AhFRDL1 mediated citrate secretion contributes to adaptation of peanuts for Fe deficiency and Al stress

Abstract

Although citrate transporters are involved in iron (Fe) translocation or aluminum (Al) tolerance in plants, none of them have been shown to confer both biological functions in Fe absorption strategy I plant species so far. We demonstrate that AhFRDL1, a citrate transporter gene from peanut (Arachis hypogaea L.) induced by both Fe-deficiency and Al-stress, participates in root-to-shoot Fe translocation and Al tolerance. Fe-deficiency induced AhFRDL1 locates in the root stele, expands to the entire root-tip cross section under Al-stress. Overexpression of AhFRDL1 restored efficient Fe translocation in Atfrd3 mutants and Al resistance in AtMATE-KO mutants. Knocking down AhFRDL1 in peanut roots resulted in reduced xylem citrate and reduced active Fe concentrations in young leaves. Furthermore, the AhFRDL1-knockdown lines had reduced root citrate exudation and were more sensitive to Al-toxicity. Compared to the Al-sensitive DBS cultivar, enhanced AhFRDL1 expression in the Fe-efficient LH11 contributes 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. Due to its dual biological function, AhFRDL1 may serve as a genetic marker for breeding for high Fe-efficiency and Al-tolerance.

Full text

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.
For permissions, please email: journals.permissions@oup.com
RESEARCH PAPER
AhFRDL1-mediated citrate secretion contributes to
adaptation to iron deficiency and aluminum stress in peanuts
Wei Qiu1, Nanqi Wang1, Jing Dai1, Tianqi Wang1, LeonV. 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: zuoym@cau.edu.cn
Received 30 November 2018; Editorial decision 18 February 2019; Accepted 17 March 2019
Editor: Karl-Josef Dietz, Bielefeld University, Germany
Abstract
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
hypogaea).
Introduction
Aluminum (Al) and iron (Fe) are, respectively, the rst and
second most abundant metal elements in the Earth’s crust,
and they have contrasting eects 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 etal., 2004; Yang etal., 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
applyparastyle "g//caption/p[1]" parastyle "FigCapt"
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

2874 | Qiu etal.
in root cells and/or translocated from the roots and stored in
vacuoles of shoot cells (Liu etal., 2014; Kochian etal., 2015;
Wang etal., 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 Ispe-
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 identied 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 ecient translocation, which is dependent on citrate eux
into the xylem in both Strategy I and II species (Morrissey
and Guerinot, 2009). This eux 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 etal., 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 etal., 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-ecient Fe–
citrate complexes are formed (Durrett etal., 2007; Morrissey
and Guerinot, 2009). Functional homologs of FRD3 involved
in facilitating root-to-shoot Fe translocation have been iden-
tied in several crop species, including soybean (Glycine max),
rice (Oryza sativa), and rye (Secale cereale) (Rogers etal., 2009;
Yokosho etal., 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 etal., 2007; Magalhaes etal., 2007; Liu etal.,
2009; Yokosho etal., 2009, 2011; Maron etal., 2010; Tovkach
etal., 2013).
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 etal., 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 dierential
physiological functions of HvAACT1 in Al resistance and Fe
nutrition are due to dierences in expression among tissues but
not due to dierent functions. Citrate transporters expressed in
the root stele facilitate root-to-shoot Fe translocation (Durrett
etal., 2007; Rogers etal., 2009; Yokosho etal., 2009), whereas
those expressed in epidermal/cortical cells are involved in Al
resistance (Liu etal., 2009; Yokosho etal., 2009, 2011; Tovkach
etal., 2013). However, among the Strategy Iplant 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 deciency 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 signicantly facilitates absorp-
tion of Fe3+-DMA by nearby peanut roots (Xiong etal., 2013).
However, the mechanism underlying root-to-shoot Fe trans-
location in peanuts is unclear.
Here, we report the identication and characterization of
AhFRDL1, a member of the MATE family, in Strategy Ipea-
nuts. It functions as a citrate transporter in Fe nutrition and Al
resistance, thus contributing to peanut ecological adaptation
under Fe-decient 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 [2mM Ca(NO3)2, 0.5mM KH2PO4, 0.75mM K2SO4,
0.1mM KCl, 0.65mM MgSO4, 1μM MnSO4, 0.1μM ZnSO4, 0.1μM
CuSO4, 0.005μM (NH4)6Mo7O24, 1μM H3BO3, and 80μM 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 80μM Fe-EDTA and grown under a 16/8h 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 45ml of 0.5mM CaCl2 (pH 4.5) solu-
tion overnight and subsequently to fresh solutions of 0.5mM CaCl2
(pH 4.5) containing 0, 40, or 80μM AlCl3. The growth conditions were
as described above. Root length was measured at 0, 6, 12, 24, 36, and
48h; at each time point, 0–1cm of the root tips was collected for RNA
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

AhFRDL1 and adaptation to Fe deficiency and Al stress | 2875
extraction, and the culture solution was decanted to measure organic acid
concentrations.
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 16/8h light/dark cycle (100μmol
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 etal. (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
etal. (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 1ml of methanol, and total chlorophyll levels
were quantied according to the method of Durrett etal. (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 400μM AlCl3 (Liu
etal., 2009; Wang etal., 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-eciency), Luhua11 (LH11,
high Fe-eciency), and Dabaisha (DBS, Al-sensitive) were subject to Fe
deciency (0μM) or Al toxicity (0, 40, 80, or 120μM AlCl3) for 7 d or
24h, respectively. Growth conditions were as described above for peanut.
After 7 d of Fe deciency, 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 2h to assay Al toxicity. The culture solution was collected for
determination of citrate secretion, and root apexes (0–1cm) were cut,
frozen immediately in liquid nitrogen, and stored at –80°C until RNA
extraction.
Collection and analysis of xylemsap
Three-week-old peanut seedlings were cut at 2cm 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 2h 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 100μM
ascorbate-reduced FeCl3. Citrate concentrations in the xylem sap were
measured using HPLC, as described by Yokosho etal. (2011).
RNA extraction and quantitative real-time PCRassays
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 specic sites were
contained. The RT-PCR fragments were sequenced before the primers
were used for qRT-PCR for quantication of AhFRDL1 expression. The
qRT-PCR program comprised an initial denaturation step at 95 °C for
10min, followed by 40 cycles at 95°C for 15s and at 60°C for 1min,
and a nal dissociation step at 95°C for 15s, 60°C for 1min, 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 etal., 2013). All primers are listed in Supplementary Table S1 at
JXB online.
Cloning and sequence analysis of AhFRDL1cDNA
A fragment of AhFRDL1 was amplied 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-specic pri-
mers were designed. The 5′- and 3′-regions of the AhFRDL1 mRNA
sequence were amplied using a SMART Rapid Amplication of cDNA
Ends (RACE) cDNA Amplication 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 amplied
using KOD DNA polymerase (Toyobo). Sequence comparisons were
conducted using DNAMAN software (Lynnon Biosoft). Aphylogenetic
tree was constructed using the MEGA ver. 7.0 software.
Citrate transport assays in Xenopus oocytes
The AhFRDL1 ORF sequence was amplied, 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 etal. (2009). For electro-
physiological studies, oocytes injected with AhFRDL1 cRNA or water
were incubated in Modied Barth’s Saline (MBS) solution at 18°C. After
incubation for 1 d, 25ml of 200mM sodium citrate was injected into the
oocytes, which were then incubated for 0.5–2h. The net current across
the oocyte membrane was measured immediately after adding 0μM or
100μM Al to the solution (Furukawa etal., 2007). Current was recorded
using a two-electrode voltage clamp system with an amplier (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 20ml of lter-sterilized exudation solution (pH 4.2) with
or without 400μM AlCl3 for 24h (see Hoekenga etal., 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, 1ml 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 inltra-
tion (Wood etal., 2009). At 2 d after transformation, CellMask™ Orange
plasma-membrane stain was inltrated into the leaves for 1h, and epi-
dermal strips were peeled from the leaves and viewed under a confocal
microscope. A35S::GFP construct served as a control.
Tissue specificity of AhFRDL1 expression
For GUS staining, a 2004-bp promoter region was PCR-amplied
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
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

2876 | Qiu etal.
roots were grown with 80μM AlCl3 in nutrient solution (pH 4.5) for
24h, 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 2h at 37°C in GUS assay
buer with 5-bromo-4-chloro-3-indoyl glucuronide as a substrate (Stone
etal., 2005). After staining, cross-sections of root samples were visualized
by microscopy. The pr imers are shown in Supplementary Table S1.
Generation of transgenicplants
To knock-down the expression of AhFRDL1, a 300-bp fragment from its
coding region was PCR-amplied 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 etal.,
2005), using an LR Clonase™ Enzyme Mix (Invitrogen). The orienta-
tion of the gene fragments was conrmed 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 hairyroots.
Surface-sterilized peanut seeds were germinated on lter papers for 3 d
until radicles grew to ~2cm in length. The root tips (~ 0.5cm) were cut
o using a sterilized blade. The radicles were then incubated for 2h with
strain K599 (Shen etal., 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-deciency 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 0.5mM CaCl2 (pH 4.5) solution over-
night, and they were then transferred to fresh solutions of 0.5mM CaCl2
(pH 4.5) containing 0, 40, or 80μM AlCl3. Each treatment comprised four
replicate pots. At 24h after treatment, solutions were collected for deter-
mination of citrate exudate using the method described above. Thirty root
tips (1cm) from one replicate pot were digested in 5ml of 1N HCl for
24h, and the Al content was assayed by ICP (Yokosho etal., 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 etal., 2009).
Statistical analysis
Statistical analyses were conducted using SAS software. The statistical sig-
nicance of dierences was determined by ANOVA and least-signicant
dierence (LSD) tests at P<0.05. Dierences between genotypes were
assessed by independent-samples t-tests.
Accession numbers
The sequences reported in this paper have been deposited in
the NCBI database (accession no. MG206076).
Results
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 81–391bp
(Fig. 1A). In the peanut genome, AhFRDL1 showed the most
similarity to a homologous AhFRDL2 gene, but had distinct
specic 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 etal., 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 identied 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 etal., 2003; Tsutsui etal., 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 thePM.
AhFRDL1 restores root-to-shoot iron translocation of
the frd3mutant
The coding region of AhFRDL1 was stably transformed into
the Arabidopsis frd3 mutant line. At total of 30 homozygous
T3 lines were identied, two of which (lines 1 and 2)were
selected for further investigation. Compared with the WT, the
frd3 mutant plants displayed signicantly 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 signicantly 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-
nicantly increased in lines 1 and 2 compared to those in frd3
(Fig. 3C, D). These results indicated that AhFRDL1 rescued
the Fe-deciency phenotype of the frd3 mutant.
Fe deficiency enhances AhFRDL1 expression and
citrate concentration in xylemsap
AhFRDL1 expression was assessed by qRT-PCR, with prim-
ers designed at specic sites in the 3´-UTR (Supplementary
Fig. S2A). In the roots, AhFRDL1 expression was signicantly
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

AhFRDL1 and adaptation to Fe deficiency and Al stress | 2877
enhanced after 4–10 d of Fe-deciency (Fig. 4A, Supplementary
S2B), but signicantly suppressed 1 d after resupply of Fe (Fig.
4A). Citrate concentrations in the xylem sap of 24-d-old peanut
seedlings were signicantly higher under Fe-deciency condi-
tions than under Fe-suciency 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.
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

2878 | Qiu etal.
Inhibition of root-to-shoot Fe translocation by silencing
AhFRDL1 expression in peanut hairyroots
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 identied 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 50μm.
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

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 specic silencing of
AhFRDL1 expression in the transgenic plants.
Under Fe-deciency conditions (0μM), 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 signicantly 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 etal., 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 roottips
Al stress aected the structure and impaired the growth of
peanut roots. Under treatment with 40μM or 80μM 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 40μM or 80μM 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 signicantly
enhanced after 12–24h 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 aected 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 24h. 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 (0μM Al) but also lacked Al-activated
root citrate exudation. The root growth of the RNAi line was
markedly inhibited under 40μM and 80 Al treatment com-
pared with the CK plants (Fig. 7D). These results indicated that
AhFRDL1 is involved in peanut Al detoxication by regulat-
ing citrate exudation from the rootapex.
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 eux in Xenopus oocytes.
Effects of Fe-deficiency and Al-stress on the tissue
specificity of AhFRDL1 expression
The tissue specicity 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 (0–1cm) and more mature root
segments (2–4cm) (Fig. 9A–C). This was signicantly enhanced
under Fe deciency, indicating that AhFRDL1 expression had
been induced (Fig. 9D–F). Under Al stress, GUS staining in the
root tip region was not only signicantly 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.
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

2880 | Qiu etal.
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 specicity 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-eciency), Luhua11 (LH11, high Fe-eciency), and
Dabaisha (DBS, Al-sensitive) were studied under Fe-deciency
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.)
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

AhFRDL1 and adaptation to Fe deficiency and Al stress | 2881
After Fe-deciency 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 signicantly higher in young leaves of LH11 than in those
of other varieties (Fig. 10B). Compared to expression under
Fe-suciency conditions, AhFRDL1 expression increased by
1.8-, 2.4-, and 1.6-fold under Fe deciency in the roots of LH14,
LH11, and DBS, respectively (Fig. 10C). Correspondingly, citrate
concentrations in the xylem were signicantly enhanced by Fe
deciency 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 80μM 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 80μM Al treatment
were signicantly increased by 2.6- and 1.9-fold, respectively
(Fig. 10G). Correspondingly, root citrate secretion from LH14
and LH11 under 80μM Al was signicantly 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 deciency
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 dierent Fe
eciencies.
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 80μM Al for 24h, as indicated by staining with 0.1% eriochrome cyanine R.The scale bar is 0.5cm. (B) Changes in relative expression
of AhFRDL1 with time in the root apex (0–1cm) under the different Al treatments. (C) Citrate exudation under the roots under the different Al treatments
as determined at 12h and 24h. 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 (0–1cm) of plants under 0, 40, or 80μM Al treatment for 24h 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 40μM 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.
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

2882 | Qiu etal.
Discussion
AhFRDL1 is involved in Fe translocation and Al
detoxification inpeanut
The 56 members of the MATE family in Arabidopsis can be
classied into ve subfamilies, among which two function-
ally characterized citrate transporters cluster together, namely
AtFRD3 and AtMATE (Li etal., 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 etal., 2009), which could also
be found in AhFRDL1 (Fig. 1B). However, the functional sig-
nicance of this structure in citrate transport has not yet been
characterized.
Although AhFRDL1 was identied 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-specic expression rather than dierential 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 etal., 2007; Sivaguru etal., 2013), whereas
AtFRD3 is expressed in the root stele and thereby facilitates
citrate release into the root xylem for ecient 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 etal.,
2007).
Several lines of evidence indicate that the AhFRDL1 cit-
rate transporter facilitates root-to-shoot Fe translocation. First,
AhFRDL1 expression was induced by Fe deciency 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-deciency
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
(Fig. 3).
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 inuenced by specic regulatory elements in the
promoters, such as SbMATE, HvAACT1, and TaMATE1B
(Magalhaes etal., 2007; Fujii etal., 2012; Tovkach etal., 2013).
In the dicot peanut, the AhFRDL1 promoter region (2029bp,
Fig. 1A) contains ve IDEF1/IDEF2 and 16 ART1 binding
sites, which regulate the expression of Fe deciency-inducible
genes in barley and Al-inducible genes in rice, respectively
(Kobayashi etal., 2003; Tsutsui etal., 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-specic or cell type-specic gene expres-
sion (Tokizawa etal., 2015; Daspute etal., 2018). In addition,
promoter analysis of AtALMT1 and a study of rice ARS5
(a transcription factor for Al-responsive genes) identied an
essential element,(AGCCCAT, that determines root tip-spe-
cic 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, +100μM Al), and AhFRDL1-expressing
oocytes with Al (AhFRDL1+Al, +100μM Al). Data are the average of
measurements from 4–6 different cells. Error bars (±SE) are shown when
values are larger than the symbol.
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

AhFRDL1 and adaptation to Fe deficiency and Al stress | 2883
patterns of AhFRDL1. Our future research will focus on the
identication of specic Fe- and Al-responsive elements in the
promoter region that determine the AhFRDL1 expression
patterns under Fe deciency 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 deciency 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 etal., 2007; Yokosho etal., 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 dierent peanut varieties, we examined its biological
functions in three varieties under Fe deciency or Al stress.
LH14 is a variety with low Fe-eciency whereas LH11 is a
variety with high Fe-eciency that is cultivated in calcareous
soils on the North China Plain (Gao etal., 2009; Guo etal.,
2014). In LH14, Fe deciency 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-ecient LH11 variety, both the AhFRDL1 expression
and xylem citrate induced by Fe deciency were signicantly
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-ecient LH11 variety facilitates nutrition by promoting
root-to-shoot translocation.
In addition to adapting to Fe deciency, 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 (0–1cm), and (C, F, I) sections of mature roots (2–4cm). 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 24h with 80μM
AlCl3 (pH 4.5). C, cortical; E, endodermis; EP, epidermis; P, pericycle. Scale bars are 200μm.
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

2884 | Qiu etal.
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 etal., 2013). We suggest that enhanced AhFRDL1
expression also contributed to Al tolerance by promoting
citrate exudation in the high Fe-ecient LH11 variety.
Similarly, higher Al-induced expression of AhFRDL1 and
higher citrate exudation confers more Al tolerance in LH14
(Fig. 10G,H).
Taken together, our results indicate that as well as facilitating
Fe translocation in Fe-ecient 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 etal., 2016); the former was adapted to
acidic soils and the latter to alkaline soils (Fischer etal., 2002;
Bertioli etal., 2016). Peanut has developed dierent 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
etal., 2014). It may be speculated that cultivated peanut has
aggregated the benecial 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 eciency (Zuo and Zhang, 2008). When pea-
nuts were domesticated in South America near the equator,
where acidic oxisol, ultisol, and alsol soils are prevalent, Al
resistance was selected for (Fischer etal., 2002). Consequently,
the AhFRDL1 expression patterns and dual functions of
AhFRDL1 in Fe eciency 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 etal., 2018; Wing etal., 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 diversied (Xiong et al., 2013;
Bertioli etal., 2016; Clevenger etal., 2016). For example, the
presence of YSL homologs implies that the cultivated dicot
peanut is more than a Fe-absorption Strategy Iplant (Xiong
etal., 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 etal., 2018). Since
the dual-function AhFRDL1 citrate transporter contributes
to the adaptation of peanut to both Fe-decient and Al-stress
conditions, the AhFRDL1 gene has the potential to serve as
an important genetic marker for breeding new varieties with
high Fe-eciency 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 2h to visualize Al toxicity. Scale bar is 0.5cm. (F) Relative net root growth (RNRG) compared with
control plants of the three cultivars grown in media containing either 40, 80, or 120μM Al for 24h. Fold-changes in (G) AhFRDL1 expression levels in
root tips (0–1cm) and (H) citrate exudation from roots of plants treated with 80μM Al for 24h. 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.)
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

AhFRDL1 and adaptation to Fe deficiency and Al stress | 2885
Supplementarydata
Supplementary data are available at JXB online.
Fig. S1. Inhibition of peanut root growth under dierent Al
concentrations.
Fig. S2. Distinct specic sites in AhFRDL1 and AhFRDL2, and
expression patterns in roots under dierent Fe and Al treatments.
Fig. S3. AhFRDL1 rescues the AtMATE-KO mutant.
Table S1. Primers used in this study.
Acknowledgements
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).
Author contributions
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.
References
Abadía J, Vázquez S, Rellán-Álvarez R, El-Jendoubi H, Abadía A,
Alvarez-FernándezA, López-Millán AF. 2011. Towards a knowledge-
based correction of iron chlorosis. Plant Physiology and Biochemistry 49,
471–482.
ArenhartRA, Bai Y, deOliveiraLF, etal. 2014. New insights into alu-
minum tolerance in rice: the ASR5 protein binds the STAR1 promoter and
other aluminum-responsive genes. Molecular Plant 7, 709–721.
Bashir K, Rasheed S, Kobayashi T, Seki M, Nishizawa NK. 2016.
Regulating subcellular metal homeostasis: the key to crop improvement.
Frontiers in Plant Science 7, 1192.
Bertioli DJ, Cannon SB, Froenicke L, et al. 2016. The genome
sequences of Arachis duranensis and Arachis ipaensis, the diploid ances-
tors of cultivated peanut. Nature Genetics 48, 438–446.
ClevengerJ, ChuY, SchefflerB, Ozias-AkinsP. 2016. A developmental
transcriptome map for allotetraploid Arachis hypogaea. Frontiers in Plant
Science 7, 1446.
CurieC, MariS. 2017. New routes for plant iron mining. New Phytologist
214, 521–525.
Daspute AA, Kobayashi Y, Panda SK, Fakrudin B, Kobayashi Y,
TokizawaM, IuchiS, ChoudharyAK, YamamotoYY, KoyamaH. 2018.
Fig. 11. A model illustrating the molecular mechanisms of AhFRDL1 involved in Fe translocation and Al detoxification in peanut. As a Strategy Iplant,
Fe3+ is reduced to Fe2+ by AhFRO1 on the surface of the root. FE2+ is then absorbed into the root cell by AhIRT1. In response to Fe deficiency, induced
expression of AhFRDL1 is restricted to the root pericycle, promoting citrate secretion into the xylem where Fe is chelated with citrate for efficient transport
to the shoot. For Al tolerance, the expression of AhFRDL1 extends from the root pericycle to the endodermis, cortex, and epidermis, which facilitates
citrate exudation to the rhizosphere for detoxification of free Al3+ ions. C, cortex; E, endodermis; EP, epidermis. (This figure is available in colour at JXB
online.)
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019

2886 | Qiu etal.
Characterization of CcSTOP1; a C2H2-type transcription factor regulates Al
tolerance gene in pigeonpea. Planta 247, 201–214.
DurrettTP, GassmannW, RogersEE. 2007. The FRD3-mediated efflux
of citrate into the root vasculature is necessary for efficient iron transloca-
tion. Plant Physiology 144, 197–205.
FischerG, VelthuizenH, ShahM, NachtergaeleF. 2002. Global agro-
ecological assessment for agriculture in the 21st century: methodology and
results. Rome, Italy/Laxenburg, Austria: FAO and IIASA.
Fujii M, YokoshoK, YamajiN, Saisho D, YamaneM, TakahashiH,
SatoK, NakazonoM, MaJF. 2012. Acquisition of aluminium tolerance
by modification of a single gene in barley. Nature Communications 3, 713.
Furukawa J, Yamaji N, Wang H, Mitani N, Murata Y, Sato K,
Katsuhara M, TakedaK, Ma JF. 2007. An aluminum-activated citrate
transporter in barley. Plant & Cell Physiology 48, 1081–1091.
GaoL, ShiYX, ZhouJM. 2009. Study on the sensitive period and screen-
ing index for iron deficiency chlorosis in peanut. Plant Nutrition and Fertilizer
Science 15, 917–922 (in Chinese).
GreenLS, RogersEE. 2004. FRD3 controls iron localization in Arabidopsis.
Plant Physiology 136, 2523–2531.
GuoX, XiongH, ShenH, QiuW, JiC, ZhangZ, ZuoY. 2014. Dynamics
in the rhizosphere and iron-uptake gene expression in peanut induced by
intercropping with maize: role in improving iron nutrition in peanut. Plant
Physiology and Biochemistry 76, 36–43.
HoekengaOA, MaronLG, PinerosMA, et al. 2006. AtALMT1, which
encodes a malate transporter, is identified as one of several genes critical for
aluminum tolerance in Arabidopsis. Proceedings of the National Academy of
Sciences, USA 103, 9738–9743.
HorstWJ, WangY, EtichaD. 2010. The role of the root apoplast in alu-
minium-induced inhibition of root elongation and in aluminium resistance of
plants: a review. Annals of Botany 106, 185–197.
JeongJ, ConnollyEL. 2009. Iron uptake mechanisms in plants: functions
of the FRO family of ferric reductases. Plant Science 176, 709–714.
KobayashiT, NakayamaY, ItaiRN, NakanishiH, YoshiharaT, MoriS,
NishizawaNK. 2003. Identification of novel cis-acting elements, IDE1 and
IDE2, of the barley IDS2 gene promoter conferring iron-deficiency-induc-
ible, root-specific expression in heterogeneous tobacco plants. The Plant
Journal 36, 780–793.
KochianLV, PiñerosMA, LiuJ, MagalhaesJV. 2015. Plant adaptation to
acid soils: the molecular basis for crop aluminum resistance. Annual Review
of Plant Biology 66, 571–598.
KumarS, Stecher G, TamuraK. 2016. MEGA7: molecular evolutionary
genetics analysis version 7.0 for bigger datasets. Molecular Biology and
Evolution 33, 1870–1874.
LiL, HeZ, PandeyGK, TsuchiyaT, LuanS. 2002. Functional cloning
and characterization of a plant efflux carrier for multidrug and heavy metal
detoxification. The Journal of Biological Chemistry 277, 5360–5368.
LiuJ, Magalhaes JV, Shaff J, Kochian LV. 2009. Aluminum-activated
citrate and malate transporters from the MATE and ALMT families function
independently to confer Arabidopsis aluminum tolerance. The Plant Journal
57, 389–399.
LiuJ, PiñerosMA, KochianLV. 2014. The role of aluminum sensing and
signaling in plant aluminum resistance. Journal of Integrative Plant Biology
56, 221–230.
Ma JF, Shen R, Nagao S, TanimotoE. 2004. Aluminum targets elon-
gating cells by reducing cell wall extensibility in wheat roots. Plant & Cell
Physiology 45, 583–589.
MagalhaesJV, LiuJ, GuimarãesCT, etal. 2007. A gene in the multidrug
and toxic compound extrusion (MATE) family confers aluminum tolerance in
sorghum. Nature Genetics 39, 1156–1161.
MaronLG, Piñeros MA, GuimarãesCT, MagalhaesJV, Pleiman JK,
MaoC, ShaffJ, BelicuasSN, KochianLV. 2010. Two functionally distinct
members of the MATE (multi-drug and toxic compound extrusion) family
of transporters potentially underlie two major aluminum tolerance QTLs in
maize. The Plant Journal 61, 728–740.
MorrisseyJ, GuerinotML. 2009. Iron uptake and transport in plants: the
good, the bad, and the ionome. Chemical Reviews 109, 4553–4567.
OmoteH, HiasaM, MatsumotoT, OtsukaM, MoriyamaY. 2006. The
MATE proteins as fundamental transporters of metabolic and xenobiotic
organic cations. Trends in Pharmacological Sciences 27, 587–593.
RömheldV, MarschnerH. 1986. Evidence for a specific uptake system for
iron phytosiderophores in roots of grasses. Plant Physiology 80, 175–180.
RogersEE, Guerinot ML. 2002. FRD3, a member of the multidrug and
toxin efflux family, controls iron deficiency responses in Arabidopsis. The
Plant Cell 14, 1787–1799.
Rogers EE, Wu X, Stacey G, Nguyen HT. 2009. Two MATE proteins
play a role in iron efficiency in soybean. Journal of Plant Physiology 166,
1453–1459.
ShenH, XiongH, GuoX, WangP, DuanP, ZhangL, ZhangF, ZuoY.
2014. AhDMT1, a Fe2+ transporter, is involved in improving iron nutrition and
N2 fixation in nodules of peanut intercropped with maize in calcareous soils.
Planta 239, 1065–1077.
SivaguruM, LiuJ, KochianLV. 2013. Targeted expression of SbMATE in
the root distal transition zone is responsible for sorghum aluminum resist-
ance. The Plant Journal 76, 297–307.
StoneC, LiuJ, ZinnKE, AllanDL, VanceCP. 2005. Transgenic proteoid
roots of white lupin: a vehicle for characterizing and silencing root genes
involved in adaptation to P stress. The Plant Journal 44, 840–853.
Tokizawa M, Kobayashi Y, Saito T, Kobayashi M, Iuchi S,
NomotoM, TadaY, YamamotoYY, KoyamaH. 2015. SENSITIVE TO
PROTON RHIZOTOXICITY1, CALMODULIN BINDING TRANSCRIPTION
ACTIVATOR2, and other transcription factors are involved in ALUMINUM-
ACTIVATED MALATE TRANSPORTER1 expression. Plant Physiology 167,
991–1003.
Tovkach A, Ryan PR, Richardson AE, Lewis DC, Rathjen TM,
RameshS, TyermanSD, DelhaizeE. 2013. Transposon-mediated altera-
tion of TaMATE1B expression in wheat confers constitutive citrate efflux
from root apices. Plant Physiology 161, 880–892.
TsutsuiT, Yamaji N, FengMaJ. 2011. Identification of a cis-acting ele-
ment of ART1, a C2H2-type zinc-finger transcription factor for aluminum
tolerance in rice. Plant Physiology 156, 925–931.
WangYQ, LiRH, Li DM, et al. 2017. NIP1;2 is a plasma membrane-
localized transporter mediating aluminum uptake, translocation, and toler-
ance in Arabidopsis. Proceedings of the National Academy of Sciences,
USA 114, 5047–5052.
WatsonA, GhoshS, WilliamsMJ, etal. 2018. Speed breeding is a pow-
erful tool to accelerate crop research and breeding. Nature Plants 4, 23–29.
WingRA, PuruggananMD, ZhangQ. 2018. The rice genome revolution:
from an ancient grain to Green Super Rice. Nature Reviews. Genetics 19,
505–517.
WoodCC, PetrieJR, ShresthaP, MansourMP, NicholsPD, GreenAG,
SinghSP. 2009. A leaf-based assay using interchangeable design principles
to rapidly assemble multistep recombinant pathways. Plant Biotechnology
Journal 7, 914–924.
WrayGA, HahnMW, AbouheifE, BalhoffJP, PizerM, RockmanMV,
RomanoLA. 2003. The evolution of transcriptional regulation in eukary-
otes. Molecular Biology and Evolution 20, 1377–1419.
XiongH, KakeiY, KobayashiT, etal. 2013. Molecular evidence for phy-
tosiderophore-induced improvement of iron nutrition of peanut intercropped
with maize in calcareous soil. Plant, Cell & Environment 36, 1888–1902.
YangJL, LiYY, ZhangYJ, ZhangSS, WuYR, WuP, ZhengSJ. 2008.
Cell wall polysaccharides are specifically involved in the exclusion of alu-
minum from the rice root apex. Plant Physiology 146, 602–611.
YangJL, ZhuXF, PengYX, ZhengC, LiGX, LiuY, ShiYZ, ZhengSJ.
2011. Cell wall hemicellulose contributes significantly to aluminum adsorp-
tion and root growth in Arabidopsis. Plant Physiology 155, 1885–1892.
YokoshoK, YamajiN, MaJF. 2010. Isolation and characterisation of two
MATE genes in rye. Functional Plant Biology 37, 296–303.
YokoshoK, YamajiN, MaJF. 2011. An Al-inducible MATE gene is involved
in external detoxification of Al in rice. The Plant Journal 68, 1061–1069.
YokoshoK, YamajiN, UenoD, MitaniN, MaJF. 2009. OsFRDL1 is a
citrate transporter required for efficient translocation of iron in rice. Plant
Physiology 149, 297–305.
ZhuXF, Shi YZ, LeiGJ, etal. 2012. XTH31, encoding an in vitro XEH/
XET-active enzyme, regulates aluminum sensitivity by modulating in vivo
XET action, cell wall xyloglucan content, and aluminum binding capacity in
Arabidopsis. The Plant Cell 24, 4731–4747.
ZhuYZ, LiYF, MiaoLG, WangYP, LiuYH, YanXJ, CuiXZ, LiHT. 2014.
Immunotoxicity of aluminum. Chemosphere 104, 1–6.
ZuoYM, ZhangFS. 2008. Effect of peanut mixed cropping with gramine-
ous species on micronutrient concentrations and iron chlorosis of peanut
plants grown in a calcareous soil. Plant and Soil 306, 23–36.
Downloaded from https://academic.oup.com/jxb/article-abstract/70/10/2873/5368416 by Cornell University Library user on 12 May 2019