*4.5. 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.

#### *4.6. 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).

#### *4.7. 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 *AcNRAMP*s under Fe and Zn deficiency conditions were analyzed by calculating the fragments per kilobase per million mapped reads (FPKM) of *AcNRAMP*s 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).

#### *4.8. 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 ddH2O. 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.

#### **5. 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.

**Supplementary Materials:** The supporting information can be downloaded at: https://www.mdpi. com/article/10.3390/ijms24087383/s1.

**Author Contributions:** W.B., Y.W. and G.Z.: conceptualization. W.B., Y.W. and G.Z.: methodology. G.Z., Z.L. and Q.A.: software, validation, and writing—original draft preparation. Z.L., G.Z. and Q.A.: investigation. W.B. and Y.W.: funding acquisition, writing—review and editing, and supervision. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Hainan Province Science and Technology Special Fund (ZDYF2021XDNY121), the Hainan Provincial Natural Science Foundation of China (320MS008), and the Initial Funds for the High-level Talents of Hainan University (KYQD(ZR)1935).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

**Acknowledgments:** Special thanks to the reviewers for their precious time and valuable suggestions.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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