Genome-wide identification of heavy-metal ATPases genes in Areca catechu: investigating their functionality under heavy metal exposure

Abstract

Heavy-metal ATPases (HMAs) play a vital role in plants, helping to transport heavy metal ions across cell membranes.However, insufficient data exists concerning HMAs genes within the Arecaceae family.In this study, 12 AcHMA genes were identified within the genome of Areca catechu, grouped into two main clusters based on their phylogenetic relationships.Genomic distribution analysis reveals that the AcHMA genes were unevenly distributed across six chromosomes. We further analyzed their physicochemical properties, collinearity, and gene structure.Furthermore, RNA-seq data analysis exhibited varied expressions in different tissues of A. catechu and found that AcHMA1, AcHMA2, and AcHMA7 were highly expressed in roots, leaves, pericarp, and male/female flowers. A total of six AcHMA candidate genes were selected based on gene expression patterns, and their expression in the roots and leaves was determined using RT-qPCR under heavy metal stress. Results showed that the expression levels of AcHMA1 and AcHMA3 genes were significantly up-regulated under Cd2 + and Zn2 + stress. Similarly, in response to Cu^2+, the AcHMA5 and AcHMA8 revealed the highest expression in roots and leaves, respectively. In conclusion, this study will offer a foundation for exploring the role of the HMAs gene family in dealing with heavy metal stress conditions in A. catechu.

Introduction

Heavy metal toxicity poses severe threats to crop yields worldwide [1]. Plants are sensitive to metal toxicity because it impacts their growth and productivity, reduces their water use efficiency, and causes various physiological and biochemical changes in plants [2]. Heavy metal contamination of soil, water, and food poses a significant health risk to the human body [3]. Plants need heavy metals such as copper, manganese, zinc, nickel, and cobalt for various biological processes [4], whereas some other non-essential toxic metals, such as lead (Pb) and cadmium (Cd), can be absorbed by plants, which are highly toxic and adversely affect crop productivity [5]. Several metabolic pathways in plants are inhibited by high concentrations of Cd²⁺ and Cu²⁺, including but not limited to photosynthesis, respiration, and enzyme activity. These ions decrease protein and chlorophyll levels, increase ROS production, and alter gene expression associated with oxidative stress responses [6, 7]. Zinc (Zn) and Iron (Fe) are essential elements involved in photosynthesis and cell respiration. Zinc is a cofactor and structural element of various transcription factors and structural proteins [8]. However, a high Zn²⁺ concentration can affect plant growth, chlorosis, necrosis in leaves, browning of roots, and the degradation of cell membrane integrity and permeability [9]. Plants employ two main strategies to mitigate the adverse effects of heavy metals and sustain normal metabolic activities: limiting the absorption of heavy metals from the soil and accumulating them within vacuoles [10]. The defensive mechanisms of various metal ion transporters control the coordination and balancing of metal ions in plants. Numerous metal transporters have been identified, including cation diffusion facilitator proteins (CDF), Zn-regulated transporter-like proteins (ZNT/ZIP), yellow stripe proteins (YSL), and ATP-binding cassette proteins (ABC). Natural resistance-associated macrophage proteins (NRAMP), Metal tolerance proteins (MTP), and heavy metal ATPases (HMA) are actively involved in metal transport. These transporters collectively contribute to effectively regulating plant metal ion levels, highlighting the complexity and cooperation in maintaining metal homeostasis [11,12,13].

Heavy-metal ATPase (HMA), or P1B-type ATPase, is one of the most important transporters of heavy metal ions. It combines ATP hydrolysis with metal ions to facilitate the absorption and translocation of heavy metal ions (such as Zn²⁺, Cd²⁺, Cu²⁺, Co²⁺, and Pb²⁺) across cell membranes [14, 15]. A typical HMAs possess 6 to 8 transmembrane helices (TMs), an H.P. locus, and a CPx/SPC motif essential for metal transport and metal binding domains at both C and N-terminal regions [16]. Based on their specific metal substrate binding properties, ATPases can be classified as Cu/Ag-ATPases or Zn/Co/Cd/Pb-ATPases [17,18,19]. At present, the HMA proteins have been characterized in many plants, i.e., A. thaliana, maize (Zea mays L.), rice (Oryza sativa), and wheat (T. aestivum) etc., at the genomic and molecular level [14, 20,21,22]. The functionality of HMA genes varies across different plant species, each contributing to specific roles. The heterologous expression of AtHMA1 in Saccharomyces cerevisiae shows its role in detoxifying excessive Zn (II) in the chloroplast [23]. The OsHMA2, AtHMA2, and AtHMA4 perform a crucial role in Zn²⁺ and Cd²⁺ translocation from the xylem of the roots to the shoots of the plant [24, 25]. Overexpression of AtHMA3 increases 2- to 3-fold tolerance against heavy metals and accumulates Cd, Zn, Pb, and Co to the vacuoles [26]. In O. sativa, OsHMA1, and OsHMA2 are supposed to be involved in Zn transportation in tonoplast [14, 15, 27]. OsHMA3 acts as a Cd, whereas OsHMA4 transports Cu to the roots cell vacuoles [28,29,30].

Areca catechu L., is a versatile plant in the palm family, holds significant medicinal and economic value, and is grown across China, India, Thailand, Indonesia, Malaysia, and Cambodia [31, 32]. The areca nut, characterized by its abundant arecoline content and regarded as the fourth most addictive globally, following alcohol, caffeine, and nicotine, has emerged as a crucial agricultural commodity in Southeast Asia and Africa. Although the complete genome sequence of Areca has been published, there remains a gap in the literature regarding a comprehensive examination of genome-wide analysis and expression profiling of the AcHMA family. In this study, we performed a genome-wide analysis of AcHMA gene family in A. catechu. A total of 12 AcHMA genes were identified, and they were analyzed for their chromosomal locations, phylogenies, conserved motifs, structures, and expression profiles, as well as their responses to heavy metals (CuSO₄, ZnSO₄, and CdCl₂). Additionally, the study of AcHMA will be of guiding significance for understanding the transport of heavy metal ions in palm plants.

Materials and methods

Retrieval of the AcHMA gene family in A. catechu

The A. catechu (Taxonomy ID: 184,783) whole-genome assembly protein sequences were obtained from NCBI (ID: JAHSVC000000000; BioSample: SAMN19591864; Accession: PRJNA735650). The conserved HMA protein domain (PF00403), E1-E2_ATPase domain (PF00122), and hydrolase domain (PF00702) were retrieved from the Pfam database (http://pfam.sanger.ac.uk/search (accessed on November 15, 2023). The HMA gene family in A. catechu was identified using two methods. In the first method, the HMAs (AtHMA1-AtHMA8) protein sequences of Arabidopsis were retrieved from TAIR (http://www.arabidopsis.org accessed on November 15, 2023) to be used as query proteins. Using BioEdit v7.2.6, a local protein sequence database of A.catechu was created. BLASTp was used to identify predictable HMA genes in A. catechu using the Arabidopsis genome.

In the second approach, we performed a Hidden Markov Model (HMM) search against the genome of A. catechu to conform the presence of HMA genes using HMMER version 3.2.1 with default settings (http://hmmer.org/ (accessed on November 15, 2023)). After merging the results, the HMA genes were further screened on the basis of their domain composition.

Subsequently, any missing conserved and duplicated sequences identified by the two methods were manually eliminated. The merged findings were further screened based on their domain composition in the NCBI Conserved Domains Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml (accessed on November 15, 2023)) and the SMART database (http://smart.embl-heidelberg.de/ (accessed on November 15, 2023)). Protein sequences without the conserved HMA Pfam domains, errors or shorter (< 100aa) were removed and those with an E-value < 1 × 10^− 10 were retained.

Analysis of physicochemical properties of AcHMAs

A variety of physiochemical properties of HMAs were identified by using the ExPASy server (http://web.expasy.org/protparam/ (accessed on November 15, 2023)), including amino acid length, predicted molecular weight, Gravy, and theoretical isoelectric points. The protein subcellular localization Prediction Tool (PSORT) (https://www.genscript.com/psort.html) was used for the subcellular localization prediction of all AcHMA genes.

Phylogenetic, gene synteny analysis and Ka/Ks calculation

The HMAs protein sequences of Arabidopsis were downloaded from TAIR (http://www.arabidopsis.org), O. sativa from Phytozome (https://phytozome.jgi.doe.gov), and Cocos nucifera from ((https://arecaceae-gdb.com/#/) (accessed on November 20, 2023). ClustalW was used for multiple sequence alignment of all identified AcHMA gene family proteins. MEGA 11.0 software generated the phylogenetic tree based on the Maximum Likelihood method with default parameters: bootstrapping to 1000, Poisson model, and complete deletion (accessed November 20, 2023). The alignment sequences were trimmed with TBtools software [33]. Evolgenius (https://www.evolgenius.info/evolview-v2/#mytrees/ia-tree6.0/6.0) was used to visualize and optimize the tree. MCScanX was set with its default parameters to identify gene duplication events and syntenic relationships. The outcomes were then graphically represented using Circos and a Dual Synteny Plot generated with TBtools. The non-synonymous and the synonymous substitution ratio (Ka/Ks) of homologous HMA gene pairs in A. thaliana, A. catechu, O. sativa, and C. nucifera were calculated using TBtools.

According to the Ka/Ks ratio, duplicate genes are detected under positive or negative selection (if the ratio is > 1, neutral selection is suggested, and Ka/Ks ratios of less than one offer negative or purifying selection) [34]. The divergence time of gene pairs was estimated using the synonymous mutation rate per synonymous site per year formula: T = Ks / 2x, where x = 6.56^10^− 9 (He et al., 2016).

Gene structure analysis, chromosomal distribution, and conserved motifs

Gene locations, chromosome lengths, exon/intron distributions were retrieved from the gene annotation file (GFF3) of the A. catechu genome and visualized via TBtools. Using the MEME online tool (http://meme-suite.org/tools/meme, accessed on November 15, 2023), the conserved motifs of AcHMA proteins were predicted and analyzed.

Cis-acting elements and transmembrane topology

The promoter sequences, obtained from the A. catechu genome, were defined as the 2,000 bases upstream from the initial ATG of the coding sequence (CDS) of AcHMA genes. PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on November 15, 2023) was used to analyze the cis-element distribution in promoter regions.

RNA-seq data analysis

The Illumina Hiseq 4000 RNA-seq data deposited at NCBI (accession number: PRJNA767949) were utilized for expression profiling analysis of AcHMA genes. FPKM values were computed for AcHMA genes based on RNA-seq data. Genes with a log2FC greater than 1, a false discovery rate of 0.05, and differentially expressed genes (DEGs) were determined based on a significance level with a p-value of less than 0.05. TBtools were utilized to plot the expression levels of AcHMA genes. Gene expression analyses were performed using BMK Cloud (www.biocloud.net).

Plant materials and treatments

This study obtained A. catechu seedlings (Reyan No. 1) from the Coconut Research Institute of the Chinese Academy of Tropical Agricultural Sciences, Wenchang, Hainan province, China. The plants were cultivated at 28℃/25℃ (14/10 h day/night) photoperiod in pots (12 cm × 12 cm) with perlite at Hainan University, Hainan province, China. The plantlets were divided into two groups: Control (CK) and Treatment (T), after being treated for two weeks with half-strength Hoagland’s solution (pH = 6). The plantlets were treated with 100 µmol/L CdCl₂, 100 µmol/L CuSO₄,100 µM ZnSO₄ [34] for 72 h to assess the expression patterns in comparison to the control group. Samples from roots and the top leaf were collected (n = 3) at four different periods (0, 12, 24, and 72 h) for each treatment, followed by quickly cryopreserved in liquid nitrogen and kept at -80 ̊C until RNA extraction.

RNA isolation and qRT-PCR

RNA isolation was conducted using the plant RNA extraction kit (Code No. DP437, TIANGEN, Beijing, China). NanoDrop 2000 (KAIAO, Beijing, China) was used to detect RNA concentration and quality. TIANScript RT Kit (Code No. KR104, TIANGEN, Beijing, China) was used to generate first-strand cDNA. The reaction mixture containing 10 µL of vazyme Master Mix (Code No. Q111-02, Vazyme, Nanjing, China), 1 µL of forward and reverse primers, 1 µL cDNA template, and 7 µL ddH₂O. Primer Premier 6.0 was used to design primer pairs, while actin was used as a control primer (Table S1). Each experiment consisted of three biological and technical replicates. The 2^−ΔΔCT method was employed to calculate the relative gene expression.

Statistical analysis

The mean expression value was calculated using technical replicates. A Two-way ANOVA was conducted using Statistix 8.1, followed by the Tukey LSD test, to evaluate the presence of significant differences (P ≥ 0.05). The data was analyzed and histograms were generated using the Origin software.

Results

In silico characterization of HMA family in A. catechu

This study identified and characterized 12 AcHMA genes from the entire genome of A. catechu. The genes were assigned as AcHMA1–AcHMA12 based on phylogenetic relations with known A. thaliana HMAs genes (Table S2). The total number of HMA family genes in the A. catechu were higher than that in the A. thaliana (8), rice (9), and maize (11) but lower than in soybean (20) Brassica napus (31). The physical and chemical properties revealed that the amino acid lengths of AcHMA proteins ranged from 428 amino acids (AcHMA2) up to 1030 (AcHMA9), whereas the average number was 812.5 amino acids. The M.W ranged from 45.76 kDa (AcHMA2) to 111.53 (AcHMA9), averaging 87.39 kDa. The theoretical isoelectric point (pI) varies from 5.41 (AcHMA8) to 8.09 (AcHMA6), with an average value of 6.68. The theoretical isoelectric point (pI) changed from 5.41 (AcHMA8) to 8.09 (AcHMA6) with an average value of 6.68, whereas the GRAVY values varied from 0.044 to 0.553 with an average range of 0.19. All genes had positive GRAVY values, indicating hydrophobic properties. The predicted subcellular localization reveals that most of the AcHAMs were located in the plasma membrane except two of them (AcHMA1 and AcHMA6) were located in cytoplasm, and only one (AcHMA12) is found in the chloroplast (Table 1).

Table 1 Details of the HMA genes identified in A. catechu and their sequence characteristics

Phylogenetic and comparative analysis of HMAs in A. catechu

In order to investigate the evolutionary relations, we used 12 HMA genes from A. catechu, eight from A. thaliana, ten from C. nucifera, and nine from O. sativa to make a phylogenetic tree using the maximum likelihood method by MEGA11. Based on substrate specificity, 39 HMA genes were classified into two main groups (Cu/Ag-ATPases and Zn/Co/Cd/Pb-ATPases). The groups are further subdivided into five subgroups (I-V). AcHMA genes are distributed in all five subgroups, although group 5 has the most (5) genes. This discrepancy in orthologous pairs suggests a closer evolutionary relationship between the same species, A. catechu, and C. nucifera, than the more distant relationship with A. thaliana and O. sativa(Fig. 1).

Fig. 1

Phylogenetic relationship of HMA genes among A. catechu, A. thaliana and O. sativa. The tree was generated using MEGA11.0 based on the Maximum Likelihood (ML) of 39 HMA protein sequences. The tree were distributed into two main clades. These clades were further classified into Zn/Cd/Co-ATPases sub-groups (I and II) and the Cu/Ag-ATPases groups (III, IV, and V). Groups and subgroups are indicated by different background colors (green represents Zn/Cd/Co-ATPases; light pink represents Cu/Ag-ATPases subgroup). The red stars, green solid circles, and light blue triangles represent the A. catechu, Arabidopsis, and O. sativa HMA proteins

Chromosomal distribution, gene synteny analysis, and duplication

The chromosomal location of 12 AcHMAs was analyzed to determine their genomic distribution. In total, twelve genes were found on six chromosomes, with chromosome twelve containing a maximum of five genes and chromosomes four and ten, each containing two genes (Fig. 2a). Synteny analysis was performed among family members of AcHMAs in A.catechu to investigate its possible evolutionary process. Three pairs of syntenic AcHMA genes (AC04G017130.1/AC05G072680.1, AC10G001250.1/AC06G001050.1 and AC12G044360.1/ AC12G047390.1) were found in A. catechu (Fig. 2a). In order to gain a better understanding of the evolution mechanisms of the AcHMA gene family, syntenic analysis were constructed among A.catechu and three other species, including A. thaliana (dicot, Brassicaceae), O. sativa (monocot, Poaceae), and C. nucifera (Arecaceae). One AcHMA homologous gene pair was identified between A. catechu and A. thaliana (AcHMA4/ AT4G30110.1). Two homologous gene pairs were determined between A. catechu and O. sativa (AcHMA5/ Os02g10290.1; AcHMA9/ Os04g46940.1), and five AcHMAs homologous gene pairs were identified between A. catechu and C. nucifera (AcHMA4/GZ01G0000730.1; AcHMA4/GZ07G0155070.1; AcHMA6/ GZ09G0200370.1; AcHMA5/ GZ05G0113160.1; AcHMA9/ GZ10G0210660.2) (Fig. 2b-d; Table S3). To understand the divergence process of AcHMA homologous genes during replication, the Ka/Ks and its ratio were calculated within A. catechu, A. thaliana, O. sativa, and C. nucifera (Table S3). The Ka/Ks values were less than one (< 1), indicating purifying selection.

Fig. 2

Synteny analysis of AcHMAs. (a)AcHMAs in the A.catechu genome and their colinear gene pairs. Chromosome numbers, gene density, and GC content are displayed from outer to inner rings. (b, c, d) Synteny analysis of HMA genes between areca and three representative plant species. Grey lines in the background indicate the collinear blocks within the orchardgrass and other plant genomes, whereas the red lines highlight the syntenic HMA gene pairs

Phylogenetic, conserved motifs and gene structure analysis

The full-length AcHMA protein sequences were aligned to create an unrooted phylogenetic tree (Fig. 3a). The 12 AcHMAs were grouped into two main categories: Zn/Co/Cd/Pb-ATPases (AcHMA 1–4) and Cu/Ag-ATPases (AcHMA 5–12) based on their characteristics. The length of amino acid residues and the number of motifs were not conserved across the AcHMA group (Fig. 3b). Some motifs (motifs 1 and 3) were highly conserved in all group members, while a few (motifs 9 and 10) were only found in 6 group members. The Zn/Co/Cd/Pb subgroup, in comparison with Cu/Ag-ATPases, lacks several motif pairs. Detailed information about these motifs is shown in (Fig. 3b). Further, we also noticed that the position of the domains in the HMA proteins follows a similar pattern reported in A. thaliana. The Cu/Ag AcHMA proteins showed two types of domains, namely HMA and E1-E2-ATPase, while the Zn/Co/Cd/Pb AcHMA proteins exhibited mostly E1-E2-ATPase and hydrolase domains (Fig. 3c).

Fig. 3

Phylogenetic analysis, conserved domains, exon-intron structure, and motif composition of HMAgenes in A. catechu (a) Phylogenetic relationships of 12 AcHMAs. (b) MEME predicted the conserved motifs of HMA proteins. The different conserved motifs of HMA proteins are displayed as other colored boxes, and non-conserved sequences are shown as gray lines. (c) Distribution of conserved protein domains. (d) Schematic for HMA intron/exon structures. The gray lines show introns, the blue boxes indicate untranslated regions, and the red boxes represent CDS regions. The scale bar is shown at the bottom

The intron-exon configurations of AcHMA genes is essential for unraveling their physiological roles. Through comparative analysis of the exon/intron ratio, it has been found that AcHMA6 has the highest number of exons/intron (17/16) while AcHMA10 has a minimum exons/intron (5/4) ratio across all AcHMA members (Table S4). In addition, the distribution of UTR numbers also varies from 1 to 5, where AcHMA4 and AcHMA8 have a maximum of 5 UTR while AcHMA9 and AcHMA10 have each 1 UTR, respectively (Fig. 3d; Table S4).The conserved motifs of AcHMA proteins were analyzed using the web tool MEME (www.meme-suiteorg/meme/tools/meme) to provide a more detailed understanding of their sequence characteristics (Table S5). The tool identified ten conserved protein motifs that exhibit significant conservation in terms of both their combination and relative position.

Characterization of cis-acting elements within AcHMA promoter regions

To better understand the complex regulatory networks that control gene expression in AcHMA genes, we identified and analyzed cis-elements in the promotor region on 2000 bp upstream of the starting site (Fig. 4a). The promoter regions of AcHMA genes contained the most stress-related cis-acting elements, which indicates their importance in stress-related pathways. This suggests that the genes may have a role in determining the plant’s reaction to external stimuli. Thus, understanding the role of these genes may provide insights into how plants adapt to their environments. Furthermore, we found that ABA (ABRE and CGTCA) and salicylic acid (SA) related cis-elements were detected in promoter regions of AcHMA genes associated with abiotic stresses (high temperature, wounds, and drought). These elements were also related to hormones (salicylic acid, gibberellin, MeJAE, Auxin, and ethylene) (Fig. 4b).

Fig. 4

(a) Predicted cis-elements in the promoter regions of AcHMA genes. Motif names were shown nearby with different colors. (b) Distribution of cis-regulatory elements of AcHMA gene members

The heatmap is divided into four sections (growth, phytohormone regulation, light-responsive, and stress), in which rows represent the AcHMA genes and columns represent the cis-regulatory elements. The bar graph and the color from yellow to dark red represent the number of elements in each row and column.

Differential expression patterns of AcHMA genes in various tissues

The transcriptomic data of A. catechu were used to calculate the gene FPKM expression values of AcHMA genes in different tissues (i.e., male and female flowers, endosperm, pericarp, leaf, and leaf vein, aerial and underground roots) (Table S6). The expression profiling reveals that AcHMA5, AcHMA8, AcHMA3, AcHMA2, and AcHMA1 were highly expressed in root while AcHMA1, AcHMA2, and AcHMA8 were upregulated in leaves and veins. Expression levels of AcHMA1, AcHMA6, AcHMA7, and AcHMA8 were higher in male and female parts of the plant. Furthermore, AcHMA1, AcHMA7, and AcHMA8 genes were highly expressed in the endosperm and pericarp. Notably, AcHMA9, AcHMA10, AcHMA11, and AcHMA12 were low expressed in all tissues of Areca (Fig. 5; Table S6).

Fig. 5

The expression heatmap of the 12 AcHMA genes in eight different tissues of the A. catechu

Abiotic stress-induced expression patterns of AcHMA genes

To reveal the response of AcHMA genes to heavy metals (Cd2+, Cu2 + and Zn2+) in roots and leaves, we detected the expression profile of AcHMA genes after heavy metal treatment. In response to Cd²⁺ treatment, AcHMA1 and AcHMA2 were highly upregulated genes; AcHMA3 and AcHMA8 exhibited moderate upregulation, while AcHMA5 was a downregulated gene in leaves after 72 h. Similarly, all genes were highly expressed in roots under Cd²⁺ treatment except AcHMA8 after 72 h (Fig. 6a, b). Furthermore, in response to Zn²⁺ treatment, in addition to AcHMA5, the expression levels of AcHMA1, AcHMA2, AcHMA3 and AcHMA7 in leaves increased gradually with the extension of treatment time (Fig. 6c). In particular, in the root, all AcHMA genes’ expression levels increased significantly after treatment (Fig. 6d). In response to Cu²⁺ treatment, AcHMA1, AcHMA2, AcHMA5, AcHMA7 and AcHMA8 were upregulated in leaves, while AcHMA3 downregulated in leaves (Fig. 6e). The gene expression levels of AcHMA5, AcHMA7, and AcHMA8 in roots substantially rose as the treatment period increased (Fig. 6f).

Fig. 6

The expression pattern of HMA genes in A. catechu leaves and roots under heavy metals. HMA genes expression in A. catechu under (a) CdCl₂ (leaf), (b) root; (c) ZnSO₄ (leaf), (d) root, (e) CuSO₄ (leaf), (f ) root at different time intervals (0 (C.K.), 24, 48, and 72 h). The data is presented in mean and standard error, n = 3, with asterisks indicating statistically significant differences (p ≤ 0.05) according to the LSD test

Discussion

Plants require a control system for managing metal homeostasis in order to balance the delivery of essential micronutrients (such as zinc, copper, and iron) to various organs while simultaneously preventing non-essential metals such as lead and cadmium from accumulating to toxic concentrations that may adversely affect the plant [35]. Heavy metal ATPase are transmembrane transporters that play a significant role in heavy metal transportation, distribution, detoxification, and accumulation in plants [36]. Previous studies have identified the HMA gene family in various plants, including 8 in A. thaliana [37], 9 in rice (Oryza sativa L.) [38], 11 in maize (Zea mays L.) [21], 11 in sorghum (Sorghum bicolor L.) [21], 17 in Populus trichocarpa [39], and 20 in soybean (Glycine max L.) [40], 31 in Brassica napus (B. napus) [41], 8 mulberry (Morus) [25], 21 barley (Hordeum vulgare L.) [42], 14 turnip (Brassica rapa) [43], 48 tomato (Solanum lycopersicum) [34], and 27 in wheat (T. aestivum) [22]. However, the heavy metal ATPase gene family within the Arecaceae family remains unreported in existing literature.

In this study, we identified 12 AcHMA genes in Areca. The number of AcHMA genes in A.catechu genome was lower than in tomato and wheat [22, 34] while almost closer to sorghum, rice and maize [21], suggesting a potential shared evolutionary or functional aspect in the context of HMA genes among these plant species. The HMA family members has been divided into several clades in different plant species based on substrate specificity and phylogenetic clustering. HMA proteins in Arabidopsis and potato were categorized into 6 clades [44], 5 in barley [42], 8 in flax [45], 7 in cotton [46], 11 in dragon fruit [47], and 3 in maize [48]. The AcHMA gene family in A.catechu was categorized into two sub-families (Cu/Ag P1B-ATPase and Zn/Co/Cd/Pb P1B-ATPase) and five sub-subfamilies (I-V) based on substrate specificity and phylogenetic clustering among A. catechu, Arabidopsis, O. sative, and C.nucifera. Our investigation into the HMA gene family in A. catechu aligns with the established pattern, suggesting a conserved organizational structure within the HMA gene family across plant species.

The similar structural pattern of HMA genes shows that they might have similar functions and connections in different plants, especially regarding their specific substances [39]. This helps us to understand the various roles these gene family play in different plants and how they adapt to their environments. It has been reported that HMA transporter activities are linked to the subcellular distribution of the HMA transporters. The AtHMA1, OsHMA1, AtHMA6, and AtHMA8 identified both in Arabidopsis and rice, exhibited a chloroplast envelop localization, and were involved in Zinc translocation into the chloroplast [23, 49]. Similarly, AtHMA2, OsHMA2, AtHMA4, AtHMA5, OsHMA5 and OsHAM9 are located in plasma membrane confers tolerance to Zn/Cd/Cu [50,51,52]. The OsHMA3 and OsHMA4 in Oryza sativa are located at the tonoplast and help in cadmium and copper accumulation in vacule [50, 53].

Furthermore, P1B-ATPases have metal binding domains (MBDs) that act as cell transport regulators [54]. The distinctive cysteine structure (CxxC) in the HMA domain enables these genes to bind metal ions through thiol groups [55, 56]. P1B-ATPases, a type of transporter, are effective in expelling essential and toxic metal ions out of cells, ensuring a balance of metals (homeostasis) within the cellular environment. This mechanism is vital for maintaining the overall health and functionality of the cell [14, 57]. Understanding the exon-intron structure is crucial in evolution and comprehending the gene family. Significant evolutionary changes occur in eukaryotic genes due to intron gain or loss, which entails inserting and deleting gene segments [58]. Analyzing the exon-intron structure of AcHMA genes reveals conservation within the gene family despite divergent exon numbers. Although, the exon/intron count varies even within the same sub-family (Cu/Ag and Zn/Co/Cd/Pb P1B-ATPase). Exon/intron ratio variations within the gene family hint at functional diversification or evolutionary changes [59].

Cis-regulatory elements play a vital role in mediating stress responses, emphasizing their significance in the regulatory network [60]. Cis- regulatory elements and plant hormones, including ethylene (ET), salicylic acid (SA), abscisic acid (ABA), and jasmonic acid (JA), act as crucial signalling molecules that empower plants to adapt to abiotic stresses [60, 61]. The analysis of cis-regulatory elements of AcHMA family genes indicates the presence of multiple abiotic stress-responsive cis-elements. Specifically, elements like MYB, Myc, and STRE, abundant in the promoter region, exhibit a robust response to heavy metal stress, implying a potential role of AcHMA in the plant’s reaction to Cd and other heavy metal stress [62]. Understanding cis-acting elements in gene promoters, like G-box and ABRE, is crucial for unravelling plant stress responses. The abundance of cis-elements suggests that these genes may be triggered in response to various environmental stresses, potentially improving its overall stress tolerance [63].

According to previous studies, HMAs are thought to play a crucial role in detoxifying heavy metals in dicots by inhibiting the efflux of these metals into the cytosol through efflux mechanisms [14]. To understand the role of AcHMAs in A.catechu, we performed RNA-seq data analysis and qPCR to investigate the expression of AcHMA under normal conditions and heavy metal stress. In normal conditions, the AcHMA1-AcHMA8 genes had high expression levels throughout many tissues. However, the expression pattern of these genes was altered in response to abiotic stimuli, thereby highlighting the diverse function of AcHMA genes. The diverse expression pattern of AcHMA genes suggests that they were widely involved in abiotic stress as well as the growth and development of A.catechu. As a result of heavy metals exposure, both roots and leaves exhibited high levels of AcHMA gene expression at 48 and 72 h. Notably, AcHMA1 and AcHMA2 genes exhibited the highest upregulation in response to Cd²⁺ and Zn²⁺ stresses in roots and leaves, while AcHMA5 and AcHMA8 showed significant upregulation in the presence of Cu²⁺.The higher expression level of AcHMA1 and AcHMA2 against Cd²⁺ stress indicates their role in cadmium transportation in roots and leaves. Previously, it was revealed that AtHMA3 in Arabidopsis, OsHMA3 in rice and ZmHMA2 and ZmHMA3 from maize has shown cadmium transport capabilities into root vacuoles of plants [1, 26, 30]. Based on these results, it appears that HMA2 and HMA3 genes play a similar role in regulating cadmium transport in diverse plant species, and this supports our conclusion regarding AcHMA1 and AcHMA2. These results provide evidence that genes closely related to each other in plant phylogeny play a similar role in regulating cadmium transport to vacuolar sequestration, as well as subsequent root-to-shoot translocation in various plant species.

Similarly, as a result of Zn²⁺ treatment, our study revealed a notable upregulation of AcHMA1, AcHMA2, AcHMA3, AcHMA7, and AcHMA8 both in leaves as well as in roots, suggesting their involvement in zinc transportation in the tonoplast. Similar gene expression patterns have been identified in both O.sativa and A.thalian, and our findings align with those earlier studies. As a result of zinc stress, previous studies reported elevated levels of AtHMA1, OsHMA1, and OsHMA2 expression in the tonoplast, indicating that these genes play a role in zinc transport [14, 23, 27]. In response to Cu²⁺ stress, most of the AcHMAs show differential expression patterns. Notably, AcHMA5 and AcHMA8 were upregulated in roots and leaves, suggesting their potential roles in Cu transport or detoxification. Additionally, AcHMA2, AcHMA5, and AcHMA7 exhibited moderate expression in leaves, while AcHMA1, AcHMA2, and AcHMA3 appeared downregulated in roots. Our findings are consistent with those of the existing literature. Noteworthy, AcHMA5 upregulation in roots corresponds to the reported involvement of AtHMA5, OsHMA5, and PtHMA5 in Cu transport from the roots to the shoots and Cu detoxification. Furthermore, the increased expression of AcHMA8 in leaves reflects the recognized roles of AtHMA8, PtHMA7, and PtHMA8 in Cu transport, emphasizing how these roles were conserved across different plant species throughout evolution. In conclusion, based on the gene expression profiles under different heavy metal stress treatments, we proposed a schematic model, which summarized the key AcHMA genes that play role in response to different heavy metal stress in Areca (Fig. 7). This study comprehensively explores the functional roles and expression patterns within the HMA gene family of A. catechu. Our results will lay a foundation for further analysis of how AcHMA gene responds to heavy metal stress in areca.

Fig. 7

Schematic model showing subcellular distribution of HMA and expression of AcHMA transporters in roots and leaves of A. catechu under Cd²⁺, Zn²⁺ and Cu²⁺ stress. The red font shows a significant overexpression, the green font indicates significant downregulation, and the black font indicates no significant gene expression change