Abstract
Background
Iron (Fe) is an essential element for plant growth and development,
whereas cadmium (Cd) is non-essential and highly toxic. Previous
studies showed that Fe deficiency enhanced Cd uptake and accumulation
in peanuts. However, the molecular mechanism underlying the increased
Cd accumulation in Fe-deficient peanut plants is poorly understood.
Results
We employed a comparative transcriptome analysis approach to identify
differentially expressed genes (DEGs) in peanut roots exposed to
Fe-sufficient without Cd, Fe-deficient without Cd, Fe-sufficient with
Cd and Fe-deficient with Cd. Compared with the control, Fe deficiency
induced 465 up-regulated and 211 down-regulated DEGs, whereas the up-
and down-regulated DEGs in Cd exposed plants were 329 and 189,
respectively. Under Fe-deficient conditions, Cd exposure resulted in
907 up-regulated DEGs and 953 down-regulated DEGs. In the presence of
Cd, Fe deficiency induced 1042 up-regulated and 847 down-regulated
genes, respectively. Based on our array data, we found that metal
transporter genes such as CAX4, COPT1, IRT1, NRAMP5, OPT3, YSL3, VIT3
and VIT4 might be involved in iron homeostasis. Moreover, combined with
quantitative real-time PCR, IRT1, NRAMP3, NRAMP5, OPT3, YSL3, ABCC3,
ZIP1, and ZIP5 were verified to be responsible for Cd uptake and
translocation in peanut plants under iron deficiency. Additionally, a
larger amount of ABC transporter genes was induced or suppressed by
iron deficiency under Cd exposure, indicating that this family may play
important roles in Fe/Cd uptake and transport.
Conclusions
The up-regulated expression of NRAMP5 and IRT1 genes induced by iron
deficiency may enhance Cd uptake in peanut roots. The decrease of Cd
translocation from roots to shoots may be resulted from the
down-regulation of ZIP1, ZIP5 and YSL3 under iron deficiency.
Electronic supplementary material
The online version of this article (10.1186/s12870-019-1654-9) contains
supplementary material, which is available to authorized users.
Keywords: Peanut, Iron deficiency, Cadmium, Transcriptome, Gene
expression
Background
Cadmium (Cd) is a non-essential and highly toxic heavy metal that is
commonly released into the arable soil due to anthropogenic activities.
The concentration of Cd in leaves greater than 5–10 μg g^− 1 dry mass
(DM) is toxic to non-tolerant crop plants [[35]1]. For humans and
animals, Cd may damage the mitochondrial and induce cell death by
apoptosis and/or necrosis, leading to tissue inflammation and fibrosis
[[36]2]. Cd exposure has been shown to be associated with cancers of
the prostate, lungs and testes in humans [[37]3]. Due to its highly
bioavailability, Cd is easily absorbed and accumulated in plants, and
subsequently transferred to humans/animals via food chains. Thus, the
presence of Cd in the arable soil can cause serious risks to human
health. The minimization of Cd in edible parts of crops is an important
demand especially in Cd-contaminated soil.
Iron (Fe) is an essential element that has multiple physiological
functions including chlorophyll biosynthesis, photosynthesis,
respiration, and redox reactions in plants. Despite its abundance in
the earth’s crust, Fe often precipitates into insoluble Fe(III) oxides
under aerobic conditions, especially in high-pH and calcareous soils
[[38]4]. Consequently, dissolved Fe in the soil solution is usually far
lower than that required to sustain plant growth [[39]5]. Iron
deficiency causes chlorosis, growth retardation, and reduced crop
productivity. It has become an important yield-limiting factor for
crops growing in calcareous and alkaline soils.
Peanut (Arachis hypogaea L.) is one of the world’s fourth largest
oilseed crop that is sensitive to Fe deficiency [[40]6]. Peanut was
also demonstrated to have a high capacity for accumulating Cd in both
the seed and vegetative tissues depending on cultivars [[41]7–[42]9].
In previous studies, we have found that the uptake and accumulation of
Cd in peanut plants is considerably enhanced by iron deficiency
[[43]10–[44]13]. However, the mechanism underlying iron
deficiency-induced increase of Cd accumulation in peanuts has not been
well understood.
Generally, the accumulation of Cd in the shoot of plants is controlled
by several biological processes, including (i) symplastic uptake by
root epidermal cells, (ii) radial transport to the vascular cylinder,
(iii) xylem loading, and (iv) root-to-shoot translocation [[45]11].
Most of these processes are regulated by the transporters of several
essential metals such as Fe, Mn, and Zn. During the past few years,
several Fe transporters belonging to natural resistance associated
macrophage proteins (Nramp), Zrt/Irt-like proteins (ZIP) and
P1B-ATPases, have been identified to be responsible for the transport
of Cd in plants [[46]14]. Thomine et al. [[47]15] found that iron
deficiency could induce the expression of AtNramp3 and AtNramp4 in
Arabidopsis. The increase of Cd uptake and accumulation under iron
deficiency has been confirmed to be mediated by Fe^2+ transporters such
as OsIRT1 and OsIRT2 in rice [[48]16]. Similarly, our results showed
that the expression of AhIRT1 and AhNramp1 genes in peanut roots can be
induced by iron deficiency, which is associated with Cd uptake and
accumulation [[49]13]. He et al. [[50]17] demonstrated that Fe supply
prevents Cd uptake in Arabidopsis by inhibiting AtIRT1 expression and
enhancing antagonism between Fe and Cd uptake.
Taken together, we hypothesize that iron deficiency-induced expression
of Fe^2+ transporter genes may be responsible for the increased uptake
and accumulation of Cd in iron-deficient peanut plants. To test this
hypothesis, a comparative transcriptome analysis was carried out on
iron-sufficient and -deficient peanut plants under Cd exposure. The
aims were: (i) to obtain the gene expression patterns in the roots
under iron deficiency; (ii) to identify the key genes related to Fe/Cd
uptake and translocation; and (iii) to elucidate the gene regulatory
network that are responsible for Cd uptake and translocation in peanut
plants under iron deficiency. The results presented here will be useful
for better understanding the mechanism of Cd accumulation induced by Fe
deficiency and facilitate gene function studies in peanut.
Results
The accumulation and translocation of cd and Fe in plants
Figure [51]1 shows plant growth as well as the accumulation and
translocation of Cd and Fe in peanut plants. Compared with normal Fe
supply, Fe-deficiency inhibited plant growth and resulted in leaf
chlorosis (Fig. [52]1 a and b), while Cd did not change plant growth in
both Fe treatments (Fig. [53]1b). It was also observed that
Fe-deficiency significantly increased root Cd concentrations but
decreased Cd concentration in xylem sap and the percentage of Cd in
shoots (Fig. [54]1 b and c), whereas Cd concentrations in the shoot
were not affected (Fig. [55]1c). These results showed good agreement
with previous findings [[56]10–[57]13], suggesting that Fe deficiency
increased uptake but decreased root-to-shoot translocation of Cd in
peanut plants. In comparison with Fe-deficient plants, Fe-sufficient
plants exhibited lower root Fe concentration and higher proportion of
Fe in shoots (Fig. [58]1d). The reverse trend in the percentage of Cd
and Fe in shoots indicates that the two ions might compete with each
other during the translocation from roots to shoots.
Fig. 1.
[59]Fig. 1
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Growth (a and b) and the accumulation and translocation of Cd (c) and
Fe (d) in Fe-sufficient (Fe[50]Cd) and Fe-deficient (Fe[0]Cd) peanut
plants exposed to 2 μM Cd for seven days. Asterisk (*) above error bars
indicate values (mean ± SE, n = 3) are significantly different between
Fe-sufficient and Fe-deficient plants according to independent-samples
t-test at 0.05 the level. Different letters above error bars indicate
significant difference at 0.05 the level according to ANOVA followed by
a Duncan multiple-range test
RNA sequencing analysis of eight cDNA libraries
To assess the global transcriptome profile of peanut roots in response
to Fe-deficiency and/or Cd exposure, RNA-Seq analysis was performed on
peanut roots exposed to Fe-sufficient without Cd (Fe[50]), Fe-deficient
without Cd (Fe[0]), Fe-sufficient with Cd (Fe[50]Cd) and Fe-deficient
with Cd (Fe[0]Cd). Two biological replicates were performed per
treatment, with a total of eight cDNA libraries constructed. A total of
58,774,869, 54,456,644, 55,033,082, and 58,221,919 raw reads were
produced from two biological replicate libraries of Fe[50], Fe[0],
Fe[50]Cd, and Fe[0]Cd respectively. After removing low quality reads
and reads containing adapter sequences, a total of 57,331,325,
53,124,279, 54,059,155, and 56,589,717 clean reads remained for Fe[50],
Fe[0], Fe[50]Cd, and Fe[0]Cd, respectively. The percentage of clean
reads in all eight libraries was more than 97.14%, and the percentage
of Q20 exceeded 97.44% (Table [61]1). Pearson’s correlation
coefficients for all biological replicates were 0.949–0.959
(Additional file [62]1: Table S1), suggesting the data were highly
reproducible.
Table 1.
Overview of raw and clean reads in Fe-sufficient (Fe[50]) and
Fe-deficient (Fe[0]) peanut plants exposed to 0 or 2 μM CdCl[2] (Cd)
for seven days
Sample Total raw reads Total clean reads Total clean bases (gb) Clean
reads q20 (%) Clean reads q30 (%) Clean reads in raw reads (%) GC
content (%)
Fe50_1 28,406,266 27,712,721 8.31 0.02 97.56 93.09 97.56
Fe50_2 30,368,603 29,618,604 8.89 0.02 97.64 93.33 97.53
Fe0_1 28,516,330 27,732,406 8.32 0.02 97.63 93.3 97.25
Fe0_2 25,940,314 25,391,873 7.62 0.02 97.44 92.86 97.89
Fe0Cd_1 28,152,785 27,619,944 8.29 0.02 97.61 93.23 98.11
Fe0Cd_2 26,880,297 26,439,211 7.93 0.02 97.69 93.47 98.36
Fe50Cd_1 33,142,927 32,227,575 9.67 0.02 97.68 93.43 97.24
Fe50Cd_2 25,078,992 24,362,142 7.31 0.02 97.63 93.31 97.14
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The high-quality clean reads were mapped to the A. duranensis genome
(reference genome) using HISAT (Hierarchical indexing for spliced
alignment of transcripts). Ultimately, more than 79% of the clean reads
were successfully mapped for all cDNA libraries, and over 77% were
observed to be unique mapped reads (Table [64]2), suggesting that the
samples were comparable.
Table 2.
Mapping results of clean reads against the peanut genomic sequence
sample Total clean reads Total mapped reads Uniquely mapped reads
Multiple mapped reads Spliced reads Unspliced reads Properly mapped
reads
Fe50_1 55,425,442 43,980,460 (79.35%) 42,937,535 (77.47%) 1,042,925
(1.88%) 16,275,516 (29.36%) 26,662,019 (48.1%) 38,544,100 (69.54%)
Fe50_2 59,237,208 46,911,319 (79.19%) 45,798,117 (77.31%) 1,113,202
(1.88%) 17,286,768 (29.18%) 28,511,349 (48.13%) 41,375,852 (69.85%)
Fe0_1 55,464,812 45,419,922 (81.89%) 44,410,704 (80.07%) 1,009,218
(1.82%) 16,915,252 (30.5%) 27,495,452 (49.57%) 40,481,232 (72.99%)
Fe0_2 50,783,746 40,804,086 (80.35%) 39,847,134 (78.46%) 956,952
(1.88%) 14,885,388 (29.31%) 24,961,746 (49.15%) 36,103,056 (71.09%)
Fe0Cd_1 55,239,888 44,105,861 (79.84%) 42,995,894 (77.83%) 1,109,967
(2.01%) 16,183,762 (29.3%) 26,812,132 (48.54%) 38,747,872 (70.14%)
Fe0Cd_2 52,878,422 42,298,150 (79.99%) 41,248,604 (78.01%) 1,049,546
(1.98%) 15,493,356 (29.3%) 25,755,248 (48.71%) 37,208,590 (70.37%)
Fe50Cd_1 64,455,150 51,650,748 (80.13%) 50,399,248 (78.19%) 1,251,500
(1.94%) 18,847,959 (29.24%) 31,551,289 (48.95%) 45,817,274 (71.08%)
Fe50Cd_2 48,724,284 38,614,623 (79.25%) 37,716,105 (77.41%) 898,518
(1.84%) 13,970,730 (28.67%) 23,745,375 (48.73%) 34,228,200 (70.25%)
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Identification of differentially expressed genes (DEGs)
A total of 63,191 genes including 34,553 known genes and 18,030 novel
genes, 8017 lncRNA, 35 misc. RNA, 580 tRNA and 1976 pseudogenes were
identified in eight cDNA libraries. Pairwise comparison analysis for
each gene were performed between Fe-sufficient and Fe-deficient
treatments (Fe[0]/Fe[50] and Fe[0]Cd/Fe[50]Cd), or between Cd-absent
and Cd-present treatments (Fe[50]Cd/Fe[50] and Fe[0]Cd/Fe[0]). DEGs
were identified by the threshold of P[adj]-value < 0.05. As a result, a
total of 3024 genes were differentially regulated in the four
comparisons, of which 676, 1889, 518, and 1860 DEGs were identified in
Fe[0] vs Fe[50], Fe[0]Cd vs Fe[50]Cd, Fe[50]Cd vs Fe[50] and Fe[0]Cd vs
Fe[0], respectively (Fig. [66]2a). Regardless of the absence or
presence of Cd, 189 genes were differentially expressed between
Fe-sufficient and Fe-deficient treatments, suggesting that these genes
were specifically induced or supressed by Fe deficiency. It was also
found that 190 genes were specifically regulated by Cd exposure under
both Fe treatments (Fig. [67]2a).
Fig. 2.
[68]Fig. 2
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Gene expression profile of in Fe-sufficient (Fe[50]) and Fe-deficient
(Fe[0]) peanut plants exposed to 0 or 2 μM CdCl[2] (Cd) for seven days.
(a) Venn diagram of DEGs. (b) The total number of up-regulated and
down-regulated genes
Compared with the control (Fe[50]), Fe deficiency (Fe[0]) induced 465
up-regulated and 211 down-regulated DEGs, whereas the up- and
down-regulated DEGs in Cd-exposed plants (Fe[50]Cd) were 329 and 189,
respectively (Fig. [70]2b). Under Fe-deficient conditions, Cd exposure
(Fe[0]Cd) resulted in 907 up-regulated DEGs and 953 down-regulated
DEGs. In the presence of Cd, Fe deficiency (Fe[0]Cd) induced 1042
up-regulated and 847 down-regulated genes (Fig. [71]2b).
Gene ontology (GO) analysis of DEGs
GO assignments were used to classify the functions of DEGs, and the
results of significantly enriched GO terms (P[adj]-value < 0.05) were
shown in Fig. [72]3. A total of 99 Fe deficiency-responsive DEGs (Fe[0]
vs Fe[50]) were assigned into 7 enriched GO terms consisting of 3
biological process (response to chemical, protein ubiquitination,
protein modification by small protein conjugation) and 4 molecular
function (nucleic acid binding transcription factor activity,
transcription factor activity, ubiquitin-protein transferase activity,
ubiquitin-like protein transferase activity) (Fig. [73]3a). Meanwhile,
66 Cd-responsive DEGs were assigned into 9 enriched GO terms, including
4 biological process (cell wall organization or biogenesis, external
encapsulating structure organization, cell wall organization, cell wall
modification), 2 cellular components (cell wall, external encapsulating
structure) and 3 molecular function (carbohydrate binding, copper ion
binding, pectinesterase activity) (Fig. [74]3b). A total of 857 DEGs
between Fe[0]Cd and Fe[0] were assigned into 46 enriched GO terms
consisting of 28 biological process (response to stress, response to
oxidative stress, metal ion transport, response to chemical, etc.) and
18 molecular function (nucleic acid binding transcription factor
activity, transcription factor activity, heme binding, tetrapyrrole
binding, etc.) subcategories (Fig. [75]3c), and 832 DEGs of Fe[0]Cd vs
Fe[50]Cd were assigned into 36 enriched GO terms including 17
biological process (response to stress, response to oxidative stress,
response to chemical, etc.), 2 cellular components (cell wall, external
encapsulating structure) and 17 molecular function (heme binding,
tetrapyrrole binding, etc.) subcategories (Fig. [76]3d).
Fig. 3.
[77]Fig. 3
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Gene ontology classification of DEGs identified. The enriched
biological process, cellular component and molecular function GO terms
of DEGs between Fe-sufficient and Fe-deficient peanut roots prior to
(a) or after (d) Cd exposure, and between Cd-free and Cd-treated peanut
roots under Fe-sufficient (b) and Fe-deficient (c) conditions
KEEG metabolic pathway analysis of DEGs
To deep insight the molecular interactions among the genes, DEGs were
further annotated with Kyoto Encyclopedia of Genes and Genomes (KEGG)
database ([79]http://www.genome.ad.jp/kegg/). A total of 76 (Fe[0] vs
Fe[50]), 256 (Fe[0]Cd vs Fe[50]Cd), 68 (Fe[50]Cd vs Fe[50]) and 240
(Fe[0]Cd vs Fe[0]) DEGs assigned to 48, 86, 47, and 88 pathways,
respectively (Additional file [80]2: Table S2). The four comparisons
differed from each other in metabolic pathways of DEGs (Fig. [81]4).
The top five pathways of Fe deficiency-responsive DEGs were
phenylpropanoid biosynthesis, plant-pathogen interaction, diterpenoid
biosynthesis, carotenoid biosynthesis, and amino sugar and nucleotide
sugar metabolism (Fig. [82]4a). Among these pathways, phenylpropanoid
biosynthesis was identified as significantly enriched (P[adj]-value
< 0.05). For Cd-responsive DEGs, pentose and glucuronate
interconversions, phenylpropanoid biosynthesis, glutathione metabolism,
galactose metabolism, and diterpenoid biosynthesis were the top five
categories, and no enriched pathway was identified (Fig. [83]4b). A
larger number of enriched pathways were found in DEGs of Fe[0]Cd vs
Fe[50]Cd (pentose and glucuronate interconversions, glutathione
metabolism, flavonoid biosynthesis, and phenylpropanoid biosynthesis)
(Fig. [84]4c) and Fe[0]Cd vs Fe[0] (phenylpropanoid biosynthesis,
cysteine and methionine metabolism, diterpenoid biosynthesis, MAPK
signaling pathway, pentose and glucuronate interconversions,
phenylalanine metabolism, and ABC transporters) (Fig. [85]4d).
Fig. 4.
[86]Fig. 4
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KEGG pathway enrichment analysis based on the differentially expressed
genes between Fe-sufficient and Fe-deficient peanut roots prior to (a)
or after (c) Cd exposure, and between Cd-free and Cd-treated peanut
roots under Fe-sufficient (b) and Fe-deficient (d) conditions
DEGs involved in heavy metal transport
According to GO functional annotation, 54 DEGs were identified to
highly similar with transporters that may be involved in the
translocation of heavy metal in plants, including ATP-binding cassette
transporters (ABC transporters), cation/H^+ antiporters (CAXs), copper
transport proteins (COPTs), Fe^2+ transport proteins (IRTs), natural
resistance-associated macrophage proteins (NRAMPs), metal tolerance
proteins (MTPs), metal-nicotianamine transporter or yellow stripe-like
transporters (YSLs), oligopeptide transporters (OTPs), vacuolar iron
transporters (VITs), and zinc transporters (ZIPs) (Table [88]3). In the
absence of Cd, ABCB19, putative ABCC15, ABCG15-like, CAX4-like, COPT1,
IRT1 (107484081 and 107494754), NRAMP5 and OPT3 were up-regulated by Fe
deficiency, while three genes (YSL3, VIT3 and VIT4) were
down-regulated. In the presence of Cd, a total of 37 DEGs encoding
metal transporters were detected between the Fe-sufficient and
Fe-deficient peanut plants, including 18 up-regulated genes (ABCA2,
ABCA7, ABCB19, ABCB21, ABCB11, ABCC3, ABCC8, ABCC15, ABCF1, ABCG6,
ABCG11, HMA5, CAX18, Nramp3, OPT3, IRT1) and 19 down-regulated genes
(ABCB11, ABCB19, ABCG5, ABCG8, ABCG32, ABCG39, COPT6, OPT7, YSL3, YSL7,
VIT4, VIT3, ZIP1, ZIP5, ZIP6). Cd exposure induced seven metal
transport genes in Fe-sufficient plants, including ABCB11, ABCB19, IRT1
(110279939), NRAMP5, HMA5, VIT4 (107468698) and ZIP11. In contrast, 32
transporter genes were induced or suppressed by Cd exposure under Fe
deficient stress, including 19 up-regulated genes (ZIP11, ABCC4, ABCC3,
ABCC8, YSL3, ABCG11, CAX20, ABCG6, NRAMP3, HMA5, OPT3, ABCB11, CAX18,
ABCG11, ABCC3, ABCB19, IRT1) and 13 down-regulated genes (ABCG15, CAX4,
VIT3, COPT6, COPT1, COPT2, ABCB19, ABCG5, ABCB2, ABCB11, ZIP6, ABCG5).
Compared with the control, exposure of Fe-deficient plants to Cd
up-regulated ABCB11, ABCB19, ABCC3, ABCC8, ABCC15, ABCF1, ABCG6,
ABCG11, CAX18-like, CAX20, IRT1, NRAMP3, NRAMP5, MTP11 and OPT3, but
down-regulated ABCG5, ABCG8, ABCG25, COPT1, VIT3, VIT4, ZIP1, ZIP5 and
ZIP6. More importantly, it was found that three up-regulated genes
(OPT3, ABCC15, IRT1) and three down-regulated genes (VIT3, VIT4, YSL3)
were specifically regulated by Fe-deficiency, regardless of Cd
treatments. Similarly, two genes (ZIP11 and HMA5) were found to be
specifically induced by Cd in both the Fe-sufficient and Fe-deficient
peanut plants.
Table 3.
DEGs possibly involved in metal transport in Fe-sufficient and
Fe-deficient peanut plants exposed to 0 or 2 μM CdCl[2] for seven days
Gene_ID Gene description Abbr. Fe0vsFe50 Fe50CdvsFe50 Fe0CdvsFe50
Fe0CdvsFe0 Fe0CdvsFe50Cd
log2Fold Change padj log2Fold Change padj log2Fold Change padj log2Fold
Change padj log2Fold Change padj
107491333 ABC transporter A family member 2 ABCA2 0.753 0.013
107491332 ABC transporter A family member 7, transcript variant × 1
ABCA7 0.784 0.006
107479052 ABC transporter B family member 2-like, transcript variant X5
ABCB2 − 1.713 0.034
107467550 ABC transporter B family member 11 ABCB11 2.600 0.000 2.761
0.000 2.126 0.000
107495097 ABC transporter B family member 11-like ABCB11 1.352 0.009
−1.652 0.008 −2.400 0.000
107471179 ABC transporter B family member 19 ABCB19 1.270 0.013 1.005
0.010 −2.171 0.000 −1.889 0.000
107476483 ABC transporter B family member 19 ABCB19 2.380 0.000 3.120
0.000 1.561 0.020
107492327 ABC transporter B family member 21-like ABCB21 2.000 0.030
107463694 ABC transporter C family member 3 ABCC3 1.313 0.000 1.045
0.004 1.339 0.000
107496250 ABC transporter C family member 3 ABCC3 3.254 0.000 3.073
0.000 2.248 0.000
107496270 ABC transporter C family member 3-like transcript variant ×2
ABCC3 −2.894 0.044
107475935 ABC transporter C family member 4 ABCC4 0.890 0.020
107464256 ABC transporter C family member 8, transcript variant ×2
ABCC8 1.918 0.000 1.132 0.010 1.548 0.000
107481980 putative ABC transporter C family member 15, transcript
variant X2 ABCC15 2.018 0.000 2.888 0.000 2.391 0.000
107476479 ABC transporter F family member 1, transcript variant ×1
ABCF1 1.661 0.000 1.193 0.007
107470452 ABC transporter G family member 5-like ABCG5 −1.958 0.000
−2.157 0.000 −1.762 0.000
107480222 ABC transporter G family member 5-like ABCG5 −1.109 0.028
−1.497 0.002 −1.326 0.002
107467717 ABC transporter G family member 6-like ABCG6 1.465 0.042
1.621 0.001 1.010 0.013
107466837 ABC transporter G family member 8-like ABCG8 −1.145 0.041
− 1.331 0.001
107481537 ABC transporter G family member 11 ABCG11 1.012 0.028 1.305
0.001 1.107 0.002
107481662 ABC transporter G family member 11, transcript variant X2
ABCG11 4.356 0.000 3.055 0.000
107492554 ABC transporter G family member 15-like ABCG15 5.706 0.032
−5.572 0.019
107467862 ABC transporter G family member 25 ABCG25 −1.042 0.018 −0.948
0.049
107469510 ABC transporter G family member 32 ABCG32 −1.273 0.020
107493900 ABC transporter G family member 39-like ABCG39 −2.463 0.017
110273497 Cation/H^+ antiporter 4-like CAX4 4.519 0.001 −4.765 0.001
107479804 Cation/H^+ antiporter 18-like, transcript variant X2 CAX18
1.903 0.000 2.838 0.000 2.334 0.000
107491686 Cation/H^+ antiporter 20 CAX20 1.106 0.048 1.366 0.003
107474428 Copper transport protein ATX1 0.981 0.006
107477078 Copper transporter 1 COPT1 1.376 0.036 −1.926 0.002 −3.309
0.000
107484791 Copper transporter 2-like, transcript variant ×1 COPT2 −2.964
0.000
107484785 Copper transporter 6 COPT6 −3.354 0.026 −3.432 0.017
107484081 Fe^2+ transport protein 1 IRT1 3.342 0.048 3.472 0.019
107491017 Fe^2+ transport protein 1 IRT1 9.778 0.000 7.200 0.000 9.773
0.000
107494754 Fe^2+ transport protein 1-like IRT1 6.608 0.023 11.806 0.000
5.210 0.000 8.432 0.000
110279939 Fe^2+ transport protein 1-like IRT1 6.658 0.020
107486418 Metal tolerance protein 11, transcript variant x2 MTP11 0.890
0.046
107480469 Metal transporter Nramp3, transcript variant ×1 Nramp3 2.912
0.000 1.898 0.000 3.565 0.000
107460699 Metal transporter Nramp5 Nramp5 1.945 0.000 1.633 0.001 1.325
0.001
107460374 Metal-nicotianamine transporter YSL3 −1.920 0.000 1.230 0.000
−0.648 0.042
107489952 Probable metal-nicotianamine transporter YSL7 −0.901 0.012
107466086 Oligopeptide transporter 3 OPT3 1.857 0.000 4.594 0.000 2.725
0.000 4.720 0.000
107482451 Oligopeptide transporter 7, transcript variant x1 OPT7 −2.576
0.014
107471962 Probable copper-transporting ATPase HMA5 1.311 0.000 2.409
0.000 1.311 0.000
107491634 Vacuolar iron transporter homolog 3 VIT3 −2.675 0.000 −6.523
0.001 −3.851 0.002 −6.562 0.001
107468698 Vacuolar iron transporter homolog 4 VIT4 2.925 0.032 −5.203
0.001
107481738 Vacuolar iron transporter homolog 4-like VIT4 −1.108 0.005
−1.493 0.000
107481739 Vacuolar iron transporter homolog 4-like VIT4 −4.145 0.000
−7.910 0.000 −8.370 0.000
107461527 Zinc transporter 1, transcript variant X1 ZIP1 −1.507 0.000
−1.671 0.000
107482454 Zinc transporter 5, transcript variant X2 ZIP5 −3.298 0.007
107494273 Zinc transporter 5-like ZIP5 −6.223 0.021
107458282 Zinc transporter 6, chloroplastic ZIP6 −0.933 0.036
107473088 Zinc transporter 6, chloroplastic ZIP6 −1.994 0.000 −1.562
0.001 −1.418 0.006
107462023 Zinc transporter 11 ZIP11 0.925 0.009 0.831 0.034 −0.636
0.036
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Data of DEGs showing a similar expression in pairwise comparisons were
not shown in the table
Verification of the DEG results
To further verify the transcriptome data, ten DEGs involved in metal
transport, including IRT1, NRAMP3, NRAMP5, OPT3, YSL3, CAX4, HMA5,
ABCC3, ZIP1, and ZIP5, were selected for RT-qPCR analysis. The RT-qPCR
results, presented in Fig. [90]5, showed a good agreement with the
RNA-Seq data (Table [91]3). Under Cd-free condition, Fe deficiency
up-regulated the expression of CAX4-like, NRAMP5 and OPT3, but
down-regulated YSL3, ZIP1 and ZIP5 in peanut roots. For Fe-sufficient
plants, the expression of IRT1, NRAMP5, HMA5 and ZIP1 was significantly
induced by Cd exposure. Compared with the control, Cd exposure with Fe
deficiency enhanced the expressions of OPT3, ABCC3, HMA5, and NRAMP3,
but decreased those of YSL3, ZIP1 and ZIP5. Pearson’s correlation
analysis showed that the relative gene expression (RT-qPCR) is
significantly correlated with the foldchange of read counts (RNA-Seq)
(r = 0.705, p = 0.000, n = 40). The high confirmation rate demonstrates
the reliability of our RNA-Seq data.
Fig. 5.
Fig. 5
[92]Open in a new tab
The qRT-PCR analysis of metal transport-related genes in peanut roots
exposed to 0 or 2 μM Cd under Fe-sufficient (Fe50) and Fe-deficient
(Fe0) conditions. The relative expression of each gene was calculated
as the 2^−ΔΔCT value and normalized by geometric mean of three stably
expressed reference genes. Data are presented as means with SE (n = 3).
Different letters above error bars indicate that values are
significantly different (P < 0.05) according to Duncan’s multiple range
test after one-way analysis of variance
Discussion
Although iron deficiency has been demonstrated to considerably enhance
the uptake and accumulation of Cd in peanut plants [[93]10–[94]13],
limited information is available about the physiological and molecular
mechanisms underlying iron deficiency-induced increase of Cd
accumulation in peanut. RNA-seq analysis has been used for revealing
the molecular mechanisms of Cd uptake, translocation and accumulation
in many plant species [[95]18–[96]20]. The complete genome sequencing
of A. duranensis and A. ipaensis, the diploid ancestors of cultivated
peanut [[97]21], has shed light to genomic studies on cultivated
peanut. However, sequence information of peanut in response to iron
deficiency and Cd exposure is scarce. In this study, we obtained
57,331,325, 53,124,279, 54,059,155, and 56,589,717 clean reads from the
peanut roots treated with Fe[50], Fe[0], Fe[50]Cd, and Fe[0]Cd
respectively. More than 79% of clean reads for all cDNA libraries were
functionally annotated in the A. duranensis genome [[98]21], and more
than 77% were unique mapped reads (Table [99]2). A total of 3024 genes
were identified to be DEGs in pairwise comparisons: Fe[0] vs Fe[50]
(676), Fe[0]Cd vs Fe[50]Cd (1889), Fe[50]Cd vs Fe[50] (518), and
Fe[0]Cd vs Fe[0] (1860) (Fig. [100]2a). Of them, 54 DEGs were
identified to highly similar with transporters that may be involved in
the uptake and translocation of Fe/Cd in plants. These results provide
clues to mechanisms underpinning Cd uptake and accumulation in
Fe-deficient peanut plants.
Genes involved in Fe uptake and translocation were greatly induced by
Fe deficiency in peanut roots. As an Fe-efficient plant that develops
strategy I mechanism in response to Fe deficiency, peanut takes up
Fe^2+ by increasing Fe^2+ transporter coupled with an increase of
ferric reductase activity and rhizosphere acidification by releasing
protons from the roots under Fe-deficient conditions [[101]6]. We found
that both the Fe^2+ transport gene IRT1 and ferric reductase FRO1 were
up-regulated by Fe deficiency in peanut roots (Table [102]3, Fig.
[103]5). The result indicates that IRT1/FRO1 system constitutes the
major pathway for Fe entry into root epidermal cells, and induction of
these genes might improve Fe nutrition under Fe-deficient stress. A
transporter gene of NRAMP family, NRAMP5 (NRAMP1) [[104]13], was also
highly induced in roots under Fe-deficient conditions (Table [105]3,
Fig. [106]5). The NRAMP5 (NRAMP1) have been shown to function in Fe
transport in several plants such as Arabidopsis [[107]22], Noccaea
caerulescens [[108]23], Malus baccata [[109]24], peanut [[110]25] and
rice [[111]26]. The induction of NRAMP5 (NRAMP1) under Fe-deficiency
indicates that this gene is involved in the uptake of Fe by peanut
roots. Interestingly, both the IRT1 and NRAMP5 are shown to be involved
in Cd transport into root cells across membrane [[112]13, [113]16,
[114]27]. The current results confirmed that iron deficiency-induced
expression of IRT1 and NRAMP5 may be responsible for the increase of Cd
uptake and accumulation in Fe-deficient peanut plants [[115]13].
We also found that large number of ABC transporter genes were
significantly induced or suppressed by Fe deficiency and/or Cd exposure
(Table [116]3). Although the detailed functions of ABC transporter
genes is poorly understood, some members have been verified to play
roles in the uptake and translocation of Fe/Cd in plants [[117]28].
ABCB19, an auxin transporter that mediates long-distance polar auxin
transport in stems and roots [[118]29, [119]30], was induced by Fe
deficiency and/or Cd exposure (Table [120]3). Fe deficiency can
increase the levels of auxin in the roots, which may promote the
formation of root hair [[121]31]. ABCC3 (MRP3), like most ABCC
transporters such as such as ABCC2 and ABCC3, is vacuolar
membrane-localized protein involved in the vacuolar transport of PC-Cd
complexes [[122]32]. The induction of ABCC3 in peanut roots by Fe
deficiency with Cd exposure (Table [123]3, Fig. [124]5) may contribute
to vacuolar Cd sequestration, enhancing Cd detoxification and reducing
root-to-shoot translocation of Cd. ABCC8 homologous to MRP6 in
Arabidopsis, which was shown to be part of a cluster (AtMRP6, AtMRP3
and AtMRP7, as well as SAT3) involved in metal tolerance [[125]33].
Besides ABC transporters, another peptide transporter OPT3 was
significantly induced under Fe-deficient conditions (Table [126]3, Fig.
[127]5). In Arabidopsis, OPT3 was shown to load iron into the phloem,
facilitate iron recirculation from the xylem to the phloem, and
regulate both shoot-to-root iron signaling and iron redistribution from
mature to developing tissues [[128]34, [129]35]. Reduced expression of
OPT3 induces an over accumulation of Fe in roots and leaves, partially
due to an elevated expression of the IRT1 [[130]36]. Therefore, the
strong induction of OPT3 in the root of Fe-deficient peanut seedlings
suggests that the gene play a role in the redistribution of Fe between
vegetative tissues. Moreover, OPT3 was also found to have an impact on
the uptake and translocation of Cd in Arabidopsis [[131]34, [132]35].
Mendoza-Cózatl et al. [[133]35] observed that an OPT3 mutant of
Arabidopsis, opt3–2, over-accumulates Cd in seeds and roots, but
under-accumulates Cd in leaves. However, Zhai et al. [[134]34]
demonstrated that OPT3 is not involved in the phloem-based
remobilization of Cd from source to sink tissues, despite it can
mediate Cd transport in vitro. They speculated that increased Cd in
seeds of the mutant is due to the impact of reduced expression of OPT3
on other transporters such as YSL1 or YSL3 [[135]34]. YSL3 is a
plasma-localized transporter delivering a broad range of
nicotianamine-metal complexes. SnYSL3 gene is shown to be involved in
the translocation and detoxification of Cd in Solanum nigrum, and its
overexpression in Arabidopsis increased the translocation of Cd and Fe
from roots to shoots [[136]37]. In the present study, YSL3 was
down-regulated in peanut roots under Fe-deficient stress (Table [137]3,
Fig. [138]5), which may be partially responsible for the reduced
root-to-shoot translocation of Cd in Fe-deficient plants.
Several other transporter genes such as CAX4-like, COPT1, VIT3 and
VIT4, were highly induced or suppressed by Fe deficiency (Table [139]3,
Fig. [140]5). CAX4 is a cation/H^+ antiporter that plays a key role in
mediating cations, such as Ca^2+ and Cd^2+, influx into the vacuole
[[141]38]. Thus, the induction of CAX4-like by Fe deficiency in peanut
roots may enhance vacuole sequestration of Cd and consequently,
reducing root-to-shoot Cd translocation. COPT1 participates in copper
acquisition and accumulation and regulates root elongation and pollen
development [[142]39]. In the current study, we found that COPT1 was
up-regulated by Fe deficiency, but down-regulated in Fe-deficient
seedlings exposed to Cd. We inferred that COPT1 indirectly effect Fe
and/or Cd acquisition by regulating root elongation. Regardless of Cd
exposure, the vacuolar iron transporters, VIT3 and VIT4, were
suppressed under Fe deficiency condition. These alterations could
reduce Fe sequestration in the vacuolar of root cells, and therefore
enhance Fe transport from roots to shoots. NRAMP3 localizes at the
vacuolar membrane, and is able to release Fe and Cd from the vacuolar
under limited Fe conditions [[143]40, [144]41]. The induction of NRAMP3
may improve the remobilization of Fe from vacuoles of root cells in
Fe-deficient peanut plants particularly under Cd stress.
Apart from abovementioned genes, three genes belonging to ZIP family,
ZIP1, ZIP5, and MTP11, differentially expressed between the control and
Cd exposure with Fe deficiency (Table [145]3, Fig. [146]5). ZIP1 shows
a high degree of homology to the AtZIP2 from Arabidopsis [[147]42] and
OsZIP1 from rice [[148]43], while ZIP5 is homologous to AtZIP1
[[149]42]. Both the AtZIP2 and OsZIP1 show a broad substrate
specificity for divalent cations including Cd [[150]42, [151]43]. In
Arabidopsis, AtZIP2 is a plasma membrane localized transporter that may
contribute to Mn/Zn movement in the stele to the xylem parenchyma, for
subsequent xylem loading and transport to the shoot [[152]44].
Similarly, OsZIP1 transcripts were localized to the epidermis and stele
of roots of zinc-deprived plants, suggesting the involvement of this
transporter in zinc absorption or transfer from the vascular tissue
[[153]43]. AtZIP1 is a vacuolar transporter that may play a role in
remobilizing Mn from the vacuole to the cytoplasm in root stellar
cells, and may contribute to radial movement to the xylem parenchyma
[[154]44]. Thus, the reduced expression of ZIP1 and ZIP5 in the roots
of Fe-deficient peanut plants under Cd stress may inhibit root-to-shoot
Cd translocation. In the case of MTP11, it was shown to function in Mn
transport and tolerance by sequestering Mn into the pre-vacuolar
compartments in Arabidopsis [[155]45]. Whether MTP11 is associated with
Fe and/or Cd transport need to be further investigated.
Conclusions
In conclusion, the current comparative study revealed that CAX4, COPT1,
IRT1, NRAMP3, NRAMP5, OPT3, YSL3, VIT3 and VIT4 might be involved in
iron homeostasis in Fe-deficient peanut plants. More importantly, some
genes such as IRT1, NRAMP3, NRAMP5, OPT3, YSL3, ABCC3, ZIP1, and ZIP5,
were identified to be responsible for the uptake, distribution, and
translocation of Cd in peanut plants under iron deficiency. Based on
our array data, we proposed a model to explain why iron deficiency
induced an increase of Cd uptake but a decrease of Cd translocation
from roots to shoots in peanut plants. The up-regulated expression of
NRAMP5 and IRT1 genes induced by iron deficiency may enhance the uptake
of cadmium by peanut roots. Iron deficiency-induced down-regulation of
ZIP1, ZIP5 and YSL3 might result in a decrease of Cd xylem (or phloem)
loading in root stele, and consequently, reducing root-to-shoot Cd
translocation in peanut plants. Additionally, although detailed
information is still unclear, larger amounts of ABC transporter genes
were induced or suppressed by iron deficiency under Cd exposure,
indicating that further study of this family would be helpful to
understand the mechanism of Fe/Cd uptake and transport.
Methods
Plant growth and treatment
Peanut plants (Arachis hypogaea cv. Fenghua 1) were cultured as the
conditions previously reported by Chen et al. [[156]13] in a growth
chamber of the Huaibei Normal University. Seeds (obtained commercially
from the Peanut Institute of Shandong Province, Qingdao) were
sterilized with 1% sodium hypochlorite for 10 min, and then they were
rinsed with tap water for 24 h and germinated in well-washed sand.
After 5 days of emergence, the uniform sized seedlings were selected
and transferred to polyethylene pot (six plants per pot) containing
3.5 L of nutrient solution (pH 5.8) [[157]11]. Seven-day-old seedlings
were pretreated with (Fe[50]) or without (Fe[0]) 50 μM FeEDTA for 7 d
in basal nutrient solution. Thereafter, 0 or 2 μM CdCl[2] were added in
nutrient solution for each Fe treatment, and continuously cultured for
one week. The Cd level (2 μM) was arranged according to the Cd
concentration in the soil solution of a Cd-contaminated farmland in
China (soil moisture at 60% of field capacity) [[158]7]. The experiment
was arranged as a completely random design with nine replications
(pots). During the growing period, the nutrient solution was renewed
twice a week, and pots were randomly arranged and moved daily to
minimize position effects.
Root samples for RNA-seq (two biological replicates) and qRT-PCR (three
biological replicates) analysis were collected separately from
Fe-sufficient and Fe-deficient plants after Cd treatment. Each
biological replicate contains a pool of six different plants growing in
each pot. All samples were immediately frozen in liquid nitrogen and
stored at − 80 °C.
Determination of cd and Fe in plants
Three pots of plants for each Fe treatment under Cd exposure were used
for collecting xylem sap excluded from the cut surface as the method
described by Uraguchi et al. [[159]46]. After weighing, the collected
sap was used for determining the Cd concentration by graphite furnace
atomic absorption spectrometry (GF-AAS). Roots and shoots were
separated and rinsed three times with deionized water. Thereafter,
plant samples were oven-dried at 70 °C for constant weight. The dried
tissues were weighed and ground into powder. The concentration of Cd
and Fe in plant samples was measured by flame AAS after digested with
HNO[3]–HClO[4] (3:1, v/v).
The translocation of Cd and Fe from roots to shoots was indicated as
the percentage of metal in shoots, which was calculated as follows
[[160]7]:
[MATH: Percentage of metal in shoots%=100×Mshoot×shoot biomass/Mroot×root biomass+Mshoot×shoot
biomass.whereMis the concentration ofCdorFein plant tissues :MATH]
cDNA library construction and RNA sequencing
Total RNA was extracted by using Trizol® Reagent (Invitrogen) and
purified using the RNeasy Plant Mini kit (Qiagen) according to the
manufacturer’s instructions. The purity and integrity of RNA were
analyzed using NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) and
Agilent 2100 Bioanalyzer (Agilent, USA), respectively. Eight cDNA
libraries named Fe[50]_1, Fe[50]_2, Fe[0]_1, Fe[0]_2, Fe[50]Cd _1,
Fe[50]Cd _2, Fe[0]Cd_1 and Fe[0]Cd_2 were constructed as the method
previously described by Hu et al. [[161]47]. The clustering of the
index-coded samples was performed on a cBot Cluster Generation System
using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the
manufacturer’s instructions. After cluster generation, the library
preparations were sequenced on an Illumina Hiseq™ 2500 platform and
125 bp/150 bp paired-end reads were generated. All library construction
and sequencing were done at the Novogene Bioinformatics Institute
(Beijing, China).
Bioinformatics analysis
Data filtering and mapping
To obtain high-quality clean reads, the raw reads from RNA-seq were
filtered by removing the adaptor sequences, ambiguous ‘N’ nucleotides
and low-quality sequences. Clean reads were aligned to the peanut
reference genome (NCBI) using HISAT2 (v2.2.4,
[162]http://www.ccb.jhu.edu/software/hisat) with default parameters.
The mapped reads of each sample were assembled by StringTie (v1.3.3b)
[[163]48] in a reference-based approach.
Identification of DEGs
The numbers of reads mapped to the reference were counted using
featureCounts v1.5.0-p3 [[164]49]. The gene expression levels were
represented by the expected number of Fragments Per Kilobase of
transcript sequence per Millions base pairs sequenced (FPKM), which was
calculated on the basis of the length of the gene and reads count
mapped to this gene. Differential expression analysis of two groups was
performed using the DESeq2 R package (1.16.1) according to the method
described by Love et al. [[165]50]. The resulting P-values were
adjusted using the Benjamini and Hochberg’s approach for controlling
the false discovery rate. Genes with an adjusted P[adj]-value < 0.05
found by DESeq2 were assigned as differentially expressed.
Gene ontology (GO) and pathway analysis of DEGs
Gene Ontology (GO) enrichment analysis of differentially expressed
genes was implemented by the clusterProfiler R package, in which gene
length bias was corrected.
GO terms with corrected P value less than 0.05 were considered
significantly enriched by differential expressed genes. KEGG pathways
were retrieved from KEGG web server ([166]http://www.genome.jp/kegg/).
The clusterProfiler R package was used to test the statistical
enrichment of differential expression genes in KEGG pathways.
qRT-PCR validation
Ten DEGs were randomly selected for qRT-PCR validation. Primer
sequences of these genes as well as reference genes are listed in
Additional file [167]3: Table S3. Total RNA (0.5 μg) from each root
sample was reverse transcribed into cDNA using Prime Script® RT reagent
Kit (Takara, Dalian, China) and random primers following manufacturer’s
instructions. Quantitative PCR reactions were performed in 20 μl
reaction volumes using a SYBR Premix EX Taq Kit (Takara) according to
the manufacturer’s instructions. Reactions were carried out on an
ABI7300 (Applied Biosystems, CA, USA). Each biological replicate was
technically replicated three times. The relative expression levels of
the selected genes were calculated using the 2^-ΔΔCT method and
normalized by geometric mean of three stably expressed housekeeping
genes (AhADH3, Ah60S and Ahactin) [[168]51, [169]52].
Additional files
[170]Additional file 1:^ (13.2KB, docx)
Table S1. Pearson correlation between samples. (DOCX 13 kb)
[171]Additional file 2:^ (21.3KB, xlsx)
Table S2. Overview of KEGG pathways for DEGs in four comparisons. (XLSX
21 kb)
[172]Additional file 3:^ (15.5KB, docx)
Table S3. The primers used in RT-qPCR analysis. (DOCX 15 kb)
Acknowledgements