2006). The exu-
date solutions were collected and the numbers of plants counted. The
exudate solutions were passed through anionic and cationic chromatog-
raphy columns to remove Al3+ and other inorganic anions. Then, 1ml of
the treated samples was subject to organic acid content analysis by HPLC
as described above.
Subcellular localization of AhFRDL1
AhFRDL1 cDNA was cloned into the pUC-GFP plasmid vector under
the control of the 35S promoter to create an AhFRDL1::GFP fusion
construct. The construct was transformed into Agrobacterium tumefaciens
(strain GV3101) by electroporation and then transiently transformed
into leaf epidermal cells of tobacco (Nicotiana benthamiana) by inltra-
tion (Wood etal., 2009). At 2 d after transformation, CellMask™ Orange
plasma-membrane stain was inltrated into the leaves for 1h, and epi-
dermal strips were peeled from the leaves and viewed under a confocal
microscope. A35S::GFP construct served as a control.
Tissue speciﬁcity of AhFRDL1 expression
For GUS staining, a 2004-bp promoter region was PCR-amplied
and subcloned into the pCAMBIA1391 vector. The resulting plasmid
AhFRDL1pro::GUS was introduced into peanut hairy roots via A.tumefa-
ciens (strain K599)-mediated transformation. After cultivation for 3 weeks,
transgenic hairy roots were used for Fe or Al treatments. For the Fe treat-
ment, the roots were grown without Fe for 1 week, while control plants
received complete nutrient solutions at pH 6.0. For the Al treatment, the
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2876 | Qiu etal.
roots were grown with 80μM AlCl3 in nutrient solution (pH 4.5) for
24h, while control plants received nutrient solution without AlCl3 at pH
4.5. Root samples were collected at the end of the treatment periods. To
detect GUS activity, samples were incubated for 2h at 37°C in GUS assay
buer with 5-bromo-4-chloro-3-indoyl glucuronide as a substrate (Stone
etal., 2005). After staining, cross-sections of root samples were visualized
by microscopy. The pr imers are shown in Supplementary Table S1.
Generation of transgenicplants
To knock-down the expression of AhFRDL1, a 300-bp fragment from its
coding region was PCR-amplied and subcloned into the pENTR/D-
TOPO vector (Invitrogen). The primers are shown in Supplementary
Table S1. The fragment of interest was then transferred through recom-
bination into pK7, which contains an RFP reporter gene (Stone etal.,
2005), using an LR Clonase™ Enzyme Mix (Invitrogen). The orienta-
tion of the gene fragments was conrmed by PCR and restriction en-
zyme analysis. The resulting plasmid and the empty pK7 vector control
were introduced into Agrobacterium rhizogenes (strain K599) for the trans-
formation of peanut hairyroots.
Surface-sterilized peanut seeds were germinated on lter papers for 3 d
until radicles grew to ~2cm in length. The root tips (~ 0.5cm) were cut
o using a sterilized blade. The radicles were then incubated for 2h with
strain K599 (Shen etal., 2014) containing the vectors detailed above. The
infected seedlings were transferred to hydroponic solutions as described
above. Screening for the transgenic hairy roots was conducted every 2
week using a stereoscope to detect the red uorescence, and the non-
transgenic roots were cut o. After two screenings, the transgenic hairy
roots were allowed to grow until being subject to Fe or Al treatment.
For the Fe-deciency treatment, three transgenic peanut plants were
grown in a 2.5-l pot containing a nutrient solution without Fe. Each
treatment comprised four replicate pots. At 7 d after treatment, xylem sap
was collected for iron and citrate determination. The chlorophyll con-
tent of a young leaf (the rst fully emerged leaf) was measured using a
SPAD-502 chlorophyll meter, and the active iron content was measured
as described above. Root samples were collected for RNA extraction and
determination of AhFRDL1 expression.
For the Al-toxicity treatment, three transgenic peanut plants were trans-
ferred to a 2.5-l pot containing 0.5mM CaCl2 (pH 4.5) solution over-
night, and they were then transferred to fresh solutions of 0.5mM CaCl2
(pH 4.5) containing 0, 40, or 80μM AlCl3. Each treatment comprised four
replicate pots. At 24h after treatment, solutions were collected for deter-
mination of citrate exudate using the method described above. Thirty root
tips (1cm) from one replicate pot were digested in 5ml of 1N HCl for
24h, and the Al content was assayed by ICP (Yokosho etal., 2011).
To analyse the function of AhFRDL1 in Arabidopsis, the
35S::AhFRDL1 construct was stably transformed into the Atfrd3 and
AtMATE-KO backgrounds via A. tumefaciens-mediated transformation
using the oral dip method (Liu etal., 2009).
Statistical analyses were conducted using SAS software. The statistical sig-
nicance of dierences was determined by ANOVA and least-signicant
dierence (LSD) tests at P<0.05. Dierences between genotypes were
assessed by independent-samples t-tests.
The sequences reported in this paper have been deposited in
the NCBI database (accession no. MG206076).
AhFRDL1 structural and phylogenetic analyses
Using homology cloning with a pair of degenerate primers
derived from the sequences of AtFRD3 and OsFRDL1, we
isolated the peanut AhFRDL1 mRNA (1563-bp), which en-
coded a 520-amino-acid putative MATE protein. The 3937-bp
genomic AhFRDL1 region contained 12 introns of 81–391bp
(Fig. 1A). In the peanut genome, AhFRDL1 showed the most
similarity to a homologous AhFRDL2 gene, but had distinct
specic sites in the 3´-UTRs (Supplementary Fig. S2A). The
AhFRDL1 protein contained 12 transmembrane domains
as predicted by the DNAMAN software (Fig. 1B) (Lynnon
Biosoft). Phylogenetic analysis indicated that AhFRDL1 shared
55.67–57.36% amino acid sequence identity with AtFRD3,
OsFRDL1, and ScFRDL1 and 51.5–68.97% with AtMATE,
SbMATE, and HvMATE (MEGA7; Kumar etal., 2016), among
which AhFRDL1 had the greatest amino acid sequence simi-
larity with AtMATE from Arabidopsis and BoMATE from
cabbage (Fig. 1C).
Sequence analyses of the 2029-bp promoter region upstream
of the predicted start codon of AhFRDL1 identied several
Fe-responsive elements, including two IDEF1 (CATGC) and
three IDEF2 (CAAGTTT) as well as 16 binding sequences
(GGNVS) for Al resistance transcription factor1 (ART1) (Fig.
1A), implying that AhFRDL1 may be responsive to both Fe de-
ciency and Al stress (Kobayashi etal., 2003; Tsutsui etal., 2011).
AhFRDL1 is localized to the plasma-membrane
To investigate the subcellular localization of AhFRDL1, an
AhFRDL1-green uorescence protein (GFP) fusion construct
with the CaMV 35S promoter was transiently expressed in
tobacco leaf epidermal cells. GFP alone, as a control, was localized
to the entire subcellular structure, including the cytosol, nucleus,
and plasma-membrane (PM; (Fig. 2A–C), whereas the AhFRDL1-
GFP fusion protein was localized to the extreme periphery of epi-
dermal cells and co-localized with the PM marker (Fig. 2D–G),
indicating that AhFRDL1 is localized to thePM.
AhFRDL1 restores root-to-shoot iron translocation of
The coding region of AhFRDL1 was stably transformed into
the Arabidopsis frd3 mutant line. At total of 30 homozygous
T3 lines were identied, two of which (lines 1 and 2)were
selected for further investigation. Compared with the WT, the
frd3 mutant plants displayed signicantly lower citrate and Fe
concentrations in the xylem sap (Fig. 3A, B), consistent with a
previous report (Green and Rogers, 2004). In contrast, citrate
and Fe concentrations in the xylem sap signicantly increased
in the AhFRDL1-overexpression lines to levels comparable to,
or higher than, those in the WT. Moreover, the free active Fe
and total chlorophyll concentrations in young leaves were sig-
nicantly increased in lines 1 and 2 compared to those in frd3
(Fig. 3C, D). These results indicated that AhFRDL1 rescued
the Fe-deciency phenotype of the frd3 mutant.
Fe deﬁciency enhances AhFRDL1 expression and
citrate concentration in xylemsap
AhFRDL1 expression was assessed by qRT-PCR, with prim-
ers designed at specic sites in the 3´-UTR (Supplementary
Fig. S2A). In the roots, AhFRDL1 expression was signicantly
AhFRDL1 and adaptation to Fe deﬁciency and Al stress | 2877
enhanced after 4–10 d of Fe-deciency (Fig. 4A, Supplementary
S2B), but signicantly suppressed 1 d after resupply of Fe (Fig.
4A). Citrate concentrations in the xylem sap of 24-d-old peanut
seedlings were signicantly higher under Fe-deciency condi-
tions than under Fe-suciency conditions, particularly after 4–10
d of Fe, and were still high after 1 d of Fe resupply (Fig. 4B).
Fig. 1. Characterization of the AhFRDL1 gene sequence. (A) AhFRDL1 gene structure. The 3937-bp genomic region of AhFRDL1 contains 12 introns
and a 2029-bp promoter with several Fe-responsive elements (CATGC, CAAGTTT) or Al-responsive elements (GGNVS), as indicated in the key. (B)
Alignment of the deduced amino acid sequences of AhFRDL1 with other known or putative plant citrate transporters. The 12 predicted transmembrane
domains in AhFRDL1 are indicated. The sequence alignments were constructed using the DNAMAN software. (C) Phylogenetic analysis of selected
citrate transporter proteins related to Fe translocation (indicated with dotted lines) or Al tolerance (solid lines). The phylogenetic tree was constructed
using the MEGA software.
2878 | Qiu etal.
Inhibition of root-to-shoot Fe translocation by silencing
AhFRDL1 expression in peanut hairyroots
To investigate the role of AhFRDL1 in Fe nutrition in pea-
nut, a construct containing the 313-bp double-stranded RNA
(dsRNA) sequence matching the 3´UTR region of AhFRDL1
or an empty vector (EV) as a control (CK line), was stably
transformed into peanut hairy roots via Agrobacterium-mediated
transformation (Fig. 5A). Because we co-transformation with a
red uorescence protein (RFP) reporter, transgenic hairy roots
could be visualized under green light (Fig. 5C) and non-trans-
formed roots could be identied and removed (Fig. 5D).
AhFRDL1 transcript levels were reduced by 84% in the
RNAi lines compared to those in the CK line (Fig. 5E) .
However, expression of a closely related endogenous homolog,
Fig. 3. AhFRDL1 rescues the Arabidopsis frd3 mutant. Concentrations of (A) citrate and (B) Fe in xylem sap, and concentrations of (C) active iron and (D)
total chlorophyll in young leaves from different transgenic lines (WT, wild-type). Data are means (±SE), n=3. Different letters indicate signiﬁcant differences
between means as determined by one-way ANOVA (P<0.05).
Fig. 2. AhFRDL1 is localized to the plasma-membrane. Confocal microscopic images of tobacco (Nicotiana benthamiana) leaf cells transiently
expressing 35S::GFP (A–C) or 35::AhFRDL1::GFP (D–G). (A, D), Green ﬂuorescence, (B, F), bright ﬁeld, (E) CellMask™ Orange plasma-membrane stain
marker, (C, G) merged. Arrows indicate the cell nucleus. Scale bars are 50μm.
AhFRDL1 and adaptation to Fe deﬁciency and Al stress | 2879
AhFRDL3, was comparable between the transgenic RNAi
lines and the CK line (Fig. 5F), indicating specic silencing of
AhFRDL1 expression in the transgenic plants.
Under Fe-deciency conditions (0μM), young leaves of the
AhFRDL1 RNAi lines showed more severe symptoms of chlo-
rosis and a lower chlorophyll content compared to CK plants,
as indicated by paler coloring around the leaf veins of RNAi
lines (Fig. 5B, J). Concentrations of citrate and Fe in the xylem
sap (Fig. 5G, H) and levels of active Fe in the young leaves (Fig.
5I) were signicantly lower in the RNAi lines than in the CK
plants, indicating that the RNAi lines mimicked the phenotypes
of the Arabidopsis frd3 mutant (Rogers and Guerinot, 2002;
Durrett etal., 2007). These results demonstrated that AhFRDL1
is involved in root-to-shoot Fe translocation in peanut.
Al induces AhFRDL1 expression and citrate secretion
from peanut roottips
Al stress aected the structure and impaired the growth of
peanut roots. Under treatment with 40μM or 80μM Al, root
apexes turned brown (an indication of damage) whereas the
roots of the CK plants displayed a healthy white color (Fig.
6A). After 4 d of treatment with 40μM or 80μM Al, relative
net root growth decreased by 25% and 64%, respectively, com-
pared to the CK line (Supplementary Fig. S1B).
Expression of AhFRDL1 in the root apex was signicantly
enhanced after 12–24h of Al treatment in an Al concentration-
dependent manner (Fig. 6B, Supplementary S2B). Al treatment
also enhanced citrate exudation from the roots in an Al con-
centration- and time-dependent manner (Fig. 6C), concomi-
tant with Al-enhanced expression of AhFRDL1 in the root
tips. Expression of AhFRDL1 was not aected by the pH of
the culture medium (Supplementary Fig. S2C).
AhFRDL1 confers Al resistance
To investigate the role of AhFRDL1 in Al tolerance in pea-
nut, transgenic AhFRDL1 RNAi plants were treated with 0,
40, or 80 μM Al for 24h. AhFRDL1 expression in root tips
was induced in the CK line in an Al concentration-dependent
manner, but it was inhibited by >71% in the RNAi lines (Fig.
7A). Correspondingly, root-tip Al concentrations were signi-
cantly higher in the RNAi lines than in the CK line (Fig. 7B) .
The CK line displayed Al concentration-dependent activation
of root citrate exudation (Fig. 7C), whereas the RNAi lines
not only showed a reduction of ~45% in root citrate secretion
under control conditions (0μM Al) but also lacked Al-activated
root citrate exudation. The root growth of the RNAi line was
markedly inhibited under 40μM and 80 Al treatment com-
pared with the CK plants (Fig. 7D). These results indicated that
AhFRDL1 is involved in peanut Al detoxication by regulat-
ing citrate exudation from the rootapex.
We then studied the role of AhFRDL1 in Al resistance in
Arabidopsis. The coding region of AhFRDL1 was stably trans-
formed into an Arabidopsis mate-KO mutant and Al resist-
ance was evaluated in two homozygous T3-overexpression
lines (OE1 and OE2). The results indicated that the transgenic
AhFRDL1 rescued the Al-induced root growth-inhibition
phenotype of mate-KO (Supplementary Fig. S3A, C). In add-
ition, root citrate exudation was 1.9- and 1-4-fold higher in
the OE1 and OE2 lines, respectively, than in the mate-KO line
(Supplementary Fig. S3B).
AhFRDL1 facilitates citrate efﬂux in Xenopus oocytes
Electrophysiological experiments were conducted to charac-
terize the transport activity of AhFRDL1 in Xenopus oocytes
pre-injected with AhFRDL1 mRNA or with H2O (CK).
After citrate injection, slightly higher negative resting mem-
brane potentials were found in AhFRDL1-expressing oocytes
compared to the control (Fig. 8). However, external Al caused
large negative currents in AhFRDL1-expressing oocytes pre-
loaded with citrate (Fig. 8), indicating that AhFRDL1 facili-
tates Al-induced citrate eux in Xenopus oocytes.
Effects of Fe-deﬁciency and Al-stress on the tissue
speciﬁcity of AhFRDL1 expression
The tissue specicity of AhFRDL1 expression in pea-
nut roots was examined using AhFRDL1-promoter–glu-
curonidase (GUS) analysis. In the CK line, GUS staining of
AhFRDL1promoter::GUS transgenic hairy roots was localized
to the pericycle of the root tips (0–1cm) and more mature root
segments (2–4cm) (Fig. 9A–C). This was signicantly enhanced
under Fe deciency, indicating that AhFRDL1 expression had
been induced (Fig. 9D–F). Under Al stress, GUS staining in the
root tip region was not only signicantly enhanced but it was
also observed across the entire root cross-section, indicating
Fig. 4. Fe deﬁciency induces AhFRDL1 expression and citrate secretion in xylem in peanut (variety Luhua14). (A) qRT-PCR analysis of AhFRDL1
expression in the roots and (B) citrate concentrations in the xylem sap during Fe deﬁciency for 10 d, followed by resupply with Fe for 1 d (+1). Data are
means (±SE), n=3. Signiﬁcant differences between means were determined by t-tests: *P<0.05.
2880 | Qiu etal.
that AhFRDL1 expression was enhanced and it was present
in the root-tip cortical and epidermal cells (Fig. 9G, H). In
contrast, in mature root regions GUS activity was only slightly
enhanced by Al stress in the pericycle, and in cortical and epi-
dermal tissues there was only weak enhancement (Fig. 9I).
Variation in the tissue specicity of AhFRDL1 expression
may therefore explain the dual function of AhFRDL1 in Fe
nutrition and Al resistance.
AhFRDL1 is induced under Fe deﬁciency and Al stress
in Fe-efﬁcient peanut varieties
To further characterize the roles of AhFRDL1 in Fe nutri-
tion and Al resistance, the peanut varieties Luhua14 (LH14,
low Fe-eciency), Luhua11 (LH11, high Fe-eciency), and
Dabaisha (DBS, Al-sensitive) were studied under Fe-deciency
and Al-stress conditions.
Fig. 5. The effect of AhFRDL1-RNAi on root-to-shoot Fe translocation in Fe-deﬁcient peanut (variety Luhua14). (A) Transgenic hairy roots (circle) initiated
from the main root tip, and the other non-transgenic lateral roots (above the circle) that were cut off. (B) AhFRDL1-RNAi plants showed more severe
symptoms of chlorosis in the young leaves than the control (CK, empty vector) under Fe-deﬁcient conditions. (C) Red ﬂuorescence of the transgenic hairy
roots. (D) Mature transgenic hairy roots with non-transgenic roots removed after 50 d.(E–J) Genetic and physiological measurements taken after 7 d
under Fe deﬁciency for control and RNAi plants. qRT-PCR analysis of (E) AhFRDL1 and (F) AhFRDL3 gene expression in the roots. Concentrations of (G)
citrate and (H) Fe in the xylem sap. (I) Active iron concentrations and (J) SPAD values in young leaves. Data are means (±SE), n=3. Signiﬁcant differences
between means were determined by t-tests: *P<0.05, **P<0.01. (This ﬁgure is available in colour at JXB online.)
AhFRDL1 and adaptation to Fe deﬁciency and Al stress | 2881
After Fe-deciency treatment for 7 d, young leaves of the
three varieties exhibited chlorosis, and SPAD values in LH11
were higher than in DBS (Fig. 10A). Active Fe concentrations
were signicantly higher in young leaves of LH11 than in those
of other varieties (Fig. 10B). Compared to expression under
Fe-suciency conditions, AhFRDL1 expression increased by
1.8-, 2.4-, and 1.6-fold under Fe deciency in the roots of LH14,
LH11, and DBS, respectively (Fig. 10C). Correspondingly, citrate
concentrations in the xylem were signicantly enhanced by Fe
deciency by 2.1-, 2.7-, and 1.9-fold in LH14, LH11, and DBS,
respectively (Fig. 10D). The levels of induced AhFRDL1 expres-
sion and enhanced xylem citrate concentrations in LH11 were
much higher than those of the other two varieties (Fig. 10C,D).
Eriochrome cyanine R staining indicated that the root tips
of LH14 and LH11 showed less damage under Al stress than
did those of DBS (Fig. 10E), in agreement with root growth
being less inhibited by 80μM Al in LH14 and LH11 than in
DBS (Fig. 10F). The relative expression levels of AhFRDL1 in
the root tips of LH14 and LH11 under 80μM Al treatment
were signicantly increased by 2.6- and 1.9-fold, respectively
(Fig. 10G). Correspondingly, root citrate secretion from LH14
and LH11 under 80μM Al was signicantly enhanced by 7.3-
and 4.2-fold, respectively (Fig. 10H). In contrast, AhFRDL1
expression and root citrate secretion were not induced by Al
treatment in DBS (Fig. 10G,H).
Thus, LH11 and LH14 exhibited inducible expression of
AhFRDL1 (Fig. 10C, G) and enhanced citrate secretion into
the xylem and rhizosphere (Fig. 10D, H) under Fe deciency
and Al stress. This implies that regulation of AhFRDL1
expression plays an important role in resistance to Fe de-
ciency and Al toxicity in peanut varieties with dierent Fe
Fig. 6. Al toxicity induces AhFRDL1 expression and root citrate exudation in peanut (variety Luhua14). (A) Damage in the root tips caused by treatment
with 0, 40, and 80μM Al for 24h, as indicated by staining with 0.1% eriochrome cyanine R.The scale bar is 0.5cm. (B) Changes in relative expression
of AhFRDL1 with time in the root apex (0–1cm) under the different Al treatments. (C) Citrate exudation under the roots under the different Al treatments
as determined at 12h and 24h. Data are means (±SE), n=4. Signiﬁcant differences between means were determined by t-tests: *P<0.05, **P<0.01.
Fig. 7. Enhanced sensitivity to Al toxicity as a result of AhFRDL1-silencing in the roots peanut (variety Luhua14). peanut root tips, (A) Relative expression
of AhFRDL1 in the root tips (0–1cm) of plants under 0, 40, or 80μM Al treatment for 24h for AhFRDL1-RNAi and empty-vector control (CK) plants. (B)
Al concentrations in the root tips of RNAi and CK plants under the different Al treatments. Data are based on 30 root tips in each replicate pot. (C) Citrate
secretion from roots under the different Al treatments. (D) Relative net root growth (RNRG) under 40μM or 80 Al treatments. Data are means (±SE), n=4.
Signiﬁcant differences between treatment means were determined by t-tests: *P<0.05, **P<0.01.
2882 | Qiu etal.
AhFRDL1 is involved in Fe translocation and Al
The 56 members of the MATE family in Arabidopsis can be
classied into ve subfamilies, among which two function-
ally characterized citrate transporters cluster together, namely
AtFRD3 and AtMATE (Li etal., 2002; Liu et al., 2009). The
known MATE-family citrate transporters share a unique topo-
logical structure, a large cytoplasmic loop between transmem-
brane domains II and III (Liu etal., 2009), which could also
be found in AhFRDL1 (Fig. 1B). However, the functional sig-
nicance of this structure in citrate transport has not yet been
Although AhFRDL1 was identied using a cloning strategy
based on its sequence homology to the Fe translocation-related
MATE genes AtFRD3 and OsFRDL1, the AhFRDL1 protein
sequence was more closely related to AtMATE, which is in-
volved in Al exclusion in Arabidopsis (Fig. 1C). The distinct
physiological functions of citrate transporters are due mainly
to their tissue-specic expression rather than dierential pro-
tein functions. For instance, the sorghum citrate transporter
SbMATE is expressed in root epidermal/cortical tissues and
facilitates Al-activated citrate exudation and Al resistance in
sorghum (Magalhaes etal., 2007; Sivaguru etal., 2013), whereas
AtFRD3 is expressed in the root stele and thereby facilitates
citrate release into the root xylem for ecient root-to-shoot
Fe translocation. However, when AtFRD3 is ectopically over-
expressed in the epidermis/cortex root tissues of transgenic
Arabidopsis plants, it facilitates Al-activated citrate exudation
to the rhizosphere and thus confers Al resistance (Durrett etal.,
Several lines of evidence indicate that the AhFRDL1 cit-
rate transporter facilitates root-to-shoot Fe translocation. First,
AhFRDL1 expression was induced by Fe deciency in the
pericycle (Figs 4A, 9F). Second, when AhFRDL1 expression
was knocked down by 80% in peanut roots, the concentrations
of citrate and Fe in the xylem sap decreased by 24% and 23%,
respectively, and young leaves exhibited severe chlorosis symp-
toms (Fig. 5). Finally, AhFRDL1 rescued the Fe-deciency
phenotype of the Arabidopsis mutant frd3, resulting in en-
hanced Fe and citrate concentrations in xylem sap and in-
creased levels of active Fe and total chlorophyll in young leaves
Expression of AhFRDL1 was also responsive to Al toxicity.
Under Al treatment, AhFRDL1 expression shifted from the
pericycle to the epidermis and cortex in root tips (Fig. 9G–I) ,
and this was associated with Al-induced exudation of citrate
from the roots (Fig. 6C). When AhFRDL1 expression was in-
hibited by 71% in root tips of a peanut RNAi line (Fig. 7A),
basal citrate secretion from the tips decreased by 45% under
control conditions (0 M AlCl3), and Al-induced citrate release
was severely suppressed (Fig. 7C). As a result, the AhFRDL1-
RNAi line accumulated more Al in root tips (Fig. 7B) and
showed much more inhibited root growth than did the control
(Fig. 7D). Additionally, when AhFRDL1-overexpression was
stably transformed into the Arabidopsis AtMATE-KO mutant
background, Al-induced root citrate exudation was restored to
the WT level, and the root growth-inhibition phenotype was
rescued (Supplementary Fig. S3). These results indicate that
AhFRDL1 is involved in both Al resistance and Fe transloca-
tion in peanut.
Various elements in the promoter region play key roles
in determining gene expression patterns (Wray et al., 2003;
Fujii et al., 2012). In graminaceous monocots, the expres-
sion patterns of the MATE-family citrate transporter genes
are strongly inuenced by specic regulatory elements in the
promoters, such as SbMATE, HvAACT1, and TaMATE1B
(Magalhaes etal., 2007; Fujii etal., 2012; Tovkach etal., 2013).
In the dicot peanut, the AhFRDL1 promoter region (2029bp,
Fig. 1A) contains ve IDEF1/IDEF2 and 16 ART1 binding
sites, which regulate the expression of Fe deciency-inducible
genes in barley and Al-inducible genes in rice, respectively
(Kobayashi etal., 2003; Tsutsui etal., 2011). Although previous
studies of the promoters of AtALMT1 (a malate transport gene
contributing to Al-tolerance) and CcMATE determined tha
the GGNVS region is essential for Al-inducible gene expres-
sion, this element has not been demonstrated to be involved in
regulation of tissue-specic or cell type-specic gene expres-
sion (Tokizawa etal., 2015; Daspute etal., 2018). In addition,
promoter analysis of AtALMT1 and a study of rice ARS5
(a transcription factor for Al-responsive genes) identied an
essential element,(AGCCCAT, that determines root tip-spe-
cic gene expression (Arenhart et al., 2014; Tokizawa et al.,
2015). However, the AGCCCAT element was not present
in the AhFRDL1 promoter region (Fig. 1A), suggesting that
other unknown cis-elements might regulate the dual-response
Fig. 8. Electrophysiological characterization of AhFRDL1 expressed in
oocytes of Xenopus laevis. Mean current to voltage relationships at pH 4.5
for controls (CK, –Al), AhFRDL1-expressing oocytes without Al (AhFRDL1,
–Al), controls with Al (CK+Al, +100μM Al), and AhFRDL1-expressing
oocytes with Al (AhFRDL1+Al, +100μM Al). Data are the average of
measurements from 4–6 different cells. Error bars (±SE) are shown when
values are larger than the symbol.
AhFRDL1 and adaptation to Fe deﬁciency and Al stress | 2883
patterns of AhFRDL1. Our future research will focus on the
identication of specic Fe- and Al-responsive elements in the
promoter region that determine the AhFRDL1 expression
patterns under Fe deciency and Al stress.
The xylem citrate concentration did not change after 1 d
of Fe resupply (Fig. 4B), which was not consistent with the
decrease of AhFRDL1 expression (Fig. 4A). The maintenance
of the xylem citrate concentration may have been due to basal
expression of AhFRDL1. Even when the expression of the gene
is decreased to basal levels, the protein will be still there, trans-
porting citrate from the roots. The initial citrate exudation at 1
d was from the constitutively expressed AhFRDL1 transporter,
but Fe deciency or Al stress further up-regulated the expression
of AhFRDL1 (Figs 4A, 6B). Therefore, the citrate transporter
AhFRDL1 was regulated at the transcriptional and/or transla-
tional levels (Magalhaes etal., 2007; Yokosho etal., 2009, 2011).
AhFRDL1 contributes to peanut adaptation under
Fe-deﬁcient and Al-stress conditions
To assess the roles of AhFRDL1 in Fe nutrition and Al resist-
ance in dierent peanut varieties, we examined its biological
functions in three varieties under Fe deciency or Al stress.
LH14 is a variety with low Fe-eciency whereas LH11 is a
variety with high Fe-eciency that is cultivated in calcareous
soils on the North China Plain (Gao etal., 2009; Guo etal.,
2014). In LH14, Fe deciency enhanced AhFRDL1 expres-
sion, which resulted in increased xylem citrate concentrations
(Fig. 4), and the citrate transporter AhFRDL1 was expressed in
the root stele (Fig. 9D–F). Knocking down AhFRDL1 in the
roots of LH14 resulted in reduced concentrations of citrate in
the xylem and Fe in young leaves (Fig. 5). These results indi-
cated that AhFRDL1 was involved in Fe translocation from
the root to the shoot in the LH14 variety. Interestingly, in the
high Fe-ecient LH11 variety, both the AhFRDL1 expression
and xylem citrate induced by Fe deciency were signicantly
higher than in LH14 (Fig. 10C, D). Whilst AhFRDL1 con-
tributes to the translocation of Fe in both LH14 and LH11,
we suggest that the higher induction of expression in the high
Fe-ecient LH11 variety facilitates nutrition by promoting
In addition to adapting to Fe deciency, we found that
LH11 also had resistant properties under Al-stress conditions.
Compared with the Al-sensitive DBS variety, Al toxicity
Fig. 9. Tissue speciﬁcity of expression of AhFRDL1 by GUS staining in peanut cultivar Luhua14. (A, D, G) Staining in the whole root proﬁle. (B, E, H),
sections of root tips (0–1cm), and (C, F, I) sections of mature roots (2–4cm). Transgenic hairy roots were grown in control nutrient solution at (A) pH 4.5
or (B, C) pH 6.0; (D–F) transgenic hairy roots were grown for 1 week without Fe (pH 6.0); and (G–I) transgenic hairy roots were grown for 24h with 80μM
AlCl3 (pH 4.5). C, cortical; E, endodermis; EP, epidermis; P, pericycle. Scale bars are 200μm.
2884 | Qiu etal.
induced a lower level of damage to the root tip in LH11,
and this was accompanied by reduced inhibition of root
growth (Fig. 10E, F). Furthermore, Al stress induced signi-
cantly higher expression of AhFRDL1 in the root tips (Fig.
10G) and higher exudation of citrate from the roots (Fig.
10H) in the LH11 variety than in DBS. Citrate transport-
ers expressed in root-apex epidermal cells are involved in
Al resistance (Liu et al., 2009; Yokosho et al., 2009, 2011;
Tovkach etal., 2013). We suggest that enhanced AhFRDL1
expression also contributed to Al tolerance by promoting
citrate exudation in the high Fe-ecient LH11 variety.
Similarly, higher Al-induced expression of AhFRDL1 and
higher citrate exudation confers more Al tolerance in LH14
Taken together, our results indicate that as well as facilitating
Fe translocation in Fe-ecient varieties, the citrate transporter
AhFRDL1 can also promote Al tolerance by increasing citrate
secretion into the rhizosphere (Fig. 11).
AhFRDL1 has the potential to serve as a genetic
marker for high Fe efﬁciency and Al tolerance
The modern cultivated peanut (Arachis hypogaea) is derived
from two wild diploid ancestors, A.duranensis (A. magna) and
A.ipaensis (Clevenger etal., 2016); the former was adapted to
acidic soils and the latter to alkaline soils (Fischer etal., 2002;
Bertioli etal., 2016). Peanut has developed dierent strategies
to cope with Al stress in acidic soils and limited Fe availabil-
ity in alkaline soils, such as exudation of organic acids (Wang
et al., 2017) or Fe-absorption Strategy I mechanisms (Guo
etal., 2014). It may be speculated that cultivated peanut has
aggregated the benecial traits of the two ancestors to make
it adaptable to complex environments. The global spread
of peanut cultivation to locations such as the North China
Plain with alkali calcareous soils has resulted in selection for
increased Fe eciency (Zuo and Zhang, 2008). When pea-
nuts were domesticated in South America near the equator,
where acidic oxisol, ultisol, and alsol soils are prevalent, Al
resistance was selected for (Fischer etal., 2002). Consequently,
the AhFRDL1 expression patterns and dual functions of
AhFRDL1 in Fe eciency and Al resistance could be a result
of human selection.
Although molecular-genetic breeding methods have been
applied to eld crops such as rice, wheat, and soybean for
many years (Watson etal., 2018; Wing etal., 2018), they have
not been readily available for use in peanut because sequenc-
ing of its genome was only accomplished in 2016 (Bertioli
et al., 2016). Compared with the Arabidopsis and rice, the
genome of cultivated peanut is more complex and gene func-
tions are likely to be more diversied (Xiong et al., 2013;
Bertioli etal., 2016; Clevenger etal., 2016). For example, the
presence of YSL homologs implies that the cultivated dicot
peanut is more than a Fe-absorption Strategy Iplant (Xiong
etal., 2013). The fact that AhYSL1 promotes Fe nutrition by
facilitating Fe3+-DMA absorption from the soil implies that
Strategy II Fe-uptake could also be active in peanut (Römheld
and Marschner, 1986). Genetic variation among domesticated
varieties and their wild relatives can be exploited for breeding
new generations of sustainable crops (Wing etal., 2018). Since
the dual-function AhFRDL1 citrate transporter contributes
to the adaptation of peanut to both Fe-decient and Al-stress
conditions, the AhFRDL1 gene has the potential to serve as
an important genetic marker for breeding new varieties with
high Fe-eciency and Al tolerance.
Fig. 10. Expression patterns of AhFRDL1 in the peanut cultivars Luhua14 (LH14, low Fe-efﬁciency), Luhua11 (LH11, high Fe-efﬁciency), and Babaisha
(DBS, Al-sensitive). (A) SPAD values and (B) active iron concentrations in young leaves (YL) and old leaves (OL) after plants were subject to Fe deﬁciency
for 7 d.(C) Fold-changes in (C) root AhFRDL1 expression levels and (D) citrate concentrations in xylem sap induced by Fe deﬁciency. (E) Images of
root tips stained with 0.1% eriochrome cyanine R for 2h to visualize Al toxicity. Scale bar is 0.5cm. (F) Relative net root growth (RNRG) compared with
control plants of the three cultivars grown in media containing either 40, 80, or 120μM Al for 24h. Fold-changes in (G) AhFRDL1 expression levels in
root tips (0–1cm) and (H) citrate exudation from roots of plants treated with 80μM Al for 24h. All data are means (±SE), n=3–9. Different letters indicate
signiﬁcant differences as determined by one-way ANOVA (P<0.05); asterisks indicate signiﬁcant differences as determined using t-tests: *P<0.05,
**P<0.01. (This ﬁgure is available in colour at JXB online.)
AhFRDL1 and adaptation to Fe deﬁciency and Al stress | 2885
Supplementary data are available at JXB online.
Fig. S1. Inhibition of peanut root growth under dierent Al
Fig. S2. Distinct specic sites in AhFRDL1 and AhFRDL2, and
expression patterns in roots under dierent Fe and Al treatments.
Fig. S3. AhFRDL1 rescues the AtMATE-KO mutant.
Table S1. Primers used in this study.
The authors wish to thank Prof. Weihua Wu and Dr Yi Wang of the State
Key Laboratory of Plant Physiology and Biochemistry of China Agricultural
University for their advice and help with electrophysiological studies, and Dr
Hiromi Nakanishi of the University of Tokyo and Dr Takanori Kobayashi of
Ishikawa Prefectural University for their critical comments. This work was
supported by the National Natural Science Foundation of China (Grant
No. 31872183) and the National Key R&D Program of China (Grant Nos.
2017YFD0202102, 2016YFD0200405, 2016YFE0101100).
WQ, LVK, and Y.Z.designed the experiments; WQ performed most of the
experiments, including generation of transgenic plants, gene expression,
and localization analysis; TW participated in the sample collection and
measurements; NW and JD conducted part of the plant nutrient analysis
and detection of organic acid; WQ, JL, and YZ wrote the paper. All of
the authors discussed the results and commented on the manuscript. YZ
provided funding for this work as cor responding author.
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