Genome-wide survey of the HMA gene family in wheat (Triticum aestivum) and its potential role in cadmium stress

Genome-wide survey of the HMA gene family in wheat (Triticum aestivum) and its potential role in cadmium stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide survey of the HMA gene family in wheat (Triticum aestivum) and its potential role in cadmium stress Leilei Shao, Xuyu Guo, Yang Wang, Tianzhen Lei, Yijie Wan, Lingjian Ma, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5264727/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in BMC Genomics → Version 1 posted 8 You are reading this latest preprint version Abstract Cadmium has been accumulating in the agricultural and ecological environment in recent years due to the release of industrial pollutants. Due to its high solubility, slow degradability and high toxicity, it is highly susceptible to occurring in agricultural fields. The presence of cadmium at low concentrations is harmful to plants. Heavy metal ATPases (HMAs) are proteins that can detoxify high concentrations of heavy metals through vacuole compartmentalization or exocytosis pathways. They have been extensively studied in plants. However, the cadmium transport function of HMAs in wheat has not been explored. In this study, a comprehensive and systematic investigation of HMA gene family members in wheat was conducted. A total of 28 putative TaHMAs were identified. Phylogenetically, these 28 putative TaHMAs were divided into two subgroups: Cu/Ag and Zn/Co/Cd/Pb. The gene structures and conserved motifs were consistent within the same branch and diverse in different branches. The TaHMA gene family is closely related to rice, B. distachyon and A. tauschii. GO analysis results suggest that TaHMAs may be involved in cation transport and membrane components. Protein interaction analysis results suggest that TaHMAs may interact with TaSOD to activate the SOD defense mechanism in wheat. Expression patterns exhibited tissue specificity. Finally, the expression patterns of TaHMAs were validated in the roots and leaves of wheat plants under cadmium stress. Our findings will be valuable for functional studies and applications of HMA gene family members in wheat. P1B-type ATPases Cd stress Cd transport Wheat Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction In recent years, the use of sewage irrigation, pesticides and chemical fertilizers has led to a large accumulation of heavy metals in the soil, posing an increasing threat to plants [1]. The harm of heavy metals to plants includes the existence of non-essential metals such as cadmium (Cd), lead (Pb) and mercury (Hg), and the excessive accumulation of essential metals such as copper (Cu), zinc (Zn) and manganese (Mn). Both the existence of non-essential metals and the excessive accumulation of essential metals are harmful to the growth and development of plants. [2]. Cadmium is one of the most toxic but non-essential heavy metals for all organisms [3]. In China, 7% of farmland is contaminated with cadmium. For example, in Dazhang Town, Xinxiang, Henan Province, at least 150,000 kilograms of grain are produced annually. However, the Cd level in local wheat is 1.7–12.8 times higher than the limit specified in the national food standards[4]. Cadmium accumulates in wheat grains. When people consume wheat products contaminated with cadmium, cadmium will enter the human body accordingly, thus causing great harm to human health. Therefore, it is crucial to reduce the accumulation of cadmium (Cd) in wheat. The stress induced by cadmium can cause damage to genomic DNA, mitochondria, chloroplasts and other organelles, and it also disrupts metabolic processes such as photosynthesis and protein hydrolysis, which are extremely adverse to plants' growth and development. [5, 6]. To cope with the stress of heavy metals, plants have various transmembrane transporters in different organelles, such as P1B-ATPases, ATP-binding cassette gene family (ABC), metal tolerance protein gene family (MTP ), family of cation diffusion promoters (CDF) and calcium exchanger gene family (CAX), which can seal and redistribute heavy metals [7]. P1B-ATPases are a type of transporter in which heavy metals are excreted from cytoplasm or transported to vacuole. P1B-ATPases are a family of genes that can transport metal cations across the membrane by hydrolyzing ATP, also known as CPx-ATPases [8], metal P-type ATPase and HMAs [9]. There are eight transmembrane helices in structure[10], CPx/SPC motif in transmembrane domain 6, and assumed transition metal binding domains at N-terminal and/or C-terminal [11]. Many HMA genes have been found in many plants, which have different heavy metal transport functions. Among the eight P1B - ATPases in Arabidopsis thaliana, AtHMA1–4 belong to the Zn/Co/Cd/Pb subgroup, and AtHMA5–8 belong to the Cu/Ag subgroup [12]. AtHMA1 can reduce the accumulation of Zn in Arabidopsis thaliana treated with a high concentration of Zn [13]. AtHMA2 and AtHMA4 can transport Zn and Cd into xylem, thus promoting the transport of Zn and Cd from roots to leaves [14]. AtHMA3 is located in the root vacuole, which can chelate Cd in the vacuole to regulate the accumulation in plants [15]. AtHMA4 may transport Zn from the cells around the root vascular tissue [16]. AtHMA5 can transport Cu into the root xylem and drive Cu to the shoot [17]. AtHMA6/PAA1 and AtHMA8/PAA2 are high-affinity Cu transporters located in chloroplasts. They control the absorption of Cu by chloroplasts, transport Cu to the chloroplast matrix and thylakoid, and maintain photosynthesis and antioxidation [18]. AtHMA7/RAN1 is the first P1B-ATPase in plants and it inputs Cu into the endoplasmic reticulum to synthesize the ethylene receptor [19]. In other plants, there are many reports about the functions of HMA genes. For example, SpHMA1 can reduce the excessive accumulation of Cd in chloroplasts and maintain leaf photosynthesis [20]. In barley, HvHMA2 is not only responsible for the transport of Cd from root to stem [21] but also improves the transport capacity of Zn under the condition of Zn deficiency [22]. OsHMA3 has the same function as AtHMA3 [23]. GmHMA3 is located in the endoplasmic reticulum of roots and can chelate Cd on the endoplasmic reticulum, reducing the transport of Cd from roots to stems [24]. In addition, members of the P1B-ATPase family have been found in different plants, including rice, corn, sorghum and barley in monocotyledons, and Arabidopsis, soybean, flax, rape and Populus tomentosa in dicotyledons. In bread wheat, the characterization of the HMA gene family under Cu stress has been reported [25], and the overexpression of the family member TaHMA2 ameliorated the translocation of zinc/cadmium from roots to buds [26]. However, analyses of the wheat HMA family are still incomplete. We conducted whole gene identification, analyzed the physical and chemical properties, gene structure and conserved motifs, performed promoter analysis, and studied the expression patterns under cadmium stress of HMA gene family members. To further provide a theoretical basis for exploring the cadmium transport function and molecular mechanism of HMA in wheat, subsequent research can utilize technologies such as gene editing and molecular-assisted breeding to study the HMA gene family, which will provide valuable insights for the development of wheat varieties with low cadmium accumulation. 2. Materials and methods 2.1 Identification of TaHMAs in wheat Whole-genome IWGSC v1.1 data and the genome annotation information Gff3 data of wheat ( Triticum aestivum L. ) was available at Ensembl Plants database ( https://plants.ensembl.org/Triticum_aestivum/Info/Index ). Tair ( https://www.arabidopsis.org/ ) and The National Rice Center ( https://www.ricedata.cn/ ) to download the known HMA family member protein sequences of Arabidopsis and Rice. The HMA-Domaining (PF00403), E1-E2_ATPase-Domaining (PF00122), Hydrolase-Domaining (PF00702) were downloaded from Pfam database ( http://pfam.xfam.org/ ). Using the HMMER3.0 program to identify proteins containing three domains in the whole wheat genome with e < 1e − 5 as threshold. Then, the protein sequences of known HMA members of Rice and Arabidopsis as query sequences using BLASTP program set threshold to e < 1e − 5 and 50% identity to search against the wheat protein dataset. By analyzing the results of HMM and BLAST preliminarily putative wheat HMA genes. The candidate HMA genes of wheat were further confirmed through the NCBI-CDD web server( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). Theoretical isoelectric points (PI) and molecular weight (MW) of TaHMAs were predicted using the ExPASy server ( https://web.expasy.org/compute_pi/ ) and the subcellular localization of these proteins was predicted using the CELLO web server ( http://cello.life.nctu.edu.tw/ ). 2.2 Phylogenetic analysis Phylogenetic analysis was performed using the HMA protein sequences of wheat, Arabidopsis and rice. A rootless neighbor joining (NJ) tree with 1000 bootstrap repeats was constructed using MEGA 6.0 software. 2.3 Chromosomal Location, Gene Structure and Conserved Motif Analysis of TaHMAs Gene The chromosomal location of the TaHMA genes was obtained from the wheat genome annotation information Gff3, and the TaHMA genes were mapped to the wheat chromosome. The gene structure and coding sequence (CDS) of TaHMAs were analyzed to study the distribution of exons. Conserved motifs of TaHMA proteins were identified using MEME ( https://meme-suite.org/meme/doc/meme.html ). The optimal motifs were set at ≥ 10 and ≤ 200 amino acids, and the maximum motifs were set at 15. The results will be displayed on evolview ( https://www.evolgenius.info/evolview-v2/#mytrees/2/2 )with the phylogenetic tree, gene structure and conserved motif of wheat HMA gene. 2.4 Gene synteny analysis of wheat with rice, A. tauschii , and B. distachyon MCScanX software was used to detect the collinear region of TaHMA gene in wheat and other three species ( Oryza sativa , Brachypodium distachyon (L.) Beauv. and Aegilops tauschii Coss. ). Circos 0.67 was used to visualize TaHMA gene duplication events and the synchronous relationship between other species. Compare the CDS and protein sequences of collinear gene pairs, and calculate the KaKs ratio by TBtools software. Finally, use [T = Ks/(2 λ × 10 − 6) Mya( λ = six point five × 10 − 9)] Calculate the divergence time of the collinear gene pair. 2.5 GO annotation and protein-protein interaction network analysis of TaHMAs GO annotations of wheat HMA proteins are available from the Plaze database ( https://bioinformatics.psb.Be/plaza/verages/plaza_v4_moncots/ ) and the Plant Transcriptional Regulation Map Database ( http://plantregmap.gao-lab.org/ ) GO annotation results were visualized using WEGO ( https://wego.genomics.cn/ ) online tool. Based on the orthologs of wheat and Arabidopsis, the interaction between TaHMA protein and other wheat proteins was constructed using ArenaNet V2 tool and string database ( https://string-db.org/cgi/input.pl ) network. Trust values > = 0.95 in the string database were used to identify the interaction network, which was graphically displayed by Cytoscape software. 2.6 Promoter Analysis of TaHMA Gene Download the upstream 2kb DNA sequence of TaHMA gene from Ensemble plant database, and then use the PLACE database to predict the cis regulatory elements of the promoter region (bioinformatics.psb.ugent.be/webtools/plantcare/html/). 2.7 Gene expression, heavy metal content determination and qRT-PCR analysis of TaHMAs For qRT-PCR, Chinese spring wheat varieties were grown in greenhouses (24 ± 2℃, 75 ± 5% relative humidity, 16/8h light/dark cycle and 125 µmol/m 2 /s photosynthetic photon flux density) in 2022. Wheat roots and shoots were harvested at the seedling stage. One leaf and one heart seedlings were treated with 40 mM and 400 mM CdCl 2 for 14 days, and then samples were collected. All samples were stored at − 80°C for use with RNAiso reagent and digestion with three biological replicates. cDNA was synthesized with RT Master Mix Perfect Real Time Kit (Takara, Beijing, China). TaHMA Quantitative reverse transcription polymerase chain reaction was performed with QuantStudio real-time qPCR software. The relative expression levels of TaHMA in nine randomly selected groups were determined through 2(−ΔΔCt) analysis. We selected TraesCS6B02G243700 as the reference gene to normalize the expression level of TaHMA gene. 3. Results 3.1 Genome-wide identification and chromosomal localization of the TaHMA gene Two methods, HMM and BLASTP, were used to obtain the TaHMA genes from the IWGSC v1.1 whole-genome data of wheat, and CD-search was used for further confirmation. There were 28 genes named TaHMA genes. According to chromosomal location, they were renamed TaHMA001 to TaHMA028. All genes were unevenly distributed on the wheat chromosomes. There were 10 genes located in the A and D genomes, respectively, and the remaining 8 genes were located in the B genome. The TaHMA genes are mainly concentrated on chromosomes 2 and 4–7, rather than on chromosomes 1, 3, and 4B. We submitted TaHMA protein sequences to the Cello website to predict their subcellular localization (Additional file 1: Table S1 ). Most of the TaHMs were located in the plasma membrane, except for TaHMA004, TaHMA005, TaHMA015, TaHMA020 and TaHMA024, which were located in the chloroplast. It is suggested that TaHMA004, TaHMA005, TaHMA015, TaHMA020 and TaHMA024 may function in the chloroplast, whereas the other TaHMs may function in the plasma membrane. Using ExPASy, we revealed that the protein lengths and relative molecular weights of TaHMA proteins were diverse (Additional file 1: Table S1 ). The relative molecular weight of TaHMA proteins ranged from 81092.87 to 110320.38. The protein lengths varied from 766 to 1023 aa. Among them, the longest was TaHMA021, and the shortest was TaHMA022 3.2 Phylogenetic analysis To find out the evolutionary relationship of wheat HMA genes, we constructed a phylogenetic tree using the HMA protein sequences of Arabidopsis, rice and wheat (Fig. 1 ). According to phylogenetic analysis, the Zn/Co/Cd/Pb subgroup and the Cu/Ag subgroup are the two major categories of HMA genes. According to previous research results and the phylogeny and classification of the wheat HMA gene family, AtHMA1 - AtHMA4, OsHMA1 - OsHMA3, TaHMA006, TaHMA007, ​​TaHMA008​​, TaHMA016, TaHMA017, TaHMA021, TaHMA022, TaHMA025, TaHMA026 belong to the Zn/Co/Cd/Pb subgroup, while AtHMA5 - AtHMA8, OsHMA4 - OsHMA9 and the rest of the TaHMAs belong to the Cu/Ag subgroup. 3.3 Gene structure and motif composition of the wheat HMA gene family In the evolution of gene families, the type and number of exons and introns play an important role. We analyzed the gene structure of TaHMAs (Fig. 2 ) and found that the number of exons in the TaHMA genes varies from 6 to 17. Each TaHMA gene has complete UTRs (non-coding regions). TaHMA001–003 and TaHMA006–008 have the least number of exons, with 6 exons each. TaHMA004, TaHMA005, TaHMA020 and TaHMA024 have the largest number of exons, with 17 exons each. Different branches have different numbers of exons and introns, while members of the same branch exhibit similar numbers of exons and introns. This suggests that TaHMA genes are conservative and diverse. To further explore the structural diversity of TaHMA genes and predict their potential functions, we used MEME Online software to analyze 28 TaHMAs, detect their motif composition, and explore motif diversity (Fig. 2 ). From the results, we found that members within the same subclass mostly share a similar structure, whereas members of different subclasses exhibit slight variations. All TaHMA genes contain motifs_1, motifs_2, motifs_3, motifs_5, motifs_6, motifs_7, motifs_9, and motifs_13, but there are specific motifs among different branches. Associated with the phylogeny, we can see that the members of the Zn/Co/Cd/Pb subgroup all have motifs ending with motif_8. The Cu/Ag subfamily members are characterized by motifs ending with motif_5 or motif_15. The motif_10 is exclusively present in the members of the Cu/Ag subfamily. Among the subgroup members, TaHMA018 and TaHMA027 have the minimum number of motifs, with 10 motifs, and both start with motif_15. TaHMA014 has the maximum number of motifs, with 20 motifs, starting at motif_13. 3.4 Collinear analysis Duplication is an essential mechanism in the evolution of genomes and genetic systems. It can provide raw materials for the creation of new genes and functions. Therefore, we analyzed the duplication events of the TaHMA gene family (Fig. 3 ), calculated the Ka/Ks ratio, and estimated the divergence time (Additional file 1: Table S3 ). After detection, 35 duplicate pairs of fragments were found in wheat varieties. All TaHMAs, except for TaHMA017, TaHMA022, and TaHMA026, had duplicate pairs, encompassing all members of the TaHMA family. This indicates that gene fragment duplication is ​​a mechanism​​ for the expansion of TaHMAs in wheat, and the presence of multiple homologous genes confirms the conservation of the gene family. The ratio of Ka/Ks within wheat varieties is lower than 1, indicating that HMA genes ​​have undergone​​ purification and selection within wheat varieties. By calculating the divergence time, it is estimated that the divergence time among wheat varieties is approximately 20 years. To understand the origin of the HMA family, collinearity analysis was conducted at the whole-genome level among wheat, Oryza sativaL , Brachypodium distachyon and Triticum crassum (Fig. 3 ). The results showed that there were 22, 24, and 26 collinear pairs between wheat and each of the other species, respectively. The ratio of Ka/Ks was lower than 1, indicating that these species had undergone purifying selection ​​along with​​ wheat. The HMA family was conserved during the evolution of these species. The divergence time between wheat and Oryza sativaL (Additional file 1: Table S4 ), Brachypodium distachyon (Additional file 1: Table S5 ) and Triticum crassum (Additional file 1: Table S6 ) is 46 Mya, 57 Mya and 15 Mya, respectively. This suggests that the HMA family in wheat is closely related to Oryza sativaL , Brachypodium distachyon and Triticum crassum .. 3.5 GO annotation and protein-protein interaction network analysis of TaHMA The members of the wheat HMA gene family were annotated using Gene Ontology (GO) (Fig. 4 , Additional file 1: Table S7 ), and a series of processes, namely Biological Process, Cellular Component, and Molecular Function, that TaHMA members might participate in were analyzed. When annotating the 28 genes, it was found that no GO terms were detected for TaHMA022, and the rest were assigned 20 GO terms. The most enriched categories were cation transport, integral component of membrane, nucleotide binding, and ATPase-coupled cation transmembrane transporter activity. All TaHMAs may be involved in these processes. In terms of biological processes, in addition to all participating in cation transport, 25 genes may participate in metal ion transport, and 9 participate in cation transmembrane transport. Only TaHMA017, TaHMA021, and TaHMA025 participate in responses to metal ions, zinc ion transport, cadmium ion transport, responses to cobalt ion, etc., while TaHMA020 and TaHMA024 are involved in copper ion homeostasis and the photosynthetic electron transport chain. In terms of cellular component, in addition to all participating in the integral component of membrane, 9 participate in the membrane, and a small number are involved in plasmodesma, plasma membrane, chloroplast envelope, etc. In terms of Molecular Function, in addition to all participating in nucleotide binding and ATPase-coupled cation transmembrane transporter activity, 25 participate in metal ion binding, 12 potentially participate in copper ion binding, 9 participate in ATP binding, and 2 participate in copper ion transmembrane transporter activity and copper chaperone activity. Prediction of protein interactions for TaHMAs (Fig. 5 , Additional file 1: Table S8 ). No interactive protein ID corresponding to TaHMA006 was found during protein mapping. Nine genes of TaHMA4, TaHMA2 and TaHMA0 were predicted to interact with TraesCS2A02G114900 and TraesCS2B02G134100. All proteins interact with TraesCS2A02G399000 and TraesCS2B02G417000. TaHMA015, TaHMA020, TaHMA024 interact with TaHMA003 in addition to TraesCS2D02G495500LC (Note: It seems there is a repetition of TaHMA024 here, you may need to double-check). Interaction was observed between six genes belonging to TaHMA6 and TaHMA9 and TraesCS5A02G030100. The five genes belonging to TaHMA1 and TaHMA7 interact with TraesCS7A02G335400, TraesCS7B02G247000 and TraesCS7D02G342900. TraesCS2A02G399000 and TraesCS2B02G417000 were predicted by NCBI CDD to be Superoxide dismutase copper chaperones. TaHMAs interact with them to activate the SOD defense mechanism in wheat. 3.6 Promoter Analysis of TaHMA Gene The expression of genes is regulated by splicing regulatory elements, which generally exist in the promoter region. Therefore, we analyzed and predicted the splicing regulatory elements in the promoter region. We analyzed the promoter regions of 28 TaHMA genes by selecting 2 kb DNA sequences upstream of the genes for prediction and obtained potential splicing regulatory elements (Fig. 6 ). By predicting the promoter regions of the TaHMA genes, we found that most of the elements are splicing regulatory elements involved in hormone response, light response, and growth and development. All TaHMAs contain light-responsive elements. For example, G-box and Sp1. There is a G-box in each TaHMA, and all 18 TaHMA genes contain Sp1, indicating that TaHMA may respond to light regulation. Except for TaHMA011, ABRE elements related to abscisic acid response are present in all other family members. The CGTCA motif and TGACG motif components associated with the MeJA reaction are not present in TaHMA023, whereas the remaining TaHMAs contain them. Anaerobic induction-related ARE elements are present in all 23 members of the HMA family. The hypoxia-specific inducing element GC-motif is present in 16 members. The low-temperature response (LTR) component is involved in 14 members. MBS, which participates in drought-induced stress, is present in 13 members. At the same time, some elements related to plant growth and development were found in the TaHMA genes, such as CAT box, circadian, and GCN4_motif, etc. This suggests that TaHMA may also be involved in regulating wheat growth and development. 3.7 Gene expression, heavy metal content determination and qRT-PCR analysis of TaHMAs Downloading the expression data of TaHMAs in four tissues (roots, stems, leaves and spikes) of Chinese Spring from the Wheatomics website reveals that most TaHMAs have higher expression levels in roots compared to the other individual tissues (Fig. 7 ). This suggests that TaHMAs have strong tissue-specific expression and may play a major role in roots. Seedlings of common wheat were treated hydroponically with 40 µM and 100 µM CdCl₂ solutions. Roots and leaves were collected at 2 h, 5 h, 12 h, 24 h, 7 d, and 14 d to determine their Cd content (Fig. 8 ). The results of Cd content determination revealed that there was no significant change in the Cd content of aboveground and belowground parts from 2 h to 12 h, whereas there was a trace increase at 24 h and a significant increase in Cd content at 7 d and 14 d. Therefore, total RNA was extracted from the roots and leaves of seedlings treated for 24 h, 7 d, and 14 d. The expression of TaHMAs was determined by fluorescence real-time quantitative PCR (Fig. 9 , Additional file 1: Table S2 ). In the roots, TaHMA4 was up-regulated at 24 h, 7 d, and 14 d, and the up-regulation became more pronounced over time, with a 24-fold up-regulation at 14 d. TaHMA2, TaHMA3, and TaHMA5 were also up-regulated, with the most pronounced up-regulation at 14 d. In contrast, the remaining TaHMAs showed down-regulation. In the leaves, TaHMA1, TaHMA6, TaHMA7, TaHMA8, and TaHMA9 all showed down-regulation, with TaHMA1 being the most significantly down-regulated. Although TaHMA2 showed some up-regulation, it was not significant. The responses of TaHMA3, TaHMA4, and TaHMA5 were not significant. The results showed that under cadmium treatment, TaHMAs exhibited different responses at different sites. 4. Discussion 4.1 Characteristics of the TaHMA family in wheat HMA genes are a kind of protein that transports heavy metals by hydrolyzing ATP. They play an important role in the transport and storage of heavy metals. They have been identified in rice, maize, sorghum and other monocotyledons[27], as well as in dicotyledons such as Arabidopsis, soybean and rapeseed[28]. The results of whole-genome sequencing of Chinese Spring wheat provide crucial foundational information for identifying the HMA gene family in wheat. Twenty-eight HMA family members of wheat were identified by protein and important domain alignment. As wheat is allohexaploid, the number of TaHMAs is significantly higher than that of other species such as rice and Arabidopsis. The genetic structure and conserved motifs of the TaHMA gene family were analyzed by constructing a phylogenetic tree. In different branches, TaHMAs have varying numbers of exons and introns, whereas in the same branch, they have similar numbers of exons and introns. Different TaHMAs contain eight conserved motifs, but there are also specific motifs, indicating that the TaHMA gene family is conserved and diverse. 4.2 The evolutionary relationship of the TaHMA family HMA is subdivided into two major subgroups: the Cu/Ag subgroup and the Zn/Cd/Co/Pb subgroup[29]. Based on the phylogenetic tree, TaHMA in the Cu/Ag subgroup is further divided into A1, A2 and B branches, while the Zn/Cd/Co/Pb subgroup is subdivided into C and D branches. In the calculation of Ka, Ks and divergence time, TaHMA009, TaHMA011 and TaHMA0013 have higher Ks values than TaHMA019, TaHMA023 and TaHMA028 in the A2 branch of the Cu/Ag subgroup. Additionally, they have a longer divergence period compared to the other gene pairs. At the same time, in order to understand the evolutionary relationship with other species, collinearity analysis was conducted, and the Ka, Ks and divergence time were calculated[30]. Most TaHMA genes are replicated in Oryza sativaL , Brachypodium distachyon and Aegilops TaHMA. The Ks of TaHMA019, TaHMA023, TaHMA028, TaHMA011 and TaHMA013 were higher than those of Aegilops tauschii, indicating a longer divergence time. In collinearity analysis with Brachypodium distachyon , the Ks of TaHMA004 and TaHMA005 with Brachypodium distachyon are greater than 1, and the divergence time is more than 200 years. In collinearity analysis with Oryza sativaL , KS is less than 1, the divergence time is about 46 years. Except for TaHMA019, TaHMA023, TaHMA028, TaHMA011 and TaHMA013, the divergence times between wheat and Aegilops for TaHMA019 and TaHMA023 are all less than 9 years or even as low as 0.1 years. It is inferred that HMA evolved slowly between wheat and Aegilops tauschii . It can be inferred that HMA is more closely related to wheat and Aegilops tauschii than to Oryza sativaL and Brachypodium distachyon . 4.3 Expression patterns and potential functions of HMA gene family in wheat In this study, we conducted Gene Ontology (GO) prediction for TaHMAs and found that all TaHMAs are involved in the cation transport process, and most of them are also involved in the metal ion transport process. Meanwhile, we retrieved the expression profiles of TaHMA genes from the published transcriptome data of Chinese Spring wheat to understand their tissue expression patterns. It was found that the expression levels of almost all TaHMA genes in roots are higher than those in stems, leaves, spikes, and seeds. Based on the GO prediction and tissue expression patterns, we inferred that TaHMA genes may mainly function as heavy metal transport proteins in roots. At the same time, we predicted the protein-protein interactions of TaHMA genes and found that all TaHMAs interact with TraesCS2A02G399000 and TraesCS2B02G417000, both of which belong to the copper chaperone of superoxide dismutase (Cu/Zn-SOD) protein family within the SOD (superoxide dismutase) gene family[31]. Therefore, we speculate that TaHMAs may interact with TraesCS2A02G399000 and TraesCS2B02G417000 during the transport of cations and metal ions, thereby activating the defense mechanism of wheat. The phylogenetic tree shows that wheat HMA family members have a higher homology with rice genes. TaHMA2 and TaHMA3 are homologous to three Arabidopsis genes (AtHMA2, AtHMA3, and AtHMA4) and two rice genes (OsHMA2 and OsHMA3). AtHMA2 and AtHMA4 in Arabidopsis have been demonstrated to be responsible for the translocation of Cd from roots to stems and for enhancing Cd tolerance in plants [14]. In Arabidopsis, rice and other plants, HMA3 has also been shown to be chelated in the vacuole to participate in Cd detoxification [32]. Under Cd treatment, TaHMA2 and TaHMA3 were up-regulated in the root system of wheat, and TaHMA2 was also expressed in the leaves. In this way, it is hypothesized that TaHMA2 and TaHMA3 may play similar roles in Cd transport as those in Arabidopsis and rice. TaHMA4 and TaHMA5 are homologous to an Arabidopsis gene (AtHMA5) and two rice genes (OsHMA4 and OsHMA5). AtHMA5 is localized in the root plasma membrane and functions as an exporter of Cu ions, mitigating copper toxicity. On the other hand, OsHMA4 acts as a sequestering agent for Cu in root vesicles, thereby restricting the accumulation of Cu in grains. OsHMA5 is involved in loading Cu into the xylem of roots and other organs [17]. AtHMA5, OsHMA4, and OsHMA5 have a Cd-transport function that remains unexplored. In wheat, TaHMA4 and TaHMA5 ​​show​​ up-regulation in response to Cd stress. Particularly, TaHMA4 showed a significant up-regulation. Therefore, further studies could be conducted to explore whether TaHMA4 and TaHMA5 have a Cd-transporting function. 5. Conclusion We identified the HMA gene family in common wheat, which was divided into two subgroups, Zn/Co/Cd/Pb and Cu/Ag, based on the phylogeny. The gene structure and conserved motifs showed some conservation within the same branch, while there was diversity among different branches. GO annotation, promoter analysis, covariance analysis and protein-protein interactions serve as the foundation for comprehending the evolutionary history, functional analysis, and other related aspects of the HMA family. The analysis of tissue expression patterns and the response to Cd stress indicated that TaHMA genes exhibit tissue-specific expression and some of them are involved in Cd ion transport in the root system. These analyses provide a basis for subsequent studies on the function of the TaHMA gene and the discovery of the molecular mechanism of cadmium transport in wheat. Abbreviations TaHMA: Wheat HMA, Cd: Cadmium, CDS: Coding sequence, HMM: Hidden Markov Model, qRT-PCR: Quantitative real-time polymerase chain reaction Declarations Acknowledgements We are very grateful to Professor Ma, Professor Niu and Professor Ding for their guidance. We thank the Instrument sharing platform of Northwest Agricultural and Forestry University and lab members for their assistance in this study. Funding This study was supported by Key Research and Development Projects of Shaanxi Province (No. 2021CDLNY01-02). The funding body was not involved in the design of the study, analysis or interpretation of data or writing the manuscript. Authors and Affiliations College of Agronomy, Northwest A&F University, Yangling 712100, China Leilei Shao, Haosen Ma, Zhan Su, Tianzhen Lei, Xuyu Guo, Yang Wang, Yijie Wan, Lingjian Ma*, Na Niu* Lingjian Ma*, [email protected] Authors’ contributions Leilei Shao conceived and designed the study. Leilei Shao and Haosen Ma performed the experiments. Zhan Su, Tianzhen Lei, Xuyu Guo, Yang Wang and Yijie Wan analysed the data. All authors read and approved the manuscript. Corresponding authors Correspondence to Lingjian Ma*, [email protected] Na Niu*, [email protected] Availability of data and materials The datasets supporting the results of this article are included in the article and Additional files. Ethics approval and consent to participate This article does not contain any studies with human participants or animals and did not involve any endangered or protected species. Competing interests The authors declare that they have no competing interests. References Qin GW, Niu ZD, Yu JD, Li ZH, Ma JY, Xiang P: Soil heavy metal pollution and food safety in China: Effects, sources and removing technology . CHEMOSPHERE 2021, 267 . Ghori NH, Ghori T, Hayat MQ, Imadi SR, Gul A, Altay V, Ozturk M: Heavy metal stress and responses in plants . INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCE AND TECHNOLOGY 2019, 16 (3):1807-1828. Siedlecka A, Krupa Z: Interaction between cadmium and iron. Accumulation and distribution of metals and changes in growth parameters of Phaseolus vulgaris L seedlings . ACTA SOCIETATIS BOTANICORUM POLONIAE 1996, 65 (3-4):277-282. Zaid IU, Zheng X, Li X: Breeding Low-Cadmium Wheat: Progress and Perspectives . 2018, 8 (11):249. 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Chao D-Y, Silva A, Baxter I, Huang YS, Nordborg M, Danku J, Lahner B, Yakubova E, Salt DE: Genome-Wide Association Studies Identify Heavy Metal ATPase3 as the Primary Determinant of Natural Variation in Leaf Cadmium in Arabidopsis thaliana . PLOS GENETICS 2012, 8 (9). Verret F, Gravot A, Auroy P, Leonhardt N, David P, Nussaume L, Vavasseur A, Richaud P: Overexpression of AtHMA4 enhances root-to-shoot translocation of zinc and cadmium and plant metal tolerance . FEBS LETTERS 2004, 576 (3):306-312. Deng FL, Yamaji N, Xia JX, Ma JF: A Member of the Heavy Metal P-Type ATPase OsHMA5 Is Involved in Xylem Loading of Copper in Rice . PLANT PHYSIOLOGY 2013, 163 (3):1353-1362. Catty P, Boutigny S, Miras R, Joyard J, Rolland N, Seigneurin-Berny D: Biochemical Characterization of AtHMA6/PAA1, a Chloroplast Envelope Cu(I)-ATPase . JOURNAL OF BIOLOGICAL CHEMISTRY 2011, 286 (42):36188-36197. Li W, Lacey RF, Ye Y, Lu J, Yeh K-C, Xiao Y, Li L, Wen C-K, Binder BM, Zhao Y: Triplin, a small molecule, reveals copper ion transport in ethylene signaling from ATX1 to RAN1 . PLOS GENETICS 2017, 13 (4). Zhao H, Wang L, Zhao F-J, Wu L, Liu A, Xu W: SpHMA1 is a chloroplast cadmium exporter protecting photochemical reactions in the Cd hyperaccumulator Sedum plumbizincicola . PLANT CELL AND ENVIRONMENT 2019, 42 (4):1112-1124. Guo Q, Tian X, Mao P, Meng L: Functional characterization of IlHMA2, a P-1B2-ATPase in Iris lactea response to Cd . ENVIRONMENTAL AND EXPERIMENTAL BOTANY 2019, 157 :131-139. Mills RF, Peaston KA, Runions J, Williams LE: HvHMA2, a P-1B-ATPase from Barley, Is Highly Conserved among Cereals and Functions in Zn and Cd Transport . PLOS ONE 2012, 7 (8). Miyadate H, Adachi S, Hiraizumi A, Tezuka K, Nakazawa N, Kawamoto T, Katou K, Kodama I, Sakurai K, Takahashi H et al : OsHMA3, a P-1B-type of ATPase affects root-to-shoot cadmium translocation in rice by mediating efflux into vacuoles . NEW PHYTOLOGIST 2011, 189 (1):190-199. Wang Y, Yu K-F, Poysa V, Shi C, Zhou Y-H: A Single Point Mutation in GmHMA3 Affects Cadimum (Cd) Translocation and Accumulation in Soybean Seeds . MOLECULAR PLANT 2012, 5 (5):1154-1156. Batool TS, Aslam R, Gul A, Paracha RZ, Ilyas M, De Abreu K, Munir F, Amir R, Williams LE: Genome-wide analysis of heavy metal ATPases (HMAs) in Poaceae species and their potential role against copper stress in Triticum aestivum . SCIENTIFIC REPORTS 2023, 13 (1). Tan JJ, Wang JW, Chai TY, Zhang YX, Feng SS, Li Y, Zhao HJ, Liu HM, Chai XP: Functional analyses of TaHMA2, a P1B-type ATPase in wheat . PLANT BIOTECHNOLOGY JOURNAL 2013, 11 (4):420-431. Zhiguo E, Li TT, Chen C, Wang L: Genome-Wide Survey and Expression Analysis of P1B-ATPases in Rice, Maize and Sorghum . RICE SCIENCE 2018, 25 (4):208-217. Fang XL, Wang L, Deng XJ, Wang P, Ma QB, Nian H, Wang YX, Yang CY: Genome-wide characterization of soybean P1B-ATPases gene family provides functional implications in cadmium responses . BMC GENOMICS 2016, 17 . Li DD, Xu XM, Hu XQ, Liu QG, Wang ZC, Zhang HZ, Wang H, Wei M, Wang HZ, Liu HM et al : Genome-Wide Analysis and Heavy Metal-Induced Expression Profiling of the HMA Gene Family in Populus trichocarpa . FRONTIERS IN PLANT SCIENCE 2015, 6 . Kong YM, Xu P, Jing XY, Chen LX, Li LG, Li X: Decipher the ancestry of the plant-specific LBD gene family . BMC GENOMICS 2017, 18 . Fukuhara R, Kageyama T: Structure, gene expression, and evolution of primate copper chaperone for superoxide dismutase . GENE 2013, 516 (1):69-75. Chao DY, Silva A, Baxter I, Huang YS, Nordborg M, Danku J, Lahner B, Yakubova E, Salt DE: Genome-Wide Association Studies Identify Heavy Metal ATPase3 as the Primary Determinant of Natural Variation in Leaf Cadmium in Arabidopsis thaliana . PLOS GENETICS 2012, 8 (9). Additional Declarations No competing interests reported. Supplementary Files TableS1.CharacteristicfeaturesoftheHMAgeneinwheat.xlsx TableS2.PrimersusedforqRTPCR.xlsx TableS3.KaKsduplicatedTaHMAgenepairs.xlsx TableS4KaKswheatandrice.xlsx TableS5KaKswheatandA.tauschii.xlsx TableS6KaKswheatandB.distachyon.xlsx TableS7.GOannotationsofTaHMAproteins.xlsx TableS8proteinproteininteraction.xlsx Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 01 May, 2025 Reviews received at journal 23 Apr, 2025 Reviews received at journal 19 Apr, 2025 Reviewers agreed at journal 13 Apr, 2025 Reviewers agreed at journal 10 Apr, 2025 Reviewers invited by journal 10 Apr, 2025 Submission checks completed at journal 09 Apr, 2025 First submitted to journal 09 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5264727","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":441234734,"identity":"bd58c529-3c80-4ce0-9a18-e02ce889625f","order_by":0,"name":"Leilei Shao","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Leilei","middleName":"","lastName":"Shao","suffix":""},{"id":441234735,"identity":"acb7a233-6355-45ef-a84d-ba94fbff0a02","order_by":1,"name":"Xuyu Guo","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Xuyu","middleName":"","lastName":"Guo","suffix":""},{"id":441234736,"identity":"075cc8f9-59d3-451e-95db-c24a85eed5bd","order_by":2,"name":"Yang Wang","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Wang","suffix":""},{"id":441234737,"identity":"1795e929-f3cf-43e7-af3a-4ad0207a5ab3","order_by":3,"name":"Tianzhen Lei","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Tianzhen","middleName":"","lastName":"Lei","suffix":""},{"id":441234738,"identity":"2f714dae-99a1-4990-b0f5-cdd2031684b6","order_by":4,"name":"Yijie Wan","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yijie","middleName":"","lastName":"Wan","suffix":""},{"id":441234739,"identity":"7e907f77-2128-4d3a-8142-be845a3e2108","order_by":5,"name":"Lingjian Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYFAC5oYDDAxsPEAGkGaQAwkZENDCCNPClgDkGROnBcrgMSBOi277wcbDBb/4ZPhn93x7zFNjkNjA3rxNgqHmDk4tZmcSGw7P7GPjkbhzdrsxzzGgFp5jZRIMx57h1nIAqIW3B+iXG7nbpHPY/iQ2SOSYSTA2HMat5fxDiBb5GznPpHP+AW2Rf0NAyw2gLTw/2HgMbuSwSee2AbVI8BDSArKlgY3H8EaamfTfPgPjNp60YouEY/gclnz4M8+fY/ZyN5KfSc74ZiDbz354440PNbi1gAFj2zEEhw1EJODXAAR/aggqGQWjYBSMghEMAMrkV43KjKOwAAAAAElFTkSuQmCC","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":true,"prefix":"","firstName":"Lingjian","middleName":"","lastName":"Ma","suffix":""},{"id":441234741,"identity":"2991cd8a-9d91-4d1c-9ecd-4140657ee95a","order_by":6,"name":"Na Niu","email":"","orcid":"","institution":"Northwest A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Niu","suffix":""}],"badges":[],"createdAt":"2024-10-15 03:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5264727/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5264727/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-025-11746-z","type":"published","date":"2025-07-01T15:58:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80401240,"identity":"58807e01-bd02-4be3-823c-51d7d4eea1d6","added_by":"auto","created_at":"2025-04-11 13:43:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":632549,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic evolutionary tree of HMA gene. The phylogenetic tree forms five branches, with branches A1, A2 and B belonging to the Cu/Ag subgroup (indicated by green brackets), and branches C and D belonging to the Zn/Cd/Co/Pb subgroup (indicated by yellow brackets). OsHMAs are shown in red font, while AtHMAs are shown in blue font.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/f80b311817bbd446fcfaa985.jpg"},{"id":80400406,"identity":"783fe961-87ce-4959-b376-5510590cbba1","added_by":"auto","created_at":"2025-04-11 13:35:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1363481,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic evolutionary tree-based gene structure and conserved motif analysis of TaHMAs. Exons are shown with red boxes, 5'UTR with green boxes and 3'UTR with blue boxes. Different motifs are represented by various colored boxes. The gene structure aligns with the length of the bottom gene sequence, whereas conserved motifs correspond to the length of the bottom protein sequence\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/adc7ae7aec2c30264fffa8c1.jpg"},{"id":80400189,"identity":"bc6cd0c7-8a26-4519-8f8b-43d4e6db9671","added_by":"auto","created_at":"2025-04-11 13:27:31","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1404191,"visible":true,"origin":"","legend":"\u003cp\u003eColinearity analysis of TaHMA genes. Duplicated gene pairs in the wheat genome (a) colinearity analysis of TaHMA genes with rice (b) B. distachyon (c) A. tauschii (d) Thin lines represent genome-wide covariate blocks for the four species, while dark lines represent colinearity gene pairs for TaHMA.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/af81e4a8592a12ffac7caedc.jpg"},{"id":80400186,"identity":"52f38cec-9a32-4f2f-b243-8c8f571b7676","added_by":"auto","created_at":"2025-04-11 13:27:31","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":491582,"visible":true,"origin":"","legend":"\u003cp\u003eGO annotations for TaHMA protein. The x-axis represents the types of GO annotations, which are mainly divided into three categories: cellular components, molecular functions and biological processes. The y-axis represents the number of genes in each GO category.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/c1582bfa3684b710d06860c2.jpg"},{"id":80400416,"identity":"7e4aaf17-10e2-4bbf-a09c-4191b7d42ae0","added_by":"auto","created_at":"2025-04-11 13:35:31","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":777474,"visible":true,"origin":"","legend":"\u003cp\u003eThe protein-protein interaction network between TaHMAs and other wheat proteins. TaHMA proteins are cyan, while other wheat proteins are yellow\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/6324ff78ef05d2cbafc98a8e.jpg"},{"id":80400193,"identity":"82aad9a2-3770-4fcc-9ff2-e739a03b757f","added_by":"auto","created_at":"2025-04-11 13:27:31","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":735729,"visible":true,"origin":"","legend":"\u003cp\u003ePromoter Analysis of the TaHMA gene. Different cis-acting elements are depicted in various colored boxes, where the lengths and positions of the cis-acting elements correspond to those of the bottom DNA sequence\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/b82bef0169e769e90c7b5795.jpg"},{"id":80400414,"identity":"9f37fa20-b6d5-4c40-bea2-e2e6dcc648bb","added_by":"auto","created_at":"2025-04-11 13:35:31","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":889145,"visible":true,"origin":"","legend":"\u003cp\u003eExpression pattern of TaHMA genes. Expression profiles of 28 TaHMA genes in roots, stems, leaves and spikes. High expression levels are shown in red, and low expression levels are shown in blue\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/ba5e4ee43e58bf0ea61cbc58.jpg"},{"id":80401245,"identity":"68db6995-b29c-42c5-a089-30716c6ae81d","added_by":"auto","created_at":"2025-04-11 13:43:31","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":312550,"visible":true,"origin":"","legend":"\u003cp\u003eCd content was determined. The Cd content of roots and leaves of seedling wheat under Cd treatment\u003c/p\u003e","description":"","filename":"Fig.8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/dd2682d63284fd2d63926366.jpg"},{"id":80401552,"identity":"21f18167-a523-4fe3-8a10-197f0245ca3f","added_by":"auto","created_at":"2025-04-11 13:51:31","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":375545,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative RT-PCR analysis of TaHMA genes. Relative expression levels of nine genes under 400 μM Cd treatment\u003c/p\u003e","description":"","filename":"Fig.9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/bccc4645a2bbcaa0e0e9ac8d.jpg"},{"id":86179751,"identity":"a372b2da-d004-41c3-a270-8b1d349d01fa","added_by":"auto","created_at":"2025-07-07 16:19:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9183529,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/fefea553-c71c-452c-b420-15d54e8807c8.pdf"},{"id":80400184,"identity":"5e68d65e-20e3-41f1-9aec-54e3fdb1c57c","added_by":"auto","created_at":"2025-04-11 13:27:31","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28910,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.CharacteristicfeaturesoftheHMAgeneinwheat.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/485d9340bac16dbbf30c8a8b.xlsx"},{"id":80400410,"identity":"c2738ad0-4880-43bd-8a88-7e788259de34","added_by":"auto","created_at":"2025-04-11 13:35:31","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8903,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.PrimersusedforqRTPCR.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/3437d9ef281908a8d9ebb6f6.xlsx"},{"id":80401241,"identity":"6907f8a1-9c84-4f58-9750-08db6d9ac93a","added_by":"auto","created_at":"2025-04-11 13:43:31","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13478,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.KaKsduplicatedTaHMAgenepairs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/1fae678349a60d7f7a0df8e6.xlsx"},{"id":80400407,"identity":"d77f2163-ff35-49d4-937f-522d30194539","added_by":"auto","created_at":"2025-04-11 13:35:31","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13591,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4KaKswheatandrice.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/9c7d706273f9e52b0f292b4d.xlsx"},{"id":80401242,"identity":"a28438ee-dd45-428f-a367-c4ef4cf6fa0c","added_by":"auto","created_at":"2025-04-11 13:43:31","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14368,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5KaKswheatandA.tauschii.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/a07c44a2903a65e30bc9d3d6.xlsx"},{"id":80401551,"identity":"a8b41b9d-b72d-4b4c-b370-e3a74c2adbd6","added_by":"auto","created_at":"2025-04-11 13:51:31","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14156,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6KaKswheatandB.distachyon.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/ad8d1590692f68be480e9fc0.xlsx"},{"id":80400202,"identity":"d07e2230-5c29-4e65-84dc-ad6f893ecbe2","added_by":"auto","created_at":"2025-04-11 13:27:31","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":17906,"visible":true,"origin":"","legend":"","description":"","filename":"TableS7.GOannotationsofTaHMAproteins.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/d86602e8eef178536e23ef3e.xlsx"},{"id":80400411,"identity":"f979fa84-4fc3-4520-88ce-3875b4d7ea28","added_by":"auto","created_at":"2025-04-11 13:35:31","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":12279,"visible":true,"origin":"","legend":"","description":"","filename":"TableS8proteinproteininteraction.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5264727/v1/c9a637624e8bf7d8b60f44ef.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide survey of the HMA gene family in wheat (Triticum aestivum) and its potential role in cadmium stress","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn recent years, the use of sewage irrigation, pesticides and chemical fertilizers has led to a large accumulation of heavy metals in the soil, posing an increasing threat to plants [1]. The harm of heavy metals to plants includes the existence of non-essential metals such as cadmium (Cd), lead (Pb) and mercury (Hg), and the excessive accumulation of essential metals such as copper (Cu), zinc (Zn) and manganese (Mn). Both the existence of non-essential metals and the excessive accumulation of essential metals are harmful to the growth and development of plants. [2]. Cadmium is one of the most toxic but non-essential heavy metals for all organisms [3]. In China, 7% of farmland is contaminated with cadmium. For example, in Dazhang Town, Xinxiang, Henan Province, at least 150,000 kilograms of grain are produced annually. However, the Cd level in local wheat is 1.7–12.8 times higher than the limit specified in the national food standards[4]. Cadmium accumulates in wheat grains. When people consume wheat products contaminated with cadmium, cadmium will enter the human body accordingly, thus causing great harm to human health. Therefore, it is crucial to reduce the accumulation of cadmium (Cd) in wheat. The stress induced by cadmium can cause damage to genomic DNA, mitochondria, chloroplasts and other organelles, and it also disrupts metabolic processes such as photosynthesis and protein hydrolysis, which are extremely adverse to plants' growth and development. [5, 6]. To cope with the stress of heavy metals, plants have various transmembrane transporters in different organelles, such as P1B-ATPases, ATP-binding cassette gene family (ABC), metal tolerance protein gene family (MTP ), family of cation diffusion promoters (CDF) and calcium exchanger gene family (CAX), which can seal and redistribute heavy metals [7]. P1B-ATPases are a type of transporter in which heavy metals are excreted from cytoplasm or transported to vacuole.\u003c/p\u003e \u003cp\u003eP1B-ATPases are a family of genes that can transport metal cations across the membrane by hydrolyzing ATP, also known as CPx-ATPases [8], metal P-type ATPase and HMAs [9]. There are eight transmembrane helices in structure[10], CPx/SPC motif in transmembrane domain 6, and assumed transition metal binding domains at N-terminal and/or C-terminal [11].\u003c/p\u003e \u003cp\u003eMany HMA genes have been found in many plants, which have different heavy metal transport functions. Among the eight P1B - ATPases in Arabidopsis thaliana, AtHMA1–4 belong to the Zn/Co/Cd/Pb subgroup, and AtHMA5–8 belong to the Cu/Ag subgroup [12]. AtHMA1 can reduce the accumulation of Zn in Arabidopsis thaliana treated with a high concentration of Zn [13]. AtHMA2 and AtHMA4 can transport Zn and Cd into xylem, thus promoting the transport of Zn and Cd from roots to leaves [14]. AtHMA3 is located in the root vacuole, which can chelate Cd in the vacuole to regulate the accumulation in plants [15]. AtHMA4 may transport Zn from the cells around the root vascular tissue [16]. AtHMA5 can transport Cu into the root xylem and drive Cu to the shoot [17]. AtHMA6/PAA1 and AtHMA8/PAA2 are high-affinity Cu transporters located in chloroplasts. They control the absorption of Cu by chloroplasts, transport Cu to the chloroplast matrix and thylakoid, and maintain photosynthesis and antioxidation [18]. AtHMA7/RAN1 is the first P1B-ATPase in plants and it inputs Cu into the endoplasmic reticulum to synthesize the ethylene receptor [19]. In other plants, there are many reports about the functions of HMA genes. For example, SpHMA1 can reduce the excessive accumulation of Cd in chloroplasts and maintain leaf photosynthesis [20]. In barley, HvHMA2 is not only responsible for the transport of Cd from root to stem [21] but also improves the transport capacity of Zn under the condition of Zn deficiency [22]. OsHMA3 has the same function as AtHMA3 [23]. GmHMA3 is located in the endoplasmic reticulum of roots and can chelate Cd on the endoplasmic reticulum, reducing the transport of Cd from roots to stems [24].\u003c/p\u003e \u003cp\u003eIn addition, members of the P1B-ATPase family have been found in different plants, including rice, corn, sorghum and barley in monocotyledons, and Arabidopsis, soybean, flax, rape and Populus tomentosa in dicotyledons. In bread wheat, the characterization of the HMA gene family under Cu stress has been reported [25], and the overexpression of the family member TaHMA2 ameliorated the translocation of zinc/cadmium from roots to buds [26]. However, analyses of the wheat HMA family are still incomplete. We conducted whole gene identification, analyzed the physical and chemical properties, gene structure and conserved motifs, performed promoter analysis, and studied the expression patterns under cadmium stress of HMA gene family members. To further provide a theoretical basis for exploring the cadmium transport function and molecular mechanism of HMA in wheat, subsequent research can utilize technologies such as gene editing and molecular-assisted breeding to study the HMA gene family, which will provide valuable insights for the development of wheat varieties with low cadmium accumulation.\u003c/p\u003e "},{"header":"2. Materials and methods","content":"\u003ch2\u003e2.1 Identification of TaHMAs in wheat\u003c/h2\u003e\u003cp\u003eWhole-genome IWGSC v1.1 data and the genome annotation information Gff3 data of wheat (\u003cem\u003eTriticum aestivum L.\u003c/em\u003e) was available at Ensembl Plants database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plants.ensembl.org/Triticum_aestivum/Info/Index\u003c/span\u003e\u003cspan address=\"https://plants.ensembl.org/Triticum_aestivum/Info/Index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Tair (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and The National Rice Center (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ricedata.cn/\u003c/span\u003e\u003cspan address=\"https://www.ricedata.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to download the known HMA family member protein sequences of Arabidopsis and Rice. The HMA-Domaining (PF00403), E1-E2_ATPase-Domaining (PF00122), Hydrolase-Domaining (PF00702) were downloaded from Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUsing the HMMER3.0 program to identify proteins containing three domains in the whole wheat genome with e \u0026lt; 1e − 5 as threshold. Then, the protein sequences of known HMA members of Rice and Arabidopsis as query sequences using BLASTP program set threshold to e \u0026lt; 1e − 5 and 50% identity to search against the wheat protein dataset. By analyzing the results of HMM and BLAST preliminarily putative wheat HMA genes. The candidate HMA genes of wheat were further confirmed through the NCBI-CDD web server(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Theoretical isoelectric points (PI) and molecular weight (MW) of TaHMAs were predicted using the ExPASy server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/compute_pi/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/compute_pi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the subcellular localization of these proteins was predicted using the CELLO web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cello.life.nctu.edu.tw/\u003c/span\u003e\u003cspan address=\"http://cello.life.nctu.edu.tw/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003e2.2 Phylogenetic analysis\u003c/h2\u003e\u003cp\u003ePhylogenetic analysis was performed using the HMA protein sequences of wheat, Arabidopsis and rice. A rootless neighbor joining (NJ) tree with 1000 bootstrap repeats was constructed using MEGA 6.0 software.\u003c/p\u003e\u003ch2\u003e2.3 Chromosomal Location, Gene Structure and Conserved Motif Analysis of TaHMAs Gene\u003c/h2\u003e\u003cp\u003eThe chromosomal location of the TaHMA genes was obtained from the wheat genome annotation information Gff3, and the TaHMA genes were mapped to the wheat chromosome. The gene structure and coding sequence (CDS) of TaHMAs were analyzed to study the distribution of exons. Conserved motifs of TaHMA proteins were identified using MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/doc/meme.html\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/doc/meme.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The optimal motifs were set at ≥ 10 and ≤ 200 amino acids, and the maximum motifs were set at 15. The results will be displayed on evolview (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.evolgenius.info/evolview-v2/#mytrees/2/2\u003c/span\u003e\u003cspan address=\"https://www.evolgenius.info/evolview-v2/#mytrees/2/2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e )with the phylogenetic tree, gene structure and conserved motif of wheat HMA gene.\u003c/p\u003e\u003ch2\u003e2.4 Gene synteny analysis of wheat with rice, \u003cem\u003eA. tauschii\u003c/em\u003e, and \u003cem\u003eB. distachyon\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eMCScanX software was used to detect the collinear region of TaHMA gene in wheat and other three species (\u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eBrachypodium distachyon (L.) Beauv.\u003c/em\u003e and \u003cem\u003eAegilops tauschii Coss.\u003c/em\u003e). Circos 0.67 was used to visualize TaHMA gene duplication events and the synchronous relationship between other species. Compare the CDS and protein sequences of collinear gene pairs, and calculate the KaKs ratio by TBtools software. Finally, use [T = Ks/(2 λ × 10 − 6) Mya( λ = six point five × 10 − 9)] Calculate the divergence time of the collinear gene pair.\u003c/p\u003e\u003ch2\u003e2.5 GO annotation and protein-protein interaction network analysis of TaHMAs\u003c/h2\u003e\u003cp\u003eGO annotations of wheat HMA proteins are available from the Plaze database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.psb.Be/plaza/verages/plaza_v4_moncots/\u003c/span\u003e\u003cspan address=\"https://bioinformatics.psb.Be/plaza/verages/plaza_v4_moncots/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the Plant Transcriptional Regulation Map Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://plantregmap.gao-lab.org/\u003c/span\u003e\u003cspan address=\"http://plantregmap.gao-lab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) GO annotation results were visualized using WEGO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wego.genomics.cn/\u003c/span\u003e\u003cspan address=\"https://wego.genomics.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) online tool.\u003c/p\u003e\u003cp\u003eBased on the orthologs of wheat and Arabidopsis, the interaction between TaHMA protein and other wheat proteins was constructed using ArenaNet V2 tool and string database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/cgi/input.pl\u003c/span\u003e\u003cspan address=\"https://string-db.org/cgi/input.pl\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) network. Trust values \u0026gt; = 0.95 in the string database were used to identify the interaction network, which was graphically displayed by Cytoscape software.\u003c/p\u003e\u003ch2\u003e2.6 Promoter Analysis of TaHMA Gene\u003c/h2\u003e\u003cp\u003eDownload the upstream 2kb DNA sequence of TaHMA gene from Ensemble plant database, and then use the PLACE database to predict the cis regulatory elements of the promoter region (bioinformatics.psb.ugent.be/webtools/plantcare/html/).\u003c/p\u003e\u003ch2\u003e2.7 Gene expression, heavy metal content determination and qRT-PCR analysis of TaHMAs\u003c/h2\u003e\u003cp\u003eFor qRT-PCR, Chinese spring wheat varieties were grown in greenhouses (24 ± 2℃, 75 ± 5% relative humidity, 16/8h light/dark cycle and 125 µmol/m\u003csup\u003e2\u003c/sup\u003e/s photosynthetic photon flux density) in 2022. Wheat roots and shoots were harvested at the seedling stage. One leaf and one heart seedlings were treated with 40 mM and 400 mM CdCl\u003csub\u003e2\u003c/sub\u003e for 14 days, and then samples were collected. All samples were stored at − 80°C for use with RNAiso reagent and digestion with three biological replicates. cDNA was synthesized with RT Master Mix Perfect Real Time Kit (Takara, Beijing, China). TaHMA Quantitative reverse transcription polymerase chain reaction was performed with QuantStudio real-time qPCR software. The relative expression levels of TaHMA in nine randomly selected groups were determined through 2(−ΔΔCt) analysis. We selected TraesCS6B02G243700 as the reference gene to normalize the expression level of TaHMA gene.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Genome-wide identification and chromosomal localization of the TaHMA gene\u003c/h2\u003e \u003cp\u003eTwo methods, HMM and BLASTP, were used to obtain the TaHMA genes from the IWGSC v1.1 whole-genome data of wheat, and CD-search was used for further confirmation. There were 28 genes named TaHMA genes. According to chromosomal location, they were renamed TaHMA001 to TaHMA028. All genes were unevenly distributed on the wheat chromosomes. There were 10 genes located in the A and D genomes, respectively, and the remaining 8 genes were located in the B genome. The TaHMA genes are mainly concentrated on chromosomes 2 and 4\u0026ndash;7, rather than on chromosomes 1, 3, and 4B.\u003c/p\u003e \u003cp\u003eWe submitted TaHMA protein sequences to the Cello website to predict their subcellular localization (Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Most of the TaHMs were located in the plasma membrane, except for TaHMA004, TaHMA005, TaHMA015, TaHMA020 and TaHMA024, which were located in the chloroplast. It is suggested that TaHMA004, TaHMA005, TaHMA015, TaHMA020 and TaHMA024 may function in the chloroplast, whereas the other TaHMs may function in the plasma membrane. Using ExPASy, we revealed that the protein lengths and relative molecular weights of TaHMA proteins were diverse (Additional file 1: Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The relative molecular weight of TaHMA proteins ranged from 81092.87 to 110320.38. The protein lengths varied from 766 to 1023 aa. Among them, the longest was TaHMA021, and the shortest was TaHMA022\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eTo find out the evolutionary relationship of wheat HMA genes, we constructed a phylogenetic tree using the HMA protein sequences of Arabidopsis, rice and wheat (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). According to phylogenetic analysis, the Zn/Co/Cd/Pb subgroup and the Cu/Ag subgroup are the two major categories of HMA genes. According to previous research results and the phylogeny and classification of the wheat HMA gene family, AtHMA1 - AtHMA4, OsHMA1 - OsHMA3, TaHMA006, TaHMA007, ​​TaHMA008​​, TaHMA016, TaHMA017, TaHMA021, TaHMA022, TaHMA025, TaHMA026 belong to the Zn/Co/Cd/Pb subgroup, while AtHMA5 - AtHMA8, OsHMA4 - OsHMA9 and the rest of the TaHMAs belong to the Cu/Ag subgroup.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Gene structure and motif composition of the wheat HMA gene family\u003c/h2\u003e \u003cp\u003eIn the evolution of gene families, the type and number of exons and introns play an important role. We analyzed the gene structure of TaHMAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and found that the number of exons in the TaHMA genes varies from 6 to 17. Each TaHMA gene has complete UTRs (non-coding regions). TaHMA001\u0026ndash;003 and TaHMA006\u0026ndash;008 have the least number of exons, with 6 exons each. TaHMA004, TaHMA005, TaHMA020 and TaHMA024 have the largest number of exons, with 17 exons each. Different branches have different numbers of exons and introns, while members of the same branch exhibit similar numbers of exons and introns. This suggests that TaHMA genes are conservative and diverse.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore the structural diversity of TaHMA genes and predict their potential functions, we used MEME Online software to analyze 28 TaHMAs, detect their motif composition, and explore motif diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). From the results, we found that members within the same subclass mostly share a similar structure, whereas members of different subclasses exhibit slight variations. All TaHMA genes contain motifs_1, motifs_2, motifs_3, motifs_5, motifs_6, motifs_7, motifs_9, and motifs_13, but there are specific motifs among different branches. Associated with the phylogeny, we can see that the members of the Zn/Co/Cd/Pb subgroup all have motifs ending with motif_8. The Cu/Ag subfamily members are characterized by motifs ending with motif_5 or motif_15. The motif_10 is exclusively present in the members of the Cu/Ag subfamily. Among the subgroup members, TaHMA018 and TaHMA027 have the minimum number of motifs, with 10 motifs, and both start with motif_15. TaHMA014 has the maximum number of motifs, with 20 motifs, starting at motif_13.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Collinear analysis\u003c/h2\u003e \u003cp\u003eDuplication is an essential mechanism in the evolution of genomes and genetic systems. It can provide raw materials for the creation of new genes and functions. Therefore, we analyzed the duplication events of the TaHMA gene family (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), calculated the Ka/Ks ratio, and estimated the divergence time (Additional file 1: Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). After detection, 35 duplicate pairs of fragments were found in wheat varieties. All TaHMAs, except for TaHMA017, TaHMA022, and TaHMA026, had duplicate pairs, encompassing all members of the TaHMA family. This indicates that gene fragment duplication is ​​a mechanism​​ for the expansion of TaHMAs in wheat, and the presence of multiple homologous genes confirms the conservation of the gene family. The ratio of Ka/Ks within wheat varieties is lower than 1, indicating that HMA genes ​​have undergone​​ purification and selection within wheat varieties. By calculating the divergence time, it is estimated that the divergence time among wheat varieties is approximately 20 years.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo understand the origin of the HMA family, collinearity analysis was conducted at the whole-genome level among wheat, \u003cem\u003eOryza sativaL\u003c/em\u003e, \u003cem\u003eBrachypodium distachyon\u003c/em\u003e and \u003cem\u003eTriticum crassum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The results showed that there were 22, 24, and 26 collinear pairs between wheat and each of the other species, respectively. The ratio of Ka/Ks was lower than 1, indicating that these species had undergone purifying selection ​​along with​​ wheat. The HMA family was conserved during the evolution of these species. The divergence time between wheat and \u003cem\u003eOryza sativaL\u003c/em\u003e (Additional file 1: Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), \u003cem\u003eBrachypodium distachyon\u003c/em\u003e (Additional file 1: Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e) and \u003cem\u003eTriticum crassum\u003c/em\u003e (Additional file 1: Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e) is 46 Mya, 57 Mya and 15 Mya, respectively. This suggests that the HMA family in wheat is closely related to \u003cem\u003eOryza sativaL\u003c/em\u003e, \u003cem\u003eBrachypodium distachyon\u003c/em\u003e and \u003cem\u003eTriticum crassum\u003c/em\u003e..\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 GO annotation and protein-protein interaction network analysis of TaHMA\u003c/h2\u003e \u003cp\u003eThe members of the wheat HMA gene family were annotated using Gene Ontology (GO) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Additional file 1: Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e), and a series of processes, namely Biological Process, Cellular Component, and Molecular Function, that TaHMA members might participate in were analyzed. When annotating the 28 genes, it was found that no GO terms were detected for TaHMA022, and the rest were assigned 20 GO terms. The most enriched categories were cation transport, integral component of membrane, nucleotide binding, and ATPase-coupled cation transmembrane transporter activity. All TaHMAs may be involved in these processes. In terms of biological processes, in addition to all participating in cation transport, 25 genes may participate in metal ion transport, and 9 participate in cation transmembrane transport. Only TaHMA017, TaHMA021, and TaHMA025 participate in responses to metal ions, zinc ion transport, cadmium ion transport, responses to cobalt ion, etc., while TaHMA020 and TaHMA024 are involved in copper ion homeostasis and the photosynthetic electron transport chain. In terms of cellular component, in addition to all participating in the integral component of membrane, 9 participate in the membrane, and a small number are involved in plasmodesma, plasma membrane, chloroplast envelope, etc. In terms of Molecular Function, in addition to all participating in nucleotide binding and ATPase-coupled cation transmembrane transporter activity, 25 participate in metal ion binding, 12 potentially participate in copper ion binding, 9 participate in ATP binding, and 2 participate in copper ion transmembrane transporter activity and copper chaperone activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrediction of protein interactions for TaHMAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Additional file 1: Table \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003e). No interactive protein ID corresponding to TaHMA006 was found during protein mapping. Nine genes of TaHMA4, TaHMA2 and TaHMA0 were predicted to interact with TraesCS2A02G114900 and TraesCS2B02G134100. All proteins interact with TraesCS2A02G399000 and TraesCS2B02G417000. TaHMA015, TaHMA020, TaHMA024 interact with TaHMA003 in addition to TraesCS2D02G495500LC (Note: It seems there is a repetition of TaHMA024 here, you may need to double-check). Interaction was observed between six genes belonging to TaHMA6 and TaHMA9 and TraesCS5A02G030100. The five genes belonging to TaHMA1 and TaHMA7 interact with TraesCS7A02G335400, TraesCS7B02G247000 and TraesCS7D02G342900. TraesCS2A02G399000 and TraesCS2B02G417000 were predicted by NCBI CDD to be Superoxide dismutase copper chaperones. TaHMAs interact with them to activate the SOD defense mechanism in wheat.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Promoter Analysis of TaHMA Gene\u003c/h2\u003e \u003cp\u003eThe expression of genes is regulated by splicing regulatory elements, which generally exist in the promoter region. Therefore, we analyzed and predicted the splicing regulatory elements in the promoter region. We analyzed the promoter regions of 28 TaHMA genes by selecting 2 kb DNA sequences upstream of the genes for prediction and obtained potential splicing regulatory elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). By predicting the promoter regions of the TaHMA genes, we found that most of the elements are splicing regulatory elements involved in hormone response, light response, and growth and development. All TaHMAs contain light-responsive elements. For example, G-box and Sp1. There is a G-box in each TaHMA, and all 18 TaHMA genes contain Sp1, indicating that TaHMA may respond to light regulation. Except for TaHMA011, ABRE elements related to abscisic acid response are present in all other family members. The CGTCA motif and TGACG motif components associated with the MeJA reaction are not present in TaHMA023, whereas the remaining TaHMAs contain them. Anaerobic induction-related ARE elements are present in all 23 members of the HMA family. The hypoxia-specific inducing element GC-motif is present in 16 members. The low-temperature response (LTR) component is involved in 14 members. MBS, which participates in drought-induced stress, is present in 13 members. At the same time, some elements related to plant growth and development were found in the TaHMA genes, such as CAT box, circadian, and GCN4_motif, etc. This suggests that TaHMA may also be involved in regulating wheat growth and development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Gene expression, heavy metal content determination and qRT-PCR analysis of TaHMAs\u003c/h2\u003e \u003cp\u003eDownloading the expression data of TaHMAs in four tissues (roots, stems, leaves and spikes) of Chinese Spring from the Wheatomics website reveals that most TaHMAs have higher expression levels in roots compared to the other individual tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This suggests that TaHMAs have strong tissue-specific expression and may play a major role in roots.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeedlings of common wheat were treated hydroponically with 40 \u0026micro;M and 100 \u0026micro;M CdCl₂ solutions. Roots and leaves were collected at 2 h, 5 h, 12 h, 24 h, 7 d, and 14 d to determine their Cd content (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The results of Cd content determination revealed that there was no significant change in the Cd content of aboveground and belowground parts from 2 h to 12 h, whereas there was a trace increase at 24 h and a significant increase in Cd content at 7 d and 14 d. Therefore, total RNA was extracted from the roots and leaves of seedlings treated for 24 h, 7 d, and 14 d. The expression of TaHMAs was determined by fluorescence real-time quantitative PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, Additional file 1: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In the roots, TaHMA4 was up-regulated at 24 h, 7 d, and 14 d, and the up-regulation became more pronounced over time, with a 24-fold up-regulation at 14 d. TaHMA2, TaHMA3, and TaHMA5 were also up-regulated, with the most pronounced up-regulation at 14 d. In contrast, the remaining TaHMAs showed down-regulation. In the leaves, TaHMA1, TaHMA6, TaHMA7, TaHMA8, and TaHMA9 all showed down-regulation, with TaHMA1 being the most significantly down-regulated. Although TaHMA2 showed some up-regulation, it was not significant. The responses of TaHMA3, TaHMA4, and TaHMA5 were not significant. The results showed that under cadmium treatment, TaHMAs exhibited different responses at different sites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Characteristics of the TaHMA family in wheat\u003c/h2\u003e \u003cp\u003eHMA genes are a kind of protein that transports heavy metals by hydrolyzing ATP. They play an important role in the transport and storage of heavy metals. They have been identified in rice, maize, sorghum and other monocotyledons[27], as well as in dicotyledons such as Arabidopsis, soybean and rapeseed[28]. The results of whole-genome sequencing of Chinese Spring wheat provide crucial foundational information for identifying the HMA gene family in wheat. Twenty-eight HMA family members of wheat were identified by protein and important domain alignment. As wheat is allohexaploid, the number of TaHMAs is significantly higher than that of other species such as rice and Arabidopsis. The genetic structure and conserved motifs of the TaHMA gene family were analyzed by constructing a phylogenetic tree. In different branches, TaHMAs have varying numbers of exons and introns, whereas in the same branch, they have similar numbers of exons and introns. Different TaHMAs contain eight conserved motifs, but there are also specific motifs, indicating that the TaHMA gene family is conserved and diverse.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.2 The evolutionary relationship of the TaHMA family\u003c/h2\u003e \u003cp\u003eHMA is subdivided into two major subgroups: the Cu/Ag subgroup and the Zn/Cd/Co/Pb subgroup[29]. Based on the phylogenetic tree, TaHMA in the Cu/Ag subgroup is further divided into A1, A2 and B branches, while the Zn/Cd/Co/Pb subgroup is subdivided into C and D branches. In the calculation of Ka, Ks and divergence time, TaHMA009, TaHMA011 and TaHMA0013 have higher Ks values than TaHMA019, TaHMA023 and TaHMA028 in the A2 branch of the Cu/Ag subgroup. Additionally, they have a longer divergence period compared to the other gene pairs. At the same time, in order to understand the evolutionary relationship with other species, collinearity analysis was conducted, and the Ka, Ks and divergence time were calculated[30]. Most TaHMA genes are replicated in \u003cem\u003eOryza sativaL\u003c/em\u003e, \u003cem\u003eBrachypodium distachyon\u003c/em\u003e and Aegilops TaHMA. The Ks of TaHMA019, TaHMA023, TaHMA028, TaHMA011 and TaHMA013 were higher than those of Aegilops tauschii, indicating a longer divergence time. In collinearity analysis with \u003cem\u003eBrachypodium distachyon\u003c/em\u003e, the Ks of TaHMA004 and TaHMA005 with \u003cem\u003eBrachypodium distachyon\u003c/em\u003e are greater than 1, and the divergence time is more than 200 years. In collinearity analysis with \u003cem\u003eOryza sativaL\u003c/em\u003e, KS is less than 1, the divergence time is about 46 years. Except for TaHMA019, TaHMA023, TaHMA028, TaHMA011 and TaHMA013, the divergence times between wheat and Aegilops for TaHMA019 and TaHMA023 are all less than 9 years or even as low as 0.1 years. It is inferred that HMA evolved slowly between wheat and \u003cem\u003eAegilops tauschii\u003c/em\u003e. It can be inferred that HMA is more closely related to wheat and \u003cem\u003eAegilops tauschii\u003c/em\u003e than to \u003cem\u003eOryza sativaL\u003c/em\u003e and \u003cem\u003eBrachypodium distachyon\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Expression patterns and potential functions of HMA gene family in wheat\u003c/h2\u003e \u003cp\u003eIn this study, we conducted Gene Ontology (GO) prediction for TaHMAs and found that all TaHMAs are involved in the cation transport process, and most of them are also involved in the metal ion transport process. Meanwhile, we retrieved the expression profiles of TaHMA genes from the published transcriptome data of Chinese Spring wheat to understand their tissue expression patterns. It was found that the expression levels of almost all TaHMA genes in roots are higher than those in stems, leaves, spikes, and seeds. Based on the GO prediction and tissue expression patterns, we inferred that TaHMA genes may mainly function as heavy metal transport proteins in roots. At the same time, we predicted the protein-protein interactions of TaHMA genes and found that all TaHMAs interact with TraesCS2A02G399000 and TraesCS2B02G417000, both of which belong to the copper chaperone of superoxide dismutase (Cu/Zn-SOD) protein family within the SOD (superoxide dismutase) gene family[31]. Therefore, we speculate that TaHMAs may interact with TraesCS2A02G399000 and TraesCS2B02G417000 during the transport of cations and metal ions, thereby activating the defense mechanism of wheat.\u003c/p\u003e \u003cp\u003eThe phylogenetic tree shows that wheat HMA family members have a higher homology with rice genes. TaHMA2 and TaHMA3 are homologous to three Arabidopsis genes (AtHMA2, AtHMA3, and AtHMA4) and two rice genes (OsHMA2 and OsHMA3). AtHMA2 and AtHMA4 in Arabidopsis have been demonstrated to be responsible for the translocation of Cd from roots to stems and for enhancing Cd tolerance in plants [14]. In Arabidopsis, rice and other plants, HMA3 has also been shown to be chelated in the vacuole to participate in Cd detoxification [32]. Under Cd treatment, TaHMA2 and TaHMA3 were up-regulated in the root system of wheat, and TaHMA2 was also expressed in the leaves. In this way, it is hypothesized that TaHMA2 and TaHMA3 may play similar roles in Cd transport as those in Arabidopsis and rice. TaHMA4 and TaHMA5 are homologous to an Arabidopsis gene (AtHMA5) and two rice genes (OsHMA4 and OsHMA5). AtHMA5 is localized in the root plasma membrane and functions as an exporter of Cu ions, mitigating copper toxicity. On the other hand, OsHMA4 acts as a sequestering agent for Cu in root vesicles, thereby restricting the accumulation of Cu in grains. OsHMA5 is involved in loading Cu into the xylem of roots and other organs [17]. AtHMA5, OsHMA4, and OsHMA5 have a Cd-transport function that remains unexplored. In wheat, TaHMA4 and TaHMA5 ​​show​​ up-regulation in response to Cd stress. Particularly, TaHMA4 showed a significant up-regulation. Therefore, further studies could be conducted to explore whether TaHMA4 and TaHMA5 have a Cd-transporting function.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eWe identified the HMA gene family in common wheat, which was divided into two subgroups, Zn/Co/Cd/Pb and Cu/Ag, based on the phylogeny. The gene structure and conserved motifs showed some conservation within the same branch, while there was diversity among different branches. GO annotation, promoter analysis, covariance analysis and protein-protein interactions serve as the foundation for comprehending the evolutionary history, functional analysis, and other related aspects of the HMA family. The analysis of tissue expression patterns and the response to Cd stress indicated that TaHMA genes exhibit tissue-specific expression and some of them are involved in Cd ion transport in the root system. These analyses provide a basis for subsequent studies on the function of the TaHMA gene and the discovery of the molecular mechanism of cadmium transport in wheat.\u003c/p\u003e"},{"header":"Abbreviations","content":"TaHMA: Wheat HMA, Cd: Cadmium, CDS: Coding sequence, HMM: Hidden Markov Model, qRT-PCR: Quantitative real-time polymerase chain reaction"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are very grateful to Professor Ma, Professor Niu and Professor Ding for their guidance. We thank the Instrument sharing platform of Northwest Agricultural and Forestry University and lab members for their assistance in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Key Research and Development Projects of Shaanxi Province (No. 2021CDLNY01-02). The funding body was not involved in the design of the study, analysis or interpretation of data or writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollege of Agronomy, Northwest A\u0026amp;F University, Yangling 712100, China\u003c/p\u003e\n\u003cp\u003eLeilei Shao, Haosen Ma, Zhan Su, Tianzhen Lei, Xuyu Guo, Yang Wang, Yijie Wan, Lingjian Ma*, Na Niu*\u003c/p\u003e\n\u003cp\u003eLingjian Ma*, [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLeilei Shao conceived and designed the study. Leilei Shao and Haosen Ma performed the experiments. Zhan Su, Tianzhen Lei, Xuyu Guo, Yang Wang and Yijie Wan analysed the data. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Lingjian Ma*, [email protected]\u003c/p\u003e\n\u003cp\u003eNa Niu*, [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the results of this article are included in the article and Additional files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals and did not involve any endangered or protected species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eQin GW, Niu ZD, Yu JD, Li ZH, Ma JY, Xiang P: \u003cstrong\u003eSoil heavy metal pollution and food safety in China: Effects, sources and removing technology\u003c/strong\u003e. \u003cem\u003eCHEMOSPHERE \u003c/em\u003e2021, \u003cstrong\u003e267\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eGhori NH, Ghori T, Hayat MQ, Imadi SR, Gul A, Altay V, Ozturk M: \u003cstrong\u003eHeavy metal stress and responses in plants\u003c/strong\u003e. \u003cem\u003eINTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCE AND TECHNOLOGY \u003c/em\u003e2019, \u003cstrong\u003e16\u003c/strong\u003e(3):1807-1828.\u003c/li\u003e\n\u003cli\u003eSiedlecka A, Krupa Z: \u003cstrong\u003eInteraction between cadmium and iron. 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IN PLANT SCIENCE \u003c/em\u003e2015, \u003cstrong\u003e6\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eKong YM, Xu P, Jing XY, Chen LX, Li LG, Li X: \u003cstrong\u003eDecipher the ancestry of the plant-specific LBD gene family\u003c/strong\u003e. \u003cem\u003eBMC GENOMICS \u003c/em\u003e2017, \u003cstrong\u003e18\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eFukuhara R, Kageyama T: \u003cstrong\u003eStructure, gene expression, and evolution of primate copper chaperone for superoxide dismutase\u003c/strong\u003e. \u003cem\u003eGENE \u003c/em\u003e2013, \u003cstrong\u003e516\u003c/strong\u003e(1):69-75.\u003c/li\u003e\n\u003cli\u003eChao DY, Silva A, Baxter I, Huang YS, Nordborg M, Danku J, Lahner B, Yakubova E, Salt DE: \u003cstrong\u003eGenome-Wide Association Studies Identify Heavy Metal ATPase3 as the Primary Determinant of Natural Variation in Leaf Cadmium in \u0026lt;i\u0026gt;Arabidopsis thaliana\u0026lt;/i\u0026gt;\u003c/strong\u003e. \u003cem\u003ePLOS GENETICS \u003c/em\u003e2012, \u003cstrong\u003e8\u003c/strong\u003e(9).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"P1B-type ATPases, Cd stress, Cd transport, Wheat","lastPublishedDoi":"10.21203/rs.3.rs-5264727/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5264727/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCadmium has been accumulating in the agricultural and ecological environment in recent years due to the release of industrial pollutants. Due to its high solubility, slow degradability and high toxicity, it is highly susceptible to occurring in agricultural fields. The presence of cadmium at low concentrations is harmful to plants. Heavy metal ATPases (HMAs) are proteins that can detoxify high concentrations of heavy metals through vacuole compartmentalization or exocytosis pathways. They have been extensively studied in plants. However, the cadmium transport function of HMAs in wheat has not been explored. In this study, a comprehensive and systematic investigation of HMA gene family members in wheat was conducted. A total of 28 putative TaHMAs were identified. Phylogenetically, these 28 putative TaHMAs were divided into two subgroups: Cu/Ag and Zn/Co/Cd/Pb. The gene structures and conserved motifs were consistent within the same branch and diverse in different branches. The TaHMA gene family is closely related to rice, B. distachyon and A. tauschii. GO analysis results suggest that TaHMAs may be involved in cation transport and membrane components. Protein interaction analysis results suggest that TaHMAs may interact with TaSOD to activate the SOD defense mechanism in wheat. Expression patterns exhibited tissue specificity. Finally, the expression patterns of TaHMAs were validated in the roots and leaves of wheat plants under cadmium stress. Our findings will be valuable for functional studies and applications of HMA gene family members in wheat.\u003c/p\u003e","manuscriptTitle":"Genome-wide survey of the HMA gene family in wheat (Triticum aestivum) and its potential role in cadmium stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-11 13:27:25","doi":"10.21203/rs.3.rs-5264727/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-01T15:24:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-23T18:44:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-19T06:36:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96065478456871829019929039675019367827","date":"2025-04-13T07:47:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"97438665096724096562421815659919556965","date":"2025-04-10T14:27:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-10T11:27:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-10T01:47:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-04-09T16:57:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"07284daf-8db2-4045-b8a6-2cdc20d96fe4","owner":[],"postedDate":"April 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:11:39+00:00","versionOfRecord":{"articleIdentity":"rs-5264727","link":"https://doi.org/10.1186/s12864-025-11746-z","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2025-07-01 15:58:01","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-04-11 13:27:25","video":"","vorDoi":"10.1186/s12864-025-11746-z","vorDoiUrl":"https://doi.org/10.1186/s12864-025-11746-z","workflowStages":[]},"version":"v1","identity":"rs-5264727","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5264727","identity":"rs-5264727","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}