*2.4. 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).

*2.4. 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, phytohormone-responsive 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 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).

**Figure 6.** Cis-acting elements of *AcNRAMPs*. The bar chart represents the total number of cis-acting elements within each AcNRAMP. The heatmap shows the number distribution of different cisacting elements within each *AcNRAMP*. **Figure 6.** Cis-acting elements of *AcNRAMPs*. The bar chart represents the total number of cis-acting elements within each AcNRAMP. The heatmap shows the number distribution of different cis-acting elements within each *AcNRAMP*.

#### *2.5. 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). *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 10 of 17

**Figure 7.** The expression patterns of 12 *AcNRAMP* genes of *A. catechu* in different tissue (**A**) and **Figure 7.** The expression patterns of 12 *AcNRAMP* genes of *A. catechu* in different tissue (**A**) and qRT-PCR validation of RNA-seq (**B**).

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 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

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

qRT-PCR validation of RNA-seq (**B**).

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.

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 11 of 17

**Figure 8.** The expression patterns of 12 *AcNRAMP* genes of *A. catechu* in Fe and Zn deficiency. FPKM values were obtained by RNA-seq. Each gene has three cartoon heatmaps that represent areca seedlings in normal, Fe-deficient, and Zn-deficient conditions. The sampling mainly targeted the first leaf (L1), third leaf (L3), and root (R) of areca seedlings. **Figure 8.** The expression patterns of 12 *AcNRAMP* genes of *A. catechu* in Fe and Zn deficiency. FPKM values were obtained by RNA-seq. Each gene has three cartoon heatmaps that represent areca seedlings in normal, Fe-deficient, and Zn-deficient conditions. The sampling mainly targeted the first leaf (L1), third leaf (L3), and root (R) of areca seedlings.

#### **3. 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 [24–26]. 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 [35–37]. 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.

#### **4. Materials and Methods**

### *4.1. 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)2EDTA), and Zn-deficient medium (CK without Zn2+), respectively.

#### *4.2. 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−<sup>5</sup> 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.

#### *4.3. Analysis of Physicochemical Properties of AcNRAMPs*

The physicochemical properties of AcNRAMP proteins were analyzed by using Ex-PASy6 (https://web.expasy.org/protparam/) (accessed on 12 January 2023) [43], and their subcellular localization was predicted using Plant-mPLoc (http://www.csbio.sjtu.edu.cn/ bioinf/plant-multi/) (accessed on 12 January 2023) [44].
