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].The heavy-metal ATPase are transmembrane transporters playing 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 were lower than 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 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 [50–52]. The OsHMA3 and OsHMA4 in Oryza sativa are located at 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 for comprehending the gene family. Significant evolutionary changes occur in eukaryotic genes as a result of intron gain or loss, which entails the insertion and deletion of 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 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 signaling 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 unraveling 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 the detoxification of 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 for investigationg the expression of AcHMA under normal condition 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 hours. Notably, AcHMA1 and AcHMA2 genes exhibited the highest upregulation in response to Cd2+ and Zn2+ stresses both 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 indicating 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 that are 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 a variety of 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 tonoplast. Similar patterns of gene expression had been identified both in O.sativa and A.Thalian, and our findings are in line with those earlier studies. As a result of zinc stress, previous study reported elevated levels of AtHMA1, OsHMA1, and OsHMA2 expression in the tonoplast, which indicates that these genes are playing 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.
The red font shows a significant overexpression, the green font indicates significant downregulation and the black fonts indicates no significant change in the gene expression.