Abstract
Cadmium (Cd) is a hazardous heavy metal, and Cd pollution has become a serious problem worldwide. Peanut (Arachis hypogaea L.) is an important oil crop in the world and has a strong capacity to accumulate Cd in soil. The natural resistant-associated macrophage protein (NRAMP) plays an important part in the absorption and transportation of Cd in plants. To date, the NRAMP family in peanut is ill-informed. In the present study, 29 AhNRAMPs were identified and were classified into three groups and fourteen proteins in group 1 (G1), ten proteins in group 2 (G2) and five proteins in group 3 (G3). There are 71-1347 amino acids in AhNRAMPs. Most of the AhNRAMPs exhibited tissue-specific expression patterns. For instance, AhNRAMP10 and AhNRAMP26 from G1 were highly expressed in roots, G2 genes in shoots and leaves and G3 genes in shoots. The transcriptional levels of AhNRAMPs in roots can be regulated by Cd. Notably, 55% of (16) AhNRAMPs genes were upregulated in peanut roots and positively responded to Cd stress. It’s worth noting that the relative expressions of AhNRAMP2 and AhNRAMP11, which were increased by 6.9-fold and 14.1-fold at 3 h in roots of Cd-enriched variety under Cd stress while decreasing by 44% and 25% at the same time in Cd sensitive variety. In a word, the comprehensive research of the AhNRAMP family provides insights into the capacity of Cd enrichment in peanut.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Cadmium (Cd) is not an essential nutrient element and is one of the most toxic heavy metals for plants and people (Akindele et al. 2020; Kumar et al. 2021). According to a survey bulletin of national soil pollution in 2014, point exceeding rates of heavy metals in arable soil was 19.4%, and Cd ranked first (7.0%). In China, about 1.3 × 104 ha of cultivated soil is contaminated by Cd (MEE and MNR 2014). Peanut (Arachis hypogaea L.) is an important oil crop globally. Peanut roots, stems and leaves had a very strong ability to accumulate Cd (Wang et al. 2018). Kernel also had a strong capacity for Cd enrichment in soil (Zhu et al. 2016; Gu et al. 2019).Planting peanut provides an effective method to repair Cd-polluted field (Chen et al. 2022). Cultivation of Cd sensitive peanut is vital to human health and food safety. Therefore, understanding absorption and transport mechanisms of Cd in peanut will be of great importance.
The natural resistant-associated macrophage protein (NRAMP) is an integral membrane transporter that usually has highly conserved 10–12 putative transmembrane domains (TMDs) (Mani and Sankaranarayanan 2018, 2022). The NRAMP protein is key in uptaking and transporting Cd in plants (Bressler et al. 2004; Mani and Sankaranarayanan 2022). AtNRAMP1, AtNRAMP5 and AtNRAMP6 are participated in the transport of Cd (Thomine et al. 2000; Cailliatte et al. 2009, 2010). OsNRAMP1, OsNRAMP2 and OsNRAMP5 proteins play vital roles in Cd transport (Takahashi et al. 2011; Ishikawa et al. 2012; Sasaki et al. 2012; Chang et al. 2020). In the Cd-hyperaccumulator Thlaspi caerulescens and Thlaspi japonicum, the NRAMP protein take charge of mobilizing Cd (Mizuno et al. 2005; Oomen et al. 2009). In barley, HvNRAMP5 was reported to uptake Cd (Wu et al. 2016). Overexpression of TtNRAMP1 improved the transport of Cd (Wang et al. 2019a). MhNRAMP1 from Malus hupehensis increased the ability of Cd uptake (Zhang et al. 2020a). Overexpressed LeNRAMP3 maintains Cd tolerance in tomato (Rahmatizadeh et al. 2021). In peanut, AhNRAMP1 was obviously induced by Fe deficiency and overexpression of AhNRAMP1 in tobacco enhanced tolerance to Fe deprivation (Xiong et al. 2012). NRAMP3 and NRAMP5 are involved in Cd uptake and translocation in peanut under Fe deficiency (Chen et al. 2019).
NRAMP proteins have been studied in Arabidopsis thaliana (Mäser et al. 2001), Oryza sativa (Chang et al. 2020), Phaseolus vulgaris (Ishida et al. 2018), Theobroma cacao (Ullah et al. 2018), Glycine Max (Qin et al. 2017), Brassica napus (Zhang et al. 2018), Spirodela polyrhiza (Chen et al. 2021), Tea (Li et al. 2021) and Populus trichocarpa (Ma et al. 2023). However, the function of the NRAMP protein in responding to Cd stress in peanut remains elusive. In the present study, we identified AhNRAMPs in the cultivated peanut genome and elucidated their sequence structures and characterization, cis-acting elements, tissue expression and expression patterns under Cd stress. Our findings are helpful to explain the transport mechanism of Cd in peanut.
Materials and methods
Discovery and physicochemical features of AhNRAMPs
The protein sequences of cultivated peanut (cv. Tifrunner) were extracted from the peanut genome database (PeanutBase, https://www.peanutbase.org/). The NRAMP genes in A. thaliana (AtNRAMPs) were acquired from TAIR (http://www.arabidopsis.org/). The hidden Markov model (HMM) profile of the NRAMP domain (PF01566) was downloaded from the Pfam database (http://pfam.xfam.org/). The AhNRAMP were identified using HMM search (E value < 10−5) and BlastP (E value < 10−5), NCBI CD-search.
Physicochemical parameters, including the number of amino acids, molecular weight (MW), theoretical isoelectric point (pI), were calculated by ExPASy6 and TBtools (Chen et al. 2020). Prediction of protein subcellular localization and transmembrane helices were used by ProtComp 9.0 (ProtComp - Predict the sub-cellular localization for Plant proteins (softberry.com)).
Conserved motifs, gene structures, and chromosome mapping of AhNRAMPs
The MEME online tool (https://meme-suite.org/meme/db/motifs) was performed to analyze conserved motifs with default parameters. TBtools was applied to analyze and map gene structures and chromosomal distribution (Chen et al. 2020).
Phylogenetic relationships, and synteny analysis of AhNRAMPs
A neighbour-joining (NJ) method in MEGA-X was used to construct a phylogenetic tree (Kumar et al. 2018). The Evolview was performed to beautify the phylogenetic tree (He et al. 2016). Syntenic relationships of AhNRAMPs in peanut were analyzed using TBtools. Gene duplication events and the non-synonymous substitution rates (Ka) and the synonymous substitution rates (Ks) of AhNRAMPs were calculated by TBtools (Chen et al. 2020).
Prediction of cis-elements in the AhNRAMP promoters
Upstream sequences (2000 bp) from the start codon of AhNRAMPs were extracted were analyzed cis-acting elements by the PlantCARE.
Prediction of putative miRNAs targeting AhNRAMPs
The CDS of all AhNRAMPs was performed to predict the miRNA target sites with psRNATarget website (https://www.zhaolab.org/psRNATarget/home) (Dai et al.2018).
Prediction of transcription factor of AhNRAMPs
The 500 bp nucleotide sequences from upstream regions of AhNRAMPs were extracted and submitted to the PlantRegMap (http://plantregmap.gao-lab.org/binding_site_prediction.php) to predict the putative transcription factors (Tian et al. 2020).
Expression of AhNRAMPs in different tissues
Expression data of AhNRAMPs in different tissues extracted from RNA-seq data (PeanutBase database: https://www.peanutbase.org/) (Clevenger et al. 2016). The transcript abundances of AhNRAMP genes were quantified by calculating TPM (Transcripts Per Kilobase of exon model per Million mapped reads). The TBtools software was used to create the heatmap (Chen et al. 2020).
Plant materials, Cd treatments, RNA extraction and qRT-PCR
Two peanut varieties came from our laboratory collection and were used for expression analysis under Cd stress. Cd-enriched variety detected 0.67 mg/kg Cd in kernel and Cd-sensitive variety detected 0.28 mg kg−1 Cd in kernel, which planted and harvested in Yichang, Jiangxi, China (Cd content in soil was 3.29 mg kg−1). Peanut seeds were cultured in soils for 21 days and four seedlings in one pot with 16 h light (350 µmol m−2 s−1 light intensity, 28 °C) and 8 h darkness (28 °C) in plant growth room in Nanchang, Jiangxi, China. The 21 day seedlings were cultivated in watered with 20 mL supplemented with CdCl2 2H2O (China National Pharmaceutical Group Co., Ltd.) to a final concentration of 2 µM Cd for 0 h (CK), 1 h, 3 h, 6 h, 12 h, 24 h, 72 h, and 168 h in one pot. The roots were sampled, quick frozen in liquid nitrogen and stored at − 80 °C. Each treatment was independently replicated three times.
TianGen RNA Plant Kit was used to extract total RNA. qRT-PCR was performed on LightCycler 96 (Roche). The 10 µL reaction volume contained 5 µL of 2×SYBR Green Mix, 2 µL of cDNA, 0.5 µL of forward and reverse primers (Supplementary Table 1), and 2 µL double distilled water (ddH2O). AhACT11 was used as a reference gene. The results were calculated by using the 2−ΔΔCt method.
Results
Identification and characterization of AhNRAMPs
In this research, a total of 29 AhNRAMP genes were identified and renamed as AhNRAMP1-AhNRAMP29 according to gene position on chromosomes. The CDS of AhNRAMP genes ranged in length from 216 bp (AhNRAMP13) to 4,044 bp (AhNRAMP25), encode deduced proteins varied from 71 (AhNRAMP13) to 1347 (AhNRAMP25) amino acids, and molecular weights between 7.88 (AhNRAMP13) and 146.97 (AhNRAMP25) KDa. Theoretical isoelectric point (pI) varied from 4.92 (AhNRAMP1 and AhNRAMP17) to 9.86 (AhNRAMP11). The putative transmembrane domains (TMDs) of AhNRAMPs varied from 0 to 14 (Table 1).
To investigate the evolutionary relationships, 6 AtNRAMP proteins from Arabidopsis thaliana, 7 OsNRAMP proteins from Oryza sativa, and 29 AhNRAMP proteins from A. hypogaea were constructed phylogenetic tree. Unlike AtNRAMPs and OsNRAMPs divided into two groups, 29 AhNRAMP proteins were divided into three groups (Fig. 1). 13 AhNRAMPs belonged to G1, (AhNRAMP3, AhNRAMP5, AhNRAMP18, AhNRAMP19, AhNRAMP22, AhNRAMP23 and AhNRAMP29 with high levels of sequence similarity to OsNRAMP3,AhNRAMP10 and AhNRAMP26 are remarkably similar to AtNRAMP1 and AtNRAMP6. AhNRAMP6, AhNRAMP12, AhNRAMP13 and AhNRAMP20 are strikingly similar to OsNRAMP1, OsNRAMP4, OsNRAMP5 and OsNRAMP6. Group2 contain 11 members. AhNRAMP1, AhNRAMP4, AhNRAMP14, AhNRAMP17 similar to AtNRAMP3 and AtNRAMP4 AhNRAMP7 and AhNRAMP21 similar to AtNRAMP2, OsNRAMP2 and OsNRAMP7, AhNRAMP2, AhNRAMP11, AhNRAMP15, AhNRAMP27 and AhNRAMP28 similar to AtNRAMP5. Group3 contains 5 members, AhNRAMP8, AhNRAMP9, AhNRAMP16, AhNRAMP24, AhNRAMP25, without homologous gene in Arabidopsis thaliana and Oryza sativa. Additionally, it was predicted that AhNRAMPs of G1 and G3 would locate on the plasma membrane and most AhNRAMPs (except AhNRAMP11 and AhNRAMP27 on the plasma membrane) of G2 on the vacuolar (Table 1). These results indicate that the conserved sequences in these genes share similar evolutionary relationships during evolution between Arabidopsis, rice and peanut, but several variations have also occurred in AhNRAMP, enabling the division of 5 AhNRAMPs into G3.
Gene structure and conserved motifs of AhNRAMPs
The result of gene structure showed AhNRAMP16 had the largest length (10.8 kb), whereas AhNRAMP13 had the minimum length (437 bp) (Tables 1, Fig. 2). The exon numbers of G1 members varied from 3–13 and nearly half of the members contain more than 10 exons. The exon numbers of G2 members varied from 3–9, and more than half members possess 4 exons. The exon numbers of G3 members are 7 (AhNRAMP8, AhNRAMP9, AhNRAMP24, AhNRAMP25) and 9 (AhNRAMP16) (Fig. 2). The most average exon numbers are G1 members (9.69), next are G3 members (7.40) and least are G2 members (4.64), indicating 29 AhNRAMP genes are highly divergent (Fig. 2). Fourteen AhNRAMP genes have a complete UTR region, five AhNRAMPs in G1, five AhNRAMPs in G2, four AhNRAMPs in G3, complete UTR regions of the genes are more highly conserved within a single subgroup. All three AhNRAMP genes in G2 have an uncomplete UTR region, AhNRAMP1 and AhNRAMP15 contain 5′-UTR, AhNRAMP27 contains 3′-UTR, showing that there are more mutations and variations occurred in G2 members during the evolutionary process (Fig. 2). The members in the same branch have a similar characteristic, which suggests a conserved relationship in group of AhNRAMPs.
To clear and definite the sequence features of the AhNRAMPs, conserved motifs were obtained by using MEME. Ten conserved motifs were detected in 29 AhNRAMP proteins and among groups detected major differences (Fig. 2) Most of the AhNRAMP proteins (23, 79%) contained motif 7, which is a characteristic NRAMP domain (GQSSTITGTY)(Fig. 2). Widespread variation was detected in the motif pattern of G1, for instance, four AhNRAMPs contain only one motif, AhNRAMP11 (motif 3), AhNRAMP13 (motif 6), AhNRAMP19 (motif 5), and AhNRAMP29 (motif 9). Motif pattern in six G1 members were motifs 1, 2, 5, 9, 6, 7, 3, 4 and 10, AhNRAMP20 and AhNRAMP26 were no motif 9, AhNRAMP5 contained two motif 5 and 10, AhNRAMP10 contained two motif 9 and no motif 1. Motif in G3 members showed two patterns: AhNRAMP16 was motif 7 and 3, others detected same motif pattern: motif 1, 2, 5, 6, 3, 4 and motif 7 at the end. The motifs of each branch exhibited similar characteristics but varied significantly between the different groups.
Chromosome location and duplication analyses of AhNRAMPs
Twenty-nine AhNRAMP genes were not evenly distributed on the 16 chromosomes in the peanut, except Chr03, Chr06, Chr14 and Chr16 (Fig. 3). The most number (4) of AhNRAMP genes were located on Chr08, Chr17 and Chr18, next (3) were distributed in Chr10 and Chr19, and other chromosomes had only one AhNRAMP.
To illuminate the evolutionary relationships of the AhNRAMPs, gene duplications were researched (Fig. 4). Twenty segmental duplication gene pairs and two tandem duplication gene pairs had been found (Fig. 4). Tandem duplication gene pairs were AhNRAMPs in G3, AhNRAMP8 and AhNRAMP9 in Chr08, AhNRAMP24 and AhNRAMP25 in Chr18 are distributed near the ends of chromosomes (Fig. 4). Ten segmental duplication gene pairs were AhNRAMPs in G1, six in G2, and four gene pairs (AhNRAMP1 and AhNRAMP17, AhNRAMP4 and AhNRAMP17, AhNRAMP7 and AhNRAMP17, AhNRAMP14 and AhNRAMP17), AhNRAMP17 is G1 member located in Chr15, the others are G2 member located in Chr01, Chr05, Chr08, Chr11, respectively (Fig. 4). The analysis of chromosome distribution showed that gene duplication mode was mainly spread on the Chr08. The Ka/Ks ratio for segmental duplication ranged from 0.04 to1.07 with an average of 0.21, while the ratios of tandem duplication were 0.29 and 0.32, indicating purifying selection played a vital role in the evolutionary process of AhNRAMP gene family. Only the Ka/Ks of one gene pair (AhNRAMP6 and AhNRAMP12) was 1.07 and showed positive selection. The segmental duplications may occur in 1.09–117.18 Mya, and tandem duplication gene pairs 5.54–5.69 Mya (Supplementary Table 2).
Cis-elements analysis in promoter of AhNRAMPs
To further study the potential function and responding mechanism of AhNRAMPs, cis-elements in the promoter of AhNRAMPs were analysed. The results showed 345 cis-elements could divided into three groups, abiotic stress-responsive elements (129), hormone-responsive elements (168), and growth and development-responsive elements (48). There were four kinds of abiotic stress-responsive elements, cis-acting regulatory element essential for the anaerobic/anoxic induction, defense and stress, drought, and low temperature. These abiotic stress-responsive elements consist of 64 ARE (50%), 3 GC-motif (2%), 23 TC-rich repeats (18%), 20 MBS (16%), and 19 L (15%) (Fig. 5). The most abundant abiotic stress-responsive elements were 12 in the promoter of AhNRAMP11, including 8 ARE, 1 GC-motif and 3 L, next were 9 in the promoters of AhNRAMP15 (8 ARE, 1 TC-rich repeats) and AhNRAMP2 (4 ARE, 1 MBS, 1 L, 3 TC-rich repeats), suggesting AhNRAMP11 and AhNRAMP15 may play important functions in anaerobic stress, AhNRAMP2 have a potential role in defense and stress.
Five hormone-responsive elements, methyl jasmonate (MeJA) responsive elements were CGTCA-motif/TGACG-motif (38%), abscisic acid (ABA) responsive element was ABRE (27%), gibberellin (GA) responsive elements were P-box/TATC-box/GARE-motif (13%), salicylic acid (SA) responsive element was TCA element (13%), and auxin-responsive elements were AuxRR-core/TGA-element (8%). The largest number of hormone-responsive elements was 11 in the promoter of AhNRAMP11, 5 ABRE, 2 CGTCA-motif, 1 GARE-motif, 1 TCA-element and 2 TGACG-motif (Fig. 5), showed AhNRAMP11 may be involved in many hormones regulation.
Five growth and development elements, the circadian control element was circadian (29%), meristem expression element was CAT-box (29%), zein metabolism element was O2-site (27%), endosperm expression elements were GCN4_motif/AACA_motif (10%), and seed-specific regulation element was RY-element (4%) (Fig. 5). The greatest number of growth and development element was 4 in the promoter of AhNRAMP1, obtained 1 AACA_motif, 1 GCN4_motif and 2 O2-site, showed AhNRAMP1 maybe involved in endosperm development and zein metabolism.
miRNAs and transcription factor targeting AhNRAMPs
To understand the miRNA and transcription factors (TFs) regulation of AhNRAMPs, we identified six miRNAs targeting 11 AhNRAMPs and 271 TFs targeting 20 AhNRAMPs (Supplementary Tables 3 and 4). Ahy-miR156a, ahy-miR156b-5p and ahy-miR156c targeted 4 AhNRAMP genes (AhNRAMP8, AhNRAMP9, AhNRAMP24 and AhNRAMP25). Ahy-miR3509-3p targeted 2 AhNRAMP genes (AhNRAMP6 and AhNRAMP20). Ahy-miR3512 targeted 2 AhNRAMP genes (AhNRAMP10 and AhNRAMP26). Ahy-miR3512 targeted 3 AhNRAMP genes (AhNRAMP3, AhNRAMP18 and AhNRAMP19). 271 TFs belong to 17 TFs families, AP2, BBR-BPC, E2F/DP, etc. The largest quantity of TFs family was ERF (143). The minimum number of TFs families were E2F/DP (1), EIL (1), G2-like (1). Notably, AhNRAMP27 was abundantly targeted by 76 TFs, including 53 MYBs, 8 C2H2s, 6 MYBs, 3 GATAs, 3 LBDs, 2 BBR-BPCs and 1 Nin-like.
Expression of AhNRAMPs in tissues
The analysis of expressions of 29 AhNRAMPs in peanut tissues revealed AhNRAMPs tissue-specifically expressed in peanut (Fig. 6). Tissue-specific expression analysis by calculating gene TPM values reveals that thirteen AhNRAMPs (ten G1 members and three G2 members), AhNRAMP2, AhNRAMP3, AhNRAMP5, AhNRAMP11, AhNRAMP12, AhNRAMP13, AhNRAMP15, AhNRAMP18, AhNRAMP19, AhNRAMP22, AhNRAMP23, AhNRAMP28 and AhNRAMP29 showed a low expression or were not express in most tissues. G1 members, AhNRAMP6 and AhNRAMP20 were little expressed in all tissues, highest expression in seed Pat.10. AhNRAMP10 and AhNRAMP26 were expressed in all tissues, highest expression in shoot, root and nodule. G2 members, AhNRAMP1 and AhNRAMP14, high expressed in almost all tissues, and highest expressed in nodule. AhNRAMP4 was expressed in all tissues. AhNRAMP17, and AhNRAMP27 were expressed in all tissues, highest expression in nodule, root and nodule, respectively. AhNRAMP7 and AhNRAMP21 were weak expressed in all tissues. G3 members, AhNRAMP16 was weak expressed in all tissues, AhNRAMP8, AhNRAMP9, AhNRAMP24 and AhNRAMP25 were similar expression patterns and represented intermediate levels of gene expression,but all G3 members were highly expressed in the shoot (Fig. 6). Tissue-specific expression of each branch exhibited similar trend but differed significantly between the different groups.
Response of AhNRAMPs to cd stress
To further explore the response of AhNRAMPs to Cd, the expression of AhNRAMPs in roots under Cd stress was analyzed. The expression patterns of AhNRAMPs differed between Cd-enriched variety and Cd-sensitive variety in response to Cd stress (Fig. 7). In comparison with the control, AhNRAMP2 and AhNRAMP11 exhibited a decreased pattern of Cd-sensitive variety and the increased trend peaked 6.9-fold and 14.1-fold at 3 h of Cd-enriched variety exposed to Cd. The expression of AhNRAMP3, AhNRAMP19, AhNRAMP27, AhNRAMP28 and AhNRAMP29 were upregulated 2.1-2.6-fold at 24 h under Cd stress of Cd-enriched variety. The expression of AhNRAMP4 was increased 6.9-fold and peaked 12.5-fold at 12 h in Cd sensitive variety, and increased 3.2-fold at 24 h of Cd-enriched variety under Cd stress. AhNRAMP5 and AhNRAMP6 in Cd sensitive variety were inhibited under Cd stress, while in Cd-enriched variety, they were not significantly changed. The expression of AhNRAMP7 was downregulated 24% at 1 h of Cd-enriched variety under Cd stress, but increased 2.1-fold at 24 h Cd treatment in Cd sensitive variety. The expression of AhNRAMP8, AhNRAMP10, AhNRAMP12, AhNRAMP21, AhNRAMP22, AhNRAMP24 and AhNRAMP25 were decreased in Cd sensitive variety and not significantly regulated in Cd-enriched variety under Cd stress. The expression of AhNRAMP1, AhNRAMP9, AhNRAMP13, AhNRAMP14, AhNRAMP15 and AhNRAMP18 were induced in two varieties under Cd stress. The expression of AhNRAMP16 and AhNRAMP17 were peaked at 24 h in Cd-enriched variety under Cd treatment. AhNRAMP19 was increased in Cd-enriched variety under Cd stress while reduced in Cd-sensitive variety. The expression of AhNRAMP20, AhNRAMP23 and AhNRAMP26 were downregulated in two varieties under Cd stress (Fig. 7). The highest level of expression was AhNRAMP11 showed 14.1-fold at 3 h Cd treatment in Cd-enriched variety. The lowest expression was AhNRAMP6 showed downregulated 6% at 72 h of Cd-sensitive variety under Cd stress. Half of the AhNRAMP genes were upregulated under Cd stress, which suggests that they could play vital roles in response to Cd.
Discussion
Characterization and evolution of AhNRAMPs
In this study, 29 AhNRAMPs were identified in peanut, which is higher than the previously studies. There are six NRAMPs in Arabidopsis thaliana (Mäser et al. 2001), seven NRAMPs in Oryza sativa (Chang et al. 2020), seven NRAMPs in Phaseolus vulgaris (Ishida et al. 2018), five NRAMPs in Theobroma cacao (Ullah et al. 2018), 13 NRAMPs in Glycine Max (Qin et al. 2017), three NRAMPs in Spirodela polyrhiza (Chen et al. 2021), 11 NRAMPs in Camellia sinensis (Li et al. 2021), 13 NRAMPs in Brassica napus (Zhang et al. 2018), 15 NRAMPs in peanut (Tan et al. 2023) and 11 NRAMPs in Populus trichocarpa (Ma et al. 2023). Based on the first and second versions of the A.hypogaea genome released on October 4, 2022, this study identified 29 AhNRAMPs, 13 AhNRAMPs in A subgenome, 16 genes in the B subgenome (Fig. 3). Previous studies mainly analyzed the functions of AhNRAMP in the Fe deficiency and Fe/Cd interactions of peanut (Xiong et al. 2012; Chen et al. 2019; Tan et al. 2023). However, this study focused on expressions of AhNRAMP genes in response to Cd in peanut.
Based on phylogenetic analysis, all 29 AhNRAMP members were divided into three groups, not similar to the two groups in Arabidopsis and rice (Fig. 1), but was consistent with a recent assessment that seven P. vulgaris NRAMP proteins and 11 PtNRAMPs into three subgroups (Ishida et al. 2018; Ma et al. 2023). Gene structure and conserved motif analysis showed that the members of same group usually shared similar exon-intron structures and motifs (Fig. 2), indicating that they have similar evolutionary relationships. The members of G3 were located in the phylogenetic tree root, and most of them had fewer motif and a long CDS region, and the results are similar to previous studies (Chen et al. 2021). The G2 included fewer exon-intron and similar motif features that are greater than those of G1 and G3. G1 had more exons, variations in motif number and similar motif arrangements (Fig. 2). There are many mutations that occurred in NRAMP conserved motif in G1 and G3 members. The NRAMP conserved motif of AhNRAMP16 is GQSSTITR, AhNRAMP8 and AhNRAMP9 are GQPPGFHN, AhNRAMP24 and AhNRAMP25 are GQYVAGFV and GQYIAGFV, respectively. AhNRAMP13, AhNRAMP18, AhNRAMP19, AhNRAMP23, AhNRAMP29 in G1 deleted NRAMP conserved motif, NRAMP conserved motif of AhNRAMP6, AhNRAMP10, AhNRAMP12, AhNRAMP20 in G1 are GQSSAITG, GQTITP, GQSSAITG and GQSSAITG, respectively. These mutations indicated AhNRAMPs may evolved new functions.The NRAMP proteins contained 10–12 TMDs and about 500 AA in plant (Mani and Sankaranarayanan 2018, 2022). Similar to other NRAMPs, In this study 16 AhNRAMP proteins contained 426–638 AA and 18 AhNRAMP proteins contained 9-14 TMDs. However, four AhNRAMP proteins in G3 contained 1341–1347 AA. three AhNRAMP proteins (AhNRAMP11, AhNRAMP16 and AhNRAMP19) did not contain TMDs. AhNRAMP13 contained one TMDs, AhNRAMP28 and AhNRAMP29 contained two TMDs, AhNRAMP27 contained 3 TMDs, AhNRAMP3, AhNRAMP12, AhNRAMP18 contained 5 TMDs, AhNRAMP23 contained 7 TMDs. The result was similar to BnNRAMPs and CsNRAMPs, Which contained 100–200 AA and 3–6 TMDs, (Zhang et al. 2018; Li et al. 2021). This may attribute to a broken and deletion NRAMP domain among species, and the effect of mutations and deletion in these AhNRAMPs needs further study.
In plants, G1 members are closer to bacteria NRAMP and G2 members are more similar to animal NRAMP (Sebastien and Schroeder 2004). The G1 proteins were a basic IP, while G2 proteins showed an acidic IP (Ishida et al. 2018). In this study, most G1 proteins (11, 85%) showed a basic IP and most G2 proteins (9, 82%) showed an acidic IP. Two G1 members (AhNRAMP18 and AhNRAMP19) showed an acidic IP and two G2 (AhNRAMP11 and AhNRAMP27) proteins showed a basic IP. The same phenomenon is also found in the organelle localization of NRAMP proteins. Most of G2 members are located on the vacuolar, except for AhNRAMP11 and AhNRAMP27, which are present on the plasma membrane. Four G3 members (AhNRAMP8, AhNRAMP9, AhNRAMP24 and AhNRAMP25) showed an acidic IP, similar to G2, and one group 3 protein (AhNRAMP16) showed a basic IP, similar to G1. The IP of these AhNRAMPs differ from other NRAMPs, suggesting AhNRAMPs may evolve a series of new functions.
Interestingly, 22 of the 29 AhNRAMP proteins further clustered into eleven branches of paired proteins (Fig. 1). In this study, 20 segmental and 2 tandem duplication pairs were present in AhNRAMPs (Fig. 4). Tandem duplication gene pairs were found in G3 members of Chr.08 and Chr.18 (Fig. 4). Previous studies indicated the presence of six duplicated pairs in GmNRAMPs, one tandem duplicated pair and one segmental duplication pair in TcNRAMPs, one syntenic block in OsNRAMPs, two segmental duplication pairs in AtNRAMPs, seven whole-genome duplication pairs and one segmental duplicated pair in AhNRAMPs (Mäser et al. 2001; Chang et al. 2020; Qin et al. 2017; Ullah et al. 2018; Tan et al. 2023). Ten segmental duplication gene pairs were AhNRAMPs in G1, six in G2 and four gene pairs, one in G1 the others in G2 (Fig. 4). Eleven homologous pairs of AhNRAMPs are the products of two tandem duplication events and nine segmental duplication events in peanut evolutionary history.
Role of NRAMPs in development tissues
GmNRAMP7 from G1 was the highest expressed GmNRAMP, GmNRAMPs from G2 highly expressed in all tissues (Qin et al. 2017). BnNRAMP1b is highly expressed in all tissues and is a constitutively expressed gene (Meng et al. 2017). SaNRAMP1 is strongly expressed in the young shoots (Zhang et al. 2020b). G1 CsNRAMPs were mainly expressed in the root and G2 genes in the stems and leaves (Li et al. 2021). In this study, G1 genes, AhNRAMP6 and AhNRAMP20 were extraordinarily expressed in seed Pat.10, AhNRAMP10 and AhNRAMP26 were highly expressed in roots, and AhNRAMP17 was the most highly expressed in nodules, AhNRAMP22 was expressed in peg tip Pat, while G2 genes highly expressed in the shoots and leaves, G3 genes were extraordinarily expressed in shoots (Fig. 6).
AhNRAMP1, AhNRAMP4, AhNRAMP8, AhNRAMP9, AhNRAMP14, AhNRAMP24 and AhNRAMP25 universally expressed in all tissues (Fig. 6), and in the promoter region of AhNRAMP1 and AhNRAMP14, there were cis-acting regulatory elements involved in endosperm expression and zein metabolism (Fig. 5), AhNRAMP4 had one cis-element related to meristem expression, AhNRAMP8 had one cis-element involved in zein metabolism regulation, AhNRAMP9 had cis-elements involved in zein metabolism regulation, circadian control and seed-specific regulation, AhNRAMP24 had cis-elements involved in zein metabolism regulation and circadian control regulation, AhNRAMP25 had cis-elements involved in zein metabolism regulation and seed-specific regulation. The constitutive expression in all tissues suggested that these genes may be necessary to maintain the basic life in peanut.
NRAMP genes involved in cd stress
AtNRAMP1, AtNRAMP6, OsNRAMP1 and OsNRAMP5 displayed Cd transport ability (Cailliatte et al. 2009, 2010; Sasaki et al. 2012; Chang et al. 2020). Six GmNRAMPs were strongly induced expressed exposure to excess Cd, while three GmNRAMPs were inhibited (Qin et al. 2017). BnNRAMP1b showed greatly enhanced expression by Cd (Meng et al. 2017). BnNRAMP2;1 and BnNRAMP4;2 showed increased expression under Cd stress (Wang et al. 2019b). All the SpNRAMP genes were negatively regulated under 50 µM Cd2+ treatments (Chen et al. 2021). PcNRAMP1 was increased expressed in roots under Cd stress (Yu et al. 2022). Under Cd stress, almost all the PtNRAMP genes were increased in roots (Ma et al. 2023). Most AhNRAMPs changed expression responses to Cd (Tan et al. 2023). In peanut AhNRAMP1 was obviously induced by Fe deprivation (Xiong et al. 2012). NRAMP3 and NRAMP5 are involved in Cd uptake and translocation in peanut plants under Fe deficiency (Chen et al. 2019).
In this study, in Cd-enriched variety there were 55% (16) AhNRAMPs upregulated and 14% (4) AhNRAMPs downregulated exposed to Cd, 28% (8) AhNRAMPs induced and 28% (8) AhNRAMPs reduced in Cd sensitive variety, 24% (7) AhNRAMPs increased and 10% (3) AhNRAMPs decreased in two varieties. AhNRAMP10 and AhNRAMP26 (ortholog of AtNRAMP1) were downregulated in two varieties under Cd stress (Fig. 7). Notably, the relative expression of AhNRAMP2 and AhNRAMP11 (G2 members, ortholog of AtNRAMP5) showed contrasting expression pattern exposed to Cd in two varieties. The two genes were upregulated 6.9-fold and 14.1-fold at 3 h by Cd in roots of Cd-enriched variety, while in roots of Cd-sensitive variety downregulated. These findings suggested that AhNRAMP2 and AhNRAMP11 might be the candidate genes for Cd regulation in peanut.
Conclusion
All in all, 29 AhNRAMPs were identified in peanut, designated as AhNRAMP1 to AhNRAMP29 according to their positions on chromosomes and classified into three groups Furthermore, 55% AhNRAMPs indicated a remarkably upregulated expression in Cd-enriched variety exposed to Cd. The expression of AhNRAMP2 and AhNRAMP11 were peaked at 3 h by Cd in roots of cCd-enriched variety, while decreasing at the same time in Cd sensitive variety. Our findings provide two important candidates AhNRAMPs, for further study in the capacity of Cd enrichment in peanut.
References
Akindele EO, Omisakin DA, Oni OA, Aliu OO, Omoniyi GE, Akinpelu OT (2020) Heavy metal toxicity in the water column and benthic sediments of a degraded tropical stream. Ecotoxicol Environ Saf 190:110153. https://doi.org/10.1016/j.ecoenv.2019.110153
Bressler JP, Olivi L, Cheong JH, Kim Y, Bannona D (2004) Divalent metal transporter 1 in lead and cadmium transport. Ann NY Acad Sci 1012:142–152. https://doi.org/10.1196/annals.1306.011
Cailliatte R, Lapeyre B, Briat JF, Mari S, Curie C (2009) The NRAMP6 metal transporter contributes to cadmium toxicity. Biochem J 422:217–228. https://doi.org/10.1042/BJ20090655
Cailliatte R, Schikora A, Briat JF, Mari S, Curie C (2010) High-affinity manganese uptake by the metal transporter NRAMP1 is essential for arabidopsis growth in low manganese conditions. Plant Cell 22:904–917. https://doi.org/10.1105/tpc.109.073023
Chang JD, Huang S, Yamaji N, Zhang W, Ma JF, Zhao FJ (2020) OsNRAMP1 transporter contributes to cadmium and manganese uptake in rice. Plant Cell Environ 43:2476–2491. https://doi.org/10.1111/pce.13843
Chen C, Cao Q, Jiang Q, Li J, Yu R, Shi G (2019) Comparative transcriptome analysis reveals gene network regulating cadmium uptake and translocation in peanut roots under iron deficiency. BMC Plant Biol 19:35. https://doi.org/10.1186/s12870-019-1654-9
Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R (2020) TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant 13:1194–1202. https://doi.org/10.1016/j.molp.2020.06.009
Chen Y, Zhao X, Li G, Kumar S, Sun Z, Li Y, Guo W, Yang J, Hou H (2021) Genome-wide identification of the Nramp gene family in Spirodela polyrhiza and expression analysis under cadmium stress. Int J Mol Sci 22:6414. https://doi.org/10.3390/ijms22126414
Chen L, Yang W, Yang Y, Tu P, Hu S (2022) Continuous rotation of three large biomass crops with high bioconcentration factor of cadmium can effectively repair contaminated farmlands. PREPRINT (Version 1) Available at research square https://doi.org/10.21203/rs.3.rs-1154155/v1
Clevenger J, Chu Y, Scheffler B, Ozias-Akins P (2016) A developmental transcriptome map for allotetraploid Arachis hypogaea. Front Plant Sci 7:1446. https://doi.org/10.3389/fpls.2016.01446
Dai X, Zhuang Z, Zhao PX (2018) psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res 46:W49–W54. https://doi.org/10.1093/nar/gky316
Gu Q, Yu T, Yang Z, Ji J, Hou Q, Wang L, Wei X, Zhang Q (2019) Prediction and risk assess⁃ment of five heavy metals in maize and peanut: a case study of Guangxi, China. Environ Toxicol Pharmacol 70:103199. https://doi.org/10.1016/j.etap.2019.103199
He Z, Zhang H, Gao S, Lercher MJ, Chen WH, Hu S (2016) Evolview v2: an online visualization and management tool for customized and annotated phylogenetic trees. Nucleic Acids Res 44:236–241. https://doi.org/10.1093/nar/gkw370
Ishida JK, Caldas DGG, Oliveira LR, Frederici GC, Leite LMP, Mui TS (2018) Genome-wide characterization of the NRAMP gene family in Phaseolus vulgaris provides insights into functional implications during common bean development. Genet Mol Biol 41:820–833. https://doi.org/10.1590/1678-4685-GMB-2017-0272
Ishikawa S, Ishimaru Y, Igura M, Kuramata M, Abe T, Senoura T, Hase Y, Arao T, Nishizawa NK, Nakanishi H (2012) Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice. Proc Natl Acad Sci USA 109:19166–19171. https://doi.org/10.1073/pnas.1806782115
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096
Kumar A, Subrahmanyam G, Mondal R, Cabral-Pinto MMS, Shabnam AA, Jigyasu DK, Malyan SK, Fagodiya RK, Khan SA, Kumar A, Yu ZG (2021) Bio-remediation approaches for alleviation of cadmium contamination in natural resources. Chemosphere 268:128855. https://doi.org/10.1016/j.chemosphere
Li J, Duan Y, Han Z, Shang X, Zhang K, Zou Z, Ma Y, Li F, Fang W, Zhu X (2021) Genome-wide identification and expression analysis of the NRAMP family genes in tea plant (Camellia sinensis). Plants (Basel) 10:1055. https://doi.org/10.3390/plants10061055
Ma X, Yang H, Bu Y, Zhang Y, Sun N, Wu X, Jing Y (2023) Genome-wide identification of the NRAMP gene family in Populus trichocarpa and their function as heavy metal transporters. Ecotoxicol Environ Saf 261:115110. https://doi.org/10.1016/j.ecoenv.2023.115110
Mani A, Sankaranarayanan K (2018) In silico analysis of natural resistance-associated macrophage protein (NRAMP) family of transporters in rice. Protein J 37:237–247. https://doi.org/10.1007/s10930-018-9773-y
Mani A, Sankaranarayanan K (2022) Natural resistance-associated macrophage proteins (NRAMPs): functional significance of metal transport in plants. In: Kumar K, Srivastava S (eds) Plant metal and metalloid transporters. Springer, Berlin. https://doi.org/10.1007/978-981-19-6103-8_5
Mäser P, Thomine S, Schroeder JI, Ward JM, Hirschi K, Sze H, Talke IN, Amtmann A, Maathuis FJ, Sanders D, Harper JF, Tchieu J, Gribskov M, Persans MW, Salt DE, Kim SA, Guerinot ML (2001) Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiol 126:1646–1667. https://doi.org/10.1104/pp.126.4.1646
Meng JG, Zhang XD, Tan SK, Zhao KX, Yang ZM (2017) Genome-wide identification of Cd-responsive NRAMP transporter genes and analyzing expression of NRAMP 1 mediated by miR167 in Brassica napus. BioMetals 30: 917–931. https://doi.org/10.1007/s10534-017-0057-3
Ministry of Ecology and Environment of the People’s Republic of China, Ministry of Natural Rsources of the People’s Republic of China (2014) National soil pollution survey bulletin. Environ Educ 6:8–10
Mizuno T, Usui K, Horie K, Nosaka S, Mizuno N and Obata H (2005) Cloning ofthree ZIP/Nramp transporter genes from a Ni hyperaccumulator plant Thlaspi japonicum and their Ni2+-transport abilities. Plant Physiol Biochem 43:793–801. https://doi.org/10.1016/j.plaphy.2005.07.006
Oomen RJ, Wu J, Lelièvre F, Blanchet S, Richaud P, Barbier-Brygoo H, Aarts MG, Thomine S (2009) Functional characterization ofNRAMP3 and NRAMP4 from the metal hyperaccumulator Thlaspi caerulescens. New Phytol 181:637–650. https://doi.org/10.1111/j.1469-8137.2008.02694.x
Qin L, Han P, Chen L, Walk TC, Li Y, Hu X, Xie L, Liao H, Liao X (2017) Genome-wide identification and expression analysis of NRAMP Family genes in soybean (Glycine Max L). Front Plant Sci 8:1436. https://doi.org/10.3389/fpls.2017.01436
Rahmatizadeh R, Jamei R, Arvin MJ, Rezanejad F (2021) Upregulation of LeNRAMP3 and LeFER genes in Solanum lycopersicum confers its cadmium tolerance. Russ J Plant Physiol 68(Suppl 1):S92–S102. https://doi.org/10.1134/S1021443721070104
Sasaki A, Yamaji N, Yokosho K, Ma JF (2012) Nramp5 is a major transporter responsible for manganese and cadmium uptake in rice. Plant Cell 24:2155–2167. https://doi.org/10.1105/tpc.112.096925
Sebastien T, Schroeder JI (2004) Plant metal transporters with homology to proteins of the NRAMP family. In: Cellier M, Gros P (eds) The NRAMP family. Kluwer Academic Publishers-Plenum Publishers, New York, pp 113–123
Takahashi R, Ishimaru Y, Senoura T, Shimo H, Ishikawa S, Arao T, Nakanishi H, Nishizawa NK (2011) The OsNRAMP1 iron transporter is involved in cd accumulation in rice. J Exp Bot 62:4843–4850. https://doi.org/10.1093/jxb/err136
Tan Z, Li J, Guan J, Wang C, Zhang Z, Shi G (2023) Genome-wide identification and expression analysis reveals roles of the NRAMP gene family in iron/cadmium interactions in peanut. Int J Mol Sci 24:1713. https://doi.org/10.3390/ijms24021713
Thomine S, Wang R, Ward JM, Crawford NM, Schroeder JI (2000) Cadmium and iron transport by members of a plant metal transporter family in arabidopsis with homology to Nramp genes. Proc Natl Acad Sci USA 97:4991–4996. https://doi.org/10.1073/pnas.97.9.4991
Tian F, Yang DC, Meng YQ, Jin J, Gao G (2020) PlantRegMap: charting functional regulatory maps in plants. Nucleic Acids Res 48:1104–1113. https://doi.org/10.1093/nar/gkz1020
Ullah I, Wang Y, Eide DJ, Dunwell JM (2018) Evolution, and functional analysis of natural resistance-associated macrophage proteins (NRAMPs) from Theobroma cacao and their role in cadmium accumulation. Sci Rep 8:14412. https://doi.org/10.1038/s41598-018-32819-y
Wang K, Wang F, Song N, Liu J, Zhang T, Wang M, Wang Y (2018) Contribution of root uptake to cadmium accumulation in two peanut cultivars: evidence from a split-column soil experiment. Environ Sci Pollut Res 25:15036–15043. https://doi.org/10.1007/s11356-018-1719-6
Wang C, Chen X, Yao Q, Long D, Fan X, Kang H, Zeng J, Sha L, Zhang H, Zhou Y, Wang Y (2019a) Overexpression of TtNRAMP6 enhances the accumulation of cd in Arabidopsis. Gene 696:225–232. https://doi.org/10.1016/j.gene.2019.02.008
Wang S, Sun J, Li S, Lu K, Meng H, Xiao Z, Zhang Z, Li J, Luo F, Li N (2019b) Physiological, genomic and transcriptomic comparison of two Brassica napus cultivars with contrasting cadmium tolerance. Plant Soil 441:71–87. https://doi.org/10.1007/s11104-019-04083-0
Wu D, Yamaji N, Yamane M, Kashino-Fujii M, Sato K, Feng Ma J (2016) The HvNramp5 transporter mediates uptake of cadmium and manganese, but not iron. Plant Physiol 172:1899–1910. https://doi.org/10.1104/pp.16.01189
Xiong H, Kobayashi T, Kakei Y, Senoura T, Nakazono M, Takahashi H, Nakanishi H, Shen H, Duan P, Guo X, Nishizawa NK, Zuo Y (2012) AhNRAMP1 iron transporter is involved in iron acquisition in peanut. J Exp Bot 63:4437–4446. https://doi.org/10.1093/jxb/ers117
Yu W, Deng S, Chen X, Cheng Y, Li Z, Wu J, Zhu D, Zhou J, Cao Y, Fayyaz P, Shi W, Luo Z (2022) PcNRAMP1 enhances cadmium uptake and accumulation in Populus × Canescens. Int J Mol Sci 23:7593. https://doi.org/10.3390/ijms23147593
Zhang XD, Meng JG, Zhao KX, Chen X, Yang ZM (2018) Annotation and characterization of Cd-responsive metal transporter genes in rapeseed (Brassica napus). Biometals 31:107–121. https://doi.org/10.1007/s10534-017-0072-4
Zhang W, Yue S, Song J, Xun M, Han M, Yang H (2020a) MhNRAMP1 from Malus hupehensis exacerbates cell death by accelerating cd uptake in tobacco and apple calli. Front Plant Sci 11:957. https://doi.org/10.3389/fpls.2020.00957
Zhang J, Zhang M, Song H, Zhao J, Shabala S, Tian S, Yang X (2020b) A novel plasma membrane-based NRAMP transporter contributes to cd and zn hyperaccumulation in Sedum Alfredii hance. Environ Exp Bot 176:104121. https://doi.org/10.1016/j.envexpbot.2020.104121
Zhu H, Chen C, Xu C, Zhu Q, Huang D (2016) Effects of soil acidification and liming on the phytoavailability of cadmium in paddy soils of central subtropical China. Environ Pollut 219:99–106. https://doi.org/10.1016/j.envpol.2016.10.043
Funding
This work was supported by agricultural collaborative innovation project of Jiangxi province, China (JXXTCXBSJJ2022007, JXXTCX202112), and the third census and collection of crop germplasm resources in China.
Author information
Authors and Affiliations
Contributions
LY performed the bioinformatic analysis and drafted the manuscript. HJ sampled the plant materials and carried out the experiments. AR and YH involved in handling figures and tables. AR, LY, DG and XZ revised the manuscript. All authors read and approved the final manuscript.
Corresponding authors
Ethics declarations
Competing interest
The authors have no relevant financial or non-financial interests to disclose.
Additional information
Communicated by Mohsin Tanveer.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Yan, L., Jin, H., Raza, A. et al. Natural resistance-associated macrophage proteins (NRAMPs) are involved in cadmium enrichment in peanut (Arachis hypogaea L.) under cadmium stress. Plant Growth Regul 102, 619–632 (2024). https://doi.org/10.1007/s10725-023-01091-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10725-023-01091-0