Systematic Analysis of NRAMP Family Genes in Areca catechu and Its Response to Zn/Fe Deficiency Stress

Areca catechu is a commercially important medicinal plant widely cultivated in tropical regions. The natural resistance-associated macrophage protein (NRAMP) is widespread in plants and plays critical roles in transporting metal ions, plant growth, and development. However, the information on NRAMPs in A. catechu is quite limited. In this study, we identified 12 NRAMPs genes in the areca genome, which were classified into five groups by phylogenetic analysis. Subcellular localization analysis reveals that, except for NRAMP2, NRAMP3, and NRAMP11, which are localized in chloroplasts, all other NRAMPs are localized on the plasma membrane. Genomic distribution analysis shows that 12 NRAMPs genes are unevenly spread on seven chromosomes. Sequence analysis shows that motif 1 and motif 6 are highly conserved motifs in 12 NRAMPs. Synteny analysis provided deep insight into the evolutionary characteristics of AcNRAMP genes. Among the A. catechu and the other three representative species, we identified a total of 19 syntenic gene pairs. Analysis of Ka/Ks values indicates that AcNRAMP genes are subjected to purifying selection in the evolutionary process. Analysis of cis-acting elements reveals that AcNRAMP genes promoter sequences contain light-responsive elements, defense- and stress-responsive elements, and plant growth/development-responsive elements. Expression profiling confirms distinct expression patterns of AcNRAMP genes in different organs and responses to Zn/Fe deficiency stress in leaves and roots. Taken together, our results lay a foundation for further exploration of the AcNRAMPs regulatory function in areca response to Fe and Zn deficiency.


Introduction
Metal ions play a key role in maintaining the stability of plant physiological and biochemical functions. For example, Fe is an essential element involved in cell respiration, photosynthesis, and the catalytic reaction of metalloproteins, while Zn is a structural cofactor of a variety of enzymes and proteins [1,2]. The balance of metal ions in the plant is also necessary for plant development. A high concentration of free iron (Fe 3+ /Fe 2+ ) generates oxygen and hydroxyl radicals through the Fenton reaction, which causes a redox reaction and accumulation of superoxide compounds, resulting in intracellular damage to DNA and lipids [3]. Meanwhile, Fe deficiency can also affect plant growth and development. Similarly, excess zinc binds to sulfur, nitrogen, and oxygen-containing functional groups in biological molecules due to its upregulated high affinity, which, in turn, interferes with their biological activities. Zn deficiency leads to the oxidative destruction of chlorophyll, lipids, and proteins [4]. 2

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The maintenance of metal ion balance in plants is mainly achieved through the synergies of various metal transporters, including zinc-regulated transporters iron-regulated transporter-like proteins (ZIP), NRAMP, cation diffusion facilitator proteins (CDF), heavy metal ATPase (HMA), yellow stripe-like (YSL), and ATP-binding cassette (ABC) transporters [5,6]. Of these, the NRAMP proteins are a vital membrane transporter family that exist widely in plants, which are mainly involved in the transport of divalent metal cations such as Zn, Fe, and Cu. Moreover, the NRAMP proteins play a principal role in regulating and maintaining the homeostasis of Mn/Fe in plants [7,8]. NRAMP was first found in mice. Subsequently, NRAMP was verified as a metal transporter in diverse organisms including fungi, animals, and plants [9,10]. In the previous study, the roles of NRAMP proteins have been explored in some plant species, including model plants, forest trees, and horticultural plants [10][11][12][13][14][15]. Different NRAMPs enable the trafficking of different essential or toxic metal ions. For instance, Arabidopsis AtNRAMP6 is considered a transporter of cadmium [16], while AtNRAMP1 is a Mn transporter. AtNRAMP3 and AtNRAMP4 are responsible for the transportation of Fe and Mn [17]. Rice OsNRAMP3 is identified as a Mn transporter, while OsNRAMP5 is identified as a Mn/Cd transporter [18,19]. In addition, OsNRAMP4 plays a key role in the trafficking of trivalent Al ions [20]. In summary, NRAMP proteins are critical members of the membrane transporter family involved in metal ion transport, absorption, intracellular transport, and detoxification.
Areca catechu (areca palm) is an evergreen tree of the genus Areca in the palm family, which is native to Malaysia and widely cultivated in tropical Asia. In our previous studies, we treated areca seedlings with Fe and Zn deficiency and found a significant decrease in iron and zinc content in the roots [21]. In this study, 12 NRAMP genes were identified from the whole genome of A. catechu, and their sequence characteristics, gene structure, phylogeny, promoter sequence, and collinearity were analyzed. Furthermore, the expression levels of these genes in different tissues and under the condition of Fe and Zn deficiency were studied. This work provides a basis for further investigating NRAMP function and areca response to Fe and Zn deficiency.
The instability index (Ii) ranged from 25.75 (AcNRAMP10) to 47.77 (AcNRAMP2). Only two AcNRAMP proteins had Ii larger than 40. The aliphatic index (Ai) analysis shows that AcNRAMP2 has the maximum Ai at 82.84, while AcN-RAMP12 has the minimum Ai at 132. 44. In addition, the grand average of hydropathicity (GRAVY) of all AcNRAMP proteins was positive except for AcNRAMP2. Prediction of subcellular localization reveals that all AcNRAMP proteins are localized to the plasma membrane except for AcNRAMP2, AcNRAMP3, and AcNRAMP11 ( Figure 1B).

Duplication Analysis and Phylogenetic Analysis of AcNRAMPs
To understand the genomic distribution of 12 AcNRAMPs, their chromosomal location was analyzed. The twelve genes were located on seven chromosomes, of which chromosome two had a maximum of three genes (Figure 2A). To reveal the AcNRAMP collinear gene pairs, the syntenic gene analysis was carried out. The results show that only one collinear AcNRAMP (AcNRAMP3-AcNRAMP4) is identified in the areca genome ( Figure 2A). To shed light on the possible evolutionary process of AcNRAMPs, the syntenic relationships of AcNRAMP genes with the other plant species were investigated, including dicot A. thaliana (belongs to the Brassicaceae), monocot O. sativa (belongs to the Poaceae), and C. nucifera (belongs to the Palmae). Three AcNRAMP homologous genes pairs are detected between A. catechu and A. thaliana (AcNRAMP1-AT5G67330.1;

Sequence Features of AcNRAMPs
To illustrate the sequence features of the AcNRAMPs, the MEME program was used to analyze their conserved motifs. In total, six conserved motifs (motif 1-6) are found in 12 AcNRAMP protein sequences. AcNRAMP proteins within the identical subgroup have similar motifs ( Figure 5A,B). Motif 1 and motif 6 are present in all proteins. Most of the AcNRAMP proteins have motifs 2 and 3. Only AcNRAMP5 proteins contain two motifs, including motif 1 and motif 6. The analysis of gene structures reveals a great change in introns number within 12 AcNRAMP genes (from 3 to 14), while the number distribution of UTR varies from 1 to 3 ( Figure 5C). Furthermore, the frequencies of amino acids on the respective position within the sequences of six motifs in the AcNRAMPs are highly different, which are worthy of further elucidation ( Figure 5D).

Sequence Features of AcNRAMPs
To illustrate the sequence features of the AcNRAMPs, the MEME program was used to analyze their conserved motifs. In total, six conserved motifs (motif 1-6) are found in 12 AcNRAMP protein sequences. AcNRAMP proteins within the identical subgroup have similar motifs ( Figure 5A,B). Motif 1 and motif 6 are present in all proteins. Most of the AcNRAMP proteins have motifs 2 and 3. Only AcNRAMP5 proteins contain two motifs, including motif 1 and motif 6. The analysis of gene structures reveals a great change in introns number within 12 AcNRAMP genes (from 3 to 14), while the number distribution of UTR varies from 1 to 3 ( Figure 5C). Furthermore, the frequencies of amino acids on the respective position within the sequences of six motifs in the AcNRAMPs are highly different, which are worthy of further elucidation ( Figure 5D).

Cis-Acting Elements of AcNRAMPs
To gain more insight into the possible regulatory factors of 12 AcNRAMP genes, the Plant CARE online program was employed to analyze their cis-acting elements. We identified 25 types of cis-acting elements and classified them into three categories, namely, phytohormoneresponsive elements (PREs), defense-and stress-responsive elements (DSREs), and plant growth/development-responsive elements (GDREs) (Figure 6). Of these, the PREs are the most abundant with eight types, containing the ABA-responsive elements such as ABRE, the MeJA-responsive elements such as CGTCA motif and TGACG motif, the auxinresponsive elements such as AuxRR-core, the gibberellin-responsive elements such as P-box and TATC-box, and the other elements such as TCA element and TGA element. The DSREs have 12 types, containing ATC motif, Box 4, GA motif, GARE motif, GATA motif, G-box, I-box, MRE, Sp1, TCCC motif, and TCT motif. However, only five types of GDREs are identified, containing ARE, LTR, MBS, GCN4 motif, and RY element. Moreover, AcNRAMP9 and AcNRAMP10 have almost identical cis-acting elements. Box 4 is present in all AcNRAMPs as the light-responsive element ( Figure 6). motif, the auxin-responsive elements such as AuxRR-core, the gibberellin-responsive elements such as P-box and TATC-box, and the other elements such as TCA element and TGA element. The DSREs have 12 types, containing ATC motif, Box4, GA motif, GARE motif, GATA motif, G-box, I-box, MRE, Sp1, TCCC motif, and TCT motif. However, only five types of GDREs are identified, containing ARE, LTR, MBS, GCN4 motif, and RY element. Moreover, AcNRAMP9 and AcNRAMP10 have almost identical cis-acting elements. Box 4 is present in all AcNRAMPs as the light-responsive element ( Figure 6).

Expression Analysis of AcNRAMPs in Different Tissues and under Fe and Zn Deficiency
To elucidate the role of AcNRAMP genes in different tissue and their response to Fe and Zn deficiency, the expression profiles of AcNRAMP genes were analyzed by calculating gene FPKM values (Table S5). Tissue-specific expression analysis reveals that AcNRAMP2, AcNRAMP8, and AcNRAMP11 are highly expressed in underground roots, and AcNRAMP4 and AcNRAMP6 are highly expressed in aerial roots. The expression levels of AcNRAMP3 are higher in the endosperm than that in the other tissues. AcNRAMP4 is generally highly expressed in most tissues of areca, especially in aerial roots. AcNRAMP5 is highly expressed in flowers and pericarp. AcNRAMP7 is absent from flowers, while the high expression level of AcNRAMP7 is found in leaves and veins. AcNRAMP8 is specifically up-regulated in underground roots. AcNRAMP9 and AcNRAMP10 are up-regulated in female flowers compared to other tissues. Furthermore, AcNRAMP11 is only up-regulated in flowers and underground roots, while AcNRAMP12 shows a higher expression level in leaves and veins ( Figure 7A, Table S5). These results suggest that AcNRAMP proteins play vital roles in metal transportation in different areca tissues. Furthermore, the relative expression of six randomly selected AcNRAMP genes in female and male flower samples was detected by qRT-PCR. The results show that the gene expression trends determined by qRT-PCR and RNA-seq are highly consistent, which indicates that transcriptome data are reliable and reproducible ( Figure 7B). To further explore the response of AcNRAMPs to Fe and Zn, the expression of AcNRAMP in leaves and roots of areca seedlings under Fe and Zn deficiency conditions was analyzed (Table S6). In roots, AcNRAMP1 and AcNRAMP4 are up-regulated under Zn/Fe deficiency stress, while AcNRAMP7 is down-regulated. AcNRAMP3 is up-regulated in Fe-deficient roots, while AcNRAMP10 is up-regulated in Zn-deficient roots (Figure 8). In leaves, we find that AcNRAMP1, AcNRAMP9, and AcNRAMP10 are highly expressed To further explore the response of AcNRAMPs to Fe and Zn, the expression of Ac-NRAMP in leaves and roots of areca seedlings under Fe and Zn deficiency conditions was analyzed (Table S6). In roots, AcNRAMP1 and AcNRAMP4 are up-regulated under Zn/Fe deficiency stress, while AcNRAMP7 is down-regulated. AcNRAMP3 is up-regulated in Fe-deficient roots, while AcNRAMP10 is up-regulated in Zn-deficient roots (Figure 8). In leaves, we find that AcNRAMP1, AcNRAMP9, and AcNRAMP10 are highly expressed in the first leaf (L1) treated with iron deficiency. Under the condition of zinc deficiency, AcNRAMP1, AcNRAMP2, AcNRAMP3, AcNRAMP6, AcNRAMP8, AcNRAMP11, and Ac-NRAMP12 also show a high expression pattern in L1 (Figure 8). In addition, compared with the third leaf (L3), Zn/Fe deficiency treatment greatly affects the expression level of the AcNRAMPs gene in L1 (Figure 8). Furthermore, our previous study also suggests that the transcriptome data are reliable and reproducible [21]. These results suggest that the AcNRAMPs family plays an important role in coping with Zn/Fe deficiency stress in Areca. in the first leaf (L1) treated with iron deficiency. Under the condition of zinc deficiency, AcNRAMP1, AcNRAMP2, AcNRAMP3, AcNRAMP6, AcNRAMP8, AcNRAMP11, and AcNRAMP12 also show a high expression pattern in L1 (Figure 8). In addition, compared with the third leaf (L3), Zn/Fe deficiency treatment greatly affects the expression level of the AcNRAMPs gene in L1 (Figure 8). Furthermore, our previous study also suggests that the transcriptome data are reliable and reproducible [21]. These results suggest that the AcNRAMPs family plays an important role in coping with Zn/Fe deficiency stress in Areca.

Discussion
The plant NRAMP gene family belongs to the conserved metal transport family in natural evolution, which is mainly responsible for the absorption, transport, and intracellular stability of Fe, Mn, and other metal ions [7,22]. In addition, the NRAMP gene family also plays a critical regulatory role in photosynthesis, protein activity maintenance, and abiotic stress response [23]. In previous studies, the members of the NRAMP gene family have been identified in diverse plant genomes, including A. thaliana [11], O. sativa [12], P. alba [10], P. vulgaris [13], T. cacao [14], and B. napus [15]. In this study, we identified 12 NRAMP genes in the A. catechu genome. Notably, the NRAMP genes in Arabidopsis were classified into two subfamilies. However, we identified five subfamilies of NRAMP genes in areca, suggesting that AcNRAMPs in areca may evolve a series of new functions.
The analysis of physicochemical properties helps decipher the potential functional natures of proteins. For instance, analysis of the protein pI value provides us with an important reference index for protein purification. Similar to other gene family members, 12 AcNRAMP proteins include both basic and acidic proteins [24][25][26]. All AcNRAMPs are considered thermostable proteins due to their Ai value being higher than 71. In contrast, most of AcNRAMP (10/12) are considered unstable proteins in a test tube due to their Ii values being higher than 40. In addition, except for AcNRAMP2, all AcNRAMP proteins have GRAVY values lower than zero, suggesting their soluble nature. Different organelle localization of NRAMP proteins shows different functions in plants. For example, a previous study shows that AtNRAMP6 is localized in the Golgi/trans-Golgi network and contributes to maintaining intracellular Fe homeostasis [27]. Additionally, AtNRAMP1 is localized on the plasma membrane and participates in Fe/Mn transportation [28]. In rice, most of the NRAMP protein members are also localized on the plasma membrane and are associated with the transport of various intracellular metal ions [17,29]. In the present study, subcellular localization predicts that most AcNRAMP genes are localized on the plasma membrane, indicating that most AcNRAMP genes transport ions on the membrane, and some AcNRAMP genes are located in chloroplast, indicating that they transport ions between organelles.
Numerous studies have found that plant NRAMP family genes are associated with the uptake and transport of various divalent metal ions [30]. In A. thaliana, AtNRAMP1 expression can be induced under iron deficiency in the root system, and overexpression lines show high tolerance to iron. These results indicate that AtNRAMP1 can regulate iron metabolism balance in roots [31]. In addition, AtNRAMP1 also has the function of absorbing and transporting manganese and iron. Similar to AtNRAMP1, AtNRAMP2 is mainly expressed in the root tip and is involved in manganese ion transport [32]. AtNRAMP3 and AtNRAMP4 expression is induced under iron deficiency stress and can regulate the homeostasis of iron and manganese ions in cells [33,34]. Furthermore, the Atnramp6 mutant shows higher cadmium tolerance than the wild type [27]. Recently, in other plants, an increasing number of NRAMP genes have been proven to play a pivotal role in maintaining the balance of iron, manganese, and zinc in cells [35][36][37]. In our study, we also analyzed the expression profiles of AcNRAMPs in different tissues of areca. We found that AcNRAMP1, AcNRAMP4, AcNRAMP6, AcNRAMP7, and AcNRAMP12 are up-regulated in leaves and veins, indicating that they may be involved in the transfer of metal elements. AcNRAMP3, AcNRAMP4, AcNRAMP11, and AcNRAMP12 are up-regulated in the underground roots, indicating that these four genes may be involved in the accumulation of metal elements in the underground roots. Overall, the expression profile of AcNRAMP family genes varies between different organs, suggesting that these genes may exert their functions in specific areca tissues.
Studies have shown that seven NRAMP genes are present in O. sativa. Of these, OsNRAMP1 is primarily located on the plasma membrane and is significantly up-regulated in response to Fe deficiency [38]. Aside from transporting Fe, OsNRAMP1 also facilitates the movement of Cd across the membrane [39]. Moreover, OsNRMAP5 plays a crucial role in the transportation of Fe, Mn, and Cd in O. sativa, thereby significantly contributing to the overall growth and development of the plant [40,41]. In this study, we further analyzed the expression profiles of AcNRAMP family genes in areca seedlings under the Zn/Fe deficiency stress. We observe that iron deficiency stress can induce the high expression of AcNRAMP1, AcNRAMP9, and AcNRAMP10 in areca L1, suggesting that these genes might be the main iron transporter in areca during the iron deficiency stress. Meanwhile, the expression level of AcNRAMP12 is higher in iron-deficient roots than in normal areca seedlings, indicating a vital role in iron uptake and transport in roots. Furthermore, the expression levels of AcNRAMP1, AcNRAMP2, AcNRAMP3, AcNRAMP6, AcNRAMP8, AcNRAMP11, and AcNRAMP12 are significantly higher in the zinc-deficient group than those in the control group, suggesting that these genes may be involved in zinc ion transport. Additionally, we identified three AcNRAMP genes (AcNRAMP2, AcNRAMP4, and AcNRAMP12) that are closely related to areca roots' tolerance to zinc deficiency stress.

Plant Material and Growth Conditions
An A. catechu cultivar "Reyan NO.1", was selected as experimental material in this study. The six-month-old areca seedlings were cultured in Hoagland solution for adaptive cultivation. After two weeks, the seed bulbs of areca seedlings were removed from the base of the stem. Subsequently, the seedlings were cultured in whole nutrient solution (CK), Fe-deficient medium (CK without Fe-(Na) 2 EDTA), and Zn-deficient medium (CK without Zn 2+ ), respectively.

Identification of AcNRAMP Genes in A. catechu
The conserved domain of NRAMP protein (PF01566) was obtained from the Pfam database. The AcNRAMP candidates were preliminarily retrieved from the A. catechu genome using hidden Markov model (HMM) search with an E-value lower than 10 −5 based on the methods by Krogh et al. [42]. Pfam Database (http://pfam.xfam.org/) (accessed on 10 January 2023) and SMART (http://smart.embl-heidelberg.de/) (accessed on 10 January 2023) were used to determine the predicted protein as a member of the transporter gene family.

Analysis of Evolutional Relationships of NRAMP Genes in A. catechu, O. sativa, and A. thaliana
The sequences of AtNRAMPs and OsNRAMPs were obtained from TAIR (https: //www.arabidopsis.org/) (accessed on 12 January 2023) and the Rice Annotation Project Database, (https://rapdb.dna.affrc.go.jp/download/irgsp1.html) (accessed on 12 January 2023), respectively. ClustalW was performed to align all sequences with default parameters, and then a phylogenetic tree was constructed using a neighbor-joining (NJ) method of MEGA-X [45]. The bootstrap replications were set as 1000 to test the NJ tree reliability. The Evolview8 program was employed to visualize the phylogenetic tree [46].

Analysis of Sequence Features, Chromosome Distribution, and Syntenic Relationships of AcNRAMPs
The MEME online (https://meme-suite.org/meme/tools/meme) (accessed on 12 January 2023) was performed to analyze conserved motifs shared among AcNRAMP genes [47,48]. The TBtools software (version. x64_1_0987657) was used to visualize the AcNRAMP gene structure according to the CDS and genomic DNA sequences of AcNRAMP genes [49]. Furthermore, the TBtools software was performed to visualize the information of genome-wide chromosomal density and the distribution of AcNRAMPs across all chromosomes of A. catechu based on the genome annotation files. The genes distributed on scaffolds were excluded. The TBtools were employed to analyze the syntenic relationships of AcNRAMPs genes in interspecies (A. catechu vs. A. thaliana, A. catechu vs. C. nucifera, and A. catechu vs. O. sativa) and intraspecies.

Analysis of Cis-Acting Elements
Upstream 2,000 bases from the first ATG of CDS of AcNRAMP genes were regarded as promoter sequences, which were acquired from the A. catechu genome. The cis-acting elements of these sequences were identified using PlantCARE (http://bioinformatics.psb. ugent.be/webtools/plantcare/html/) (accessed on 18 January 2023).

Transcriptomic Data Analysis of AcNRAMPs
In this experiment, three individual areca seedlings from each group were separately collected as three biological replicates. RNA from a total of 27 samples was extracted and subjected to high-throughput sequencing on the Illumina Hiseq 4000 platform at Biomarker Technologies Co. (Beijing, China). After filtering out low-quality reads, clean reads were aligned to the areca reference genome. Gene expression analysis was performed using BMKCloud (www.biocloud.net) (accessed on 18 January 2023). The expression profiles of AcNRAMPs under Fe and Zn deficiency conditions were analyzed by calculating the fragments per kilobase per million mapped reads (FPKM) of AcNRAMPs based on the RNA-seq data. The genes with expression levels that met the standards (|log2FC| > 1, FDR < 0.05, and p-value < 0.05) were considered differentially expressed genes (DEGs). The heatmap representing the expression levels of AcNRAMP genes was plotted using TBtools. The RNA-seq raw data were deposited at NCBI (accession number: PRJNA767949).

qRT-PCR Analysis
The RNA quick isolation kit (Tiangen, Beijing, China) was used to extract the highquality total RNA from the areca samples, and the One-Step gDNA Removal and cDNA Synthesis SuperMix (Tiangen, Beijing, China) was used to synthesize cDNA. The qRT-PCR was conducted to analyze the relative expression of AcNRAMPs. Primer Premier 5.0 was used to design the qRT-PCR primers of the AcNRAMP genes (Table S1). The qRT-PCR used β-actin as an internal reference gene. The 20 µL qRT-PCR reaction mixture contained 10 µL of 2 × SYBR Green Master Mix (Tiangen, Beijing, China), 1 µL of forward and reverse primers, 1 µL of forward and reverse primers, 1 µL cDNA template, and 7 µL ddH 2 O. The qRT-PCR program follows 94°C hold for 30 s, 40 cycles at 94 • C, hold for 30 s, and 60 • C hold for 30 s. The final results were analyzed by using the 2 −∆∆Ct method. All experiments were repeated with three biological and technical replicates.

Conclusions
NRAMP proteins play pivotal roles in plant biological processes, including the trafficking of metal ions, plant growth, and development. However, the information on NRAMPs in A. catechu, a commercially important medicinal plant of the palm family, is still unclear. In this study, the total members of the NRAMP gene family in the areca genome were identified. Subsequently, the sequence characteristics, gene structure, phylogeny, promoter sequence, and collinearity of all AcNRAMP genes were comprehensively analyzed. Furthermore, the expression levels of these genes in different tissues and under the condition of Fe and Zn deficiency were studied. The results reveal that AcNRAMP genes exert their functions in specific areca tissues and play key roles in areca response to Fe and Zn deficiency. This work provides a valuable reference for the in-depth study of the function of the AcNRAMP gene in coping with Fe and Zn deficiency stress in areca.