**1. 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 (Fe3+/Fe2+) 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].

**Citation:** Zhou, G.; An, Q.; Liu, Z.; Wan, Y.; Bao, W. Systematic Analysis of NRAMP Family Genes in *Areca catechu* and Its Response to Zn/Fe Deficiency Stress. *Int. J. Mol. Sci.* **2023**, *24*, 7383. https://doi.org/ 10.3390/ijms24087383

Academic Editors: Jian Zhang and Zhiyong Li

Received: 15 March 2023 Revised: 12 April 2023 Accepted: 15 April 2023 Published: 17 April 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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 [10–15]. 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.

#### **2. Results**

#### *2.1. Identification and Characterization of AcNRAMPs*

Twelve *AcNRAMP* candidates were identified in the *A. catechu* genome (Figure 1A, Table S2). The amino acid lengths of 12 AcNRAMP proteins altered from 120 (AcNRAMP5) to 971 (AcNRAMP2), while the molecular weight changed from 13.86–106.01 KDa. The theoretical isoelectric point (pI) of AcNRAMP proteins varied from 4.5 (AcNRAMP10)–9.14 (Ac-NRAMP5). 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).

### *2.2. Duplication Analysis and Phylogenetic Analysis of AcNRAMPs*

To understand the genomic distribution of 12 *AcNRAMP*s, 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 *AcNRAMP*s, 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*;

*AcNRAMP4*–*AT2G23150.1*; *AcNRAMP4*–*AT5G67330.1*) (Figure 2B, Table S3). Five *Ac-NRAMP* homologous gene pairs are identified between *A. catechu* and *O. sativa* (*AcN-RAMP1*–*Os12t0581600-01*; *AcNRAMP1*–*Os03t0208500-01*; *AcNRAMP2*–*Os07t0155600-01*; *AcNRAMP4*–*Os03t0208500-01*; *AcNRAMP6*–*Os06t0676000-01*) (Figure 2C, Table S3). A total of 11 *AcNRAMP* homologous gene pairs are identified between *A. catechu* and *C. nucifera* (*Ac-NRAMP1*–*GZ08G0188430.1*; *AcNRAMP1*–*GZ09G0194900.1*; *AcNRAMP1*–*GZ15G0285060.1*; *AcNRAMP2*–*GZ03G0077500.1*; *AcNRAMP4*–*GZ08G0188430.1*; *AcNRAMP4*–*GZ09G0194900.1*; *AcNRAMP4*–*GZ15G0285060.1*; *AcNRAMP6*–*GZ01G0002400.1*; *AcNRAMP7*–*GZ07G0166110.1*; *AcNRAMP12*–*GZ02G0049210.1*; *AcNRAMP14*–*GZ08G0185160.1*) (Figure 2D, Table S3). *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 3 of 17

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 4 of 17

(**B**).

*GZ01G0002400.1*; *AcNRAMP7*–*GZ07G0166110.1*; *AcNRAMP12*–*GZ02G0049210.1*; *AcNRAMP14*–*GZ08G0185160.1*) (Figure 2D, Table S3). **Figure 2.** Chromosomal location of *AcNRAMP* genes. (**A**) Chromosomal location of *AcNRAMP*s and their collinear gene pairs in *A. catechu* genome. The outer ring to the inner ring represents 16 chromosomes, gene density, and GC contents, respectively. (**B**–**D**) Syntenic analysis of *AcNRAMP*s between *A. catechu* and *A. thaliana*, *A. catechu* and *O. sativa*, and *A. catechu* and *C. nucifera*, respectively. The gray lines among each chromosome represent all gene pairs between *A. catechu* and other plant species, while the red lines represent the gene pairs associated with *AcNRAMP*  genes. **Figure 2.** Chromosomal location of *AcNRAMP* genes. (**A**) Chromosomal location of *AcNRAMP*s and their collinear gene pairs in *A. catechu* genome. The outer ring to the inner ring represents 16 chromosomes, gene density, and GC contents, respectively. (**B**–**D**) Syntenic analysis of *AcNRAMP*s between *A. catechu* and *A. thaliana*, *A. catechu* and *O. sativa*, and *A. catechu* and *C. nucifera*, respectively. The gray lines among each chromosome represent all gene pairs between *A. catechu* and other plant species, while the red lines represent the gene pairs associated with *AcNRAMP* genes.

To uncover the selection pressure on *NRAMP* genes during evolution, we calculated the non-synonymous substitution rate (Ka), the synonymous substitution rate (Ks), and

0.0797 to 0.6172) but are less than 1, suggesting purifying selection on these genes. Generally, the Ka/Ks ratios of *AcNRAMPs* and *OsNRAMPs* gene pairs are larger than those of *AcNRAMPs* and *CnNRAMPs* gene pairs, implying faster evolutionary rates of

*AcNRAMP4*–*GZ09G0194900.1*; *AcNRAMP4*–*GZ15G0285060.1*; *AcNRAMP6*–

*AcNRAMP* genes on *O. sativa* than on *C. nucifera* (Table S4).

To uncover the selection pressure on *NRAMP* genes during evolution, we calculated the non-synonymous substitution rate (Ka), the synonymous substitution rate (Ks), and their ratio (Ka/Ks) of homologous *NRAMP* genes in *A. catechu*, *A. thaliana*, *O. sativa*, and *C. nucifera* (Figure 3; Table S4). The results show that the Ka/Ks values vary greatly (from 0.0797 to 0.6172) but are less than 1, suggesting purifying selection on these genes. Generally, the Ka/Ks ratios of *AcNRAMPs* and *OsNRAMPs* gene pairs are larger than those of *AcNRAMPs* and *CnNRAMPs* gene pairs, implying faster evolutionary rates of *AcNRAMP* genes on *O. sativa* than on *C. nucifera* (Table S4). *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 5 of 17

**Figure 3.** Comparison of *NRAMP* homologous genes substitution rate. (**A**) The number distribution of the collinear gene pairs in three species. Ac: *A. catechu*; At: *A. thaliana*; Os: *O. sativa*; Cn: *C. nucifera*. (**B**–**D**) The Ka, Ks, and Ka/Ks distribution of the *NRAMP* duplicated genes. of the collinear gene pairs in three species. Ac: *A. catechu*; At: *A. thaliana*; Os: *O. sativa*; Cn: *C. nucifera*. (**B**–**D**) The Ka, Ks, and Ka/Ks distribution of the *NRAMP* duplicated genes.

To clarify the phylogenetic relationships among *NRAMP* homologs in different plant species, a total of 25 *NRAMP* proteins from *A. thaliana* (6), *O. sativa* (7), and *A. catechu* (12) were used to establish a phylogenetic tree (Figure 4). According to the topology of the phylogenetic tree, 25 NRAMP were subdivided into five subgroups (group 1–group 5). *AcNRAMP* genes are distributed in all five subgroups, among which group 2 has the largest number (Figure 4). **Figure 3.** Comparison of *NRAMP* homologous genes substitution rate. (**A**) The number distribution To clarify the phylogenetic relationships among *NRAMP* homologs in different plant species, a total of 25 *NRAMP* proteins from *A. thaliana* (6), *O. sativa* (7), and *A. catechu* (12) were used to establish a phylogenetic tree (Figure 4). According to the topology of the phylogenetic tree, 25 NRAMP were subdivided into five subgroups (group 1–group 5). *AcNRAMP* genes are distributed in all five subgroups, among which group 2 has the largest number (Figure 4).

#### *2.3. 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).

**Figure 4.** Phylogenetic relationships of NRAMP proteins in *A. catechu*, *O. sativa*, and *A. thaliana*. (**A**) The phylogenetic tree has 25 NRAMP proteins, including 12 AcNRAMP, 7 OsNRAMP, and 6 AtNRAMP). Star-marked 12 NRAMP genes of *A. catechu*. (**B**) The number of AcNRAMPs, OsNRAMP, and AtNRAMP in the different subgroups. **Figure 4.** Phylogenetic relationships of NRAMP proteins in *A. catechu*, *O. sativa*, and *A. thaliana*. (**A**) The phylogenetic tree has 25 NRAMP proteins, including 12 AcNRAMP, 7 OsNRAMP, and 6 AtNRAMP). Star-marked 12 NRAMP genes of *A. catechu*. (**B**) The number of AcNRAMPs, Os-NRAMP, and AtNRAMP in the different subgroups.

To illustrate the sequence features of the *AcNRAMPs*, the MEME program was used

12 AcNRAMP protein sequences. AcNRAMP proteins within the identical subgroup have

*2.3. Sequence Features of AcNRAMPs* 

different, which are worthy of further elucidation (Figure 5D).

**Figure 5.** The sequence features of the *AcNRAMP* genes. (**A**) The phylogenetic tree has 12 AcNRAMPs. (**B**) The motif distribution in the 12 AcNRAMPs. The various colored rectangles represent motifs 1 to 6; the black solid lines indicate the sequences outside motifs; the bar scale below represents the number of amino acids. (**C**) The gene structures of the 12 *AcNRAMP*s. The green ellipses and orange ellipses represent exons and untranslated regions (UTR), respectively; the black solid lines linked with the ellipse indicate the introns; the bar scale below represents the gene length. (**D**) Sequence logos of conserved amino acid residues sequences. The X-axis represents the position of the different amino acids in each motif, while the Y-axis represents the bit value of each amino acid. **Figure 5.** The sequence features of the *AcNRAMP* genes. (**A**) The phylogenetic tree has 12 AcNRAMPs. (**B**) The motif distribution in the 12 AcNRAMPs. The various colored rectangles represent motifs 1 to 6; the black solid lines indicate the sequences outside motifs; the bar scale below represents the number of amino acids. (**C**) The gene structures of the 12 *AcNRAMP*s. The green ellipses and orange ellipses represent exons and untranslated regions (UTR), respectively; the black solid lines linked with the ellipse indicate the introns; the bar scale below represents the gene length. (**D**) Sequence logos of conserved amino acid residues sequences. The X-axis represents the position of the different amino acids in each motif, while the Y-axis represents the bit value of each amino acid.

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
