Genomic characterisation of the metal tolerance protein gene family and elucidation of functional role in heavy metal tolerance and accumulation in Coptis chinensis

Metal tolerance protein (MTP) family members, functioning as plant divalent cation transporters, play essential roles in maintaining heavy metal homeostasis and tolerance. This study presents a novel comprehensive genomic characterisation of the MTP gene family in Coptis chinensis, involving systematic identification and functional annotation. A total of 25 CcMTP genes were identified and classified into three major subfamilies based on phylogenetic analysis. Conserved motif profiling and gene structure annotation revealed both conserved and divergent features among the subfamilies. Genomic collinearity analysis identified one tandemly duplicated gene pair (CcMTP12/CcMTP20) in C. chinensis. The promoter regions of CcMTP genes were enriched with cis-acting elements associated with phytohormones, light, and growth and development. Gene expression analysis showed that several CcMTPs were significantly upregulated in response to cadmium stress across different tissues. CcMTP11, CcMTP16, and CcMTP24, which exhibited high expression in roots, stems, and leaves, conferred enhanced tolerance to multiple heavy metals. Notably, CcMTP24 promoted Δycf1 yeast growth under Cd, iron, zinc, and manganese stress and reduced Cd accumulation in yeast. Collectively, pivotal CcMTPs, such as CcMTP24 identified in this study, may provide mechanistic insights to elucidate the roles of MTPs in heavy metal tolerance in C. chinensis.

Introduction

Heavy metal pollution poses a great threat to plant growth and development, human health, and environmental safety (Bari et al., 2021; Fan et al., 2023). Some essential heavy metals, such as iron (Fe), zinc (Zn), and manganese (Mn), play vital roles at low concentrations in plant metabolism and development (Fan et al., 2023). However, excessive levels of these micronutrients result in toxic effects on plants and can even impact human health (Lefèvre et al., 2018). In contrast, other heavy metals, such as arsenic (As), lead (Pb), cobalt (Co), and cadmium (Cd) are considered non-essential elements that can cause toxicity in crop plants even at very low concentrations and pose a severe health threat to humans through the food chain (Dai and Zhou, 2025). Among all heavy metals, Cd ranks as the most prevalent and hazardous inorganic pollutant in arable soil (Sun et al., 2014). In plants, Cd exposure results in easily recognisable symptoms, such as chlorosis and stunted growth. Cd contamination also causes a decline in leaf transpiration, inhibition of nutrient element absorption and distribution, accumulation of reactive oxygen species (ROS), and disruption of stomatal movements, leading to physiological and morphological damage in plants. Moreover, an excessive amount of Cd in plants typically hinders growth and may result in death (Lal et al., 2019; Zhao et al., 2022).

To overcome Cd toxicity, plants have evolved complex and sophisticated regulatory mechanisms (Shahid et al., 2017; Yokosho et al., 2021). Among these mechanisms, two crucial strategies for reducing or eliminating the accumulation and toxicity of Cd in plant cells are hindering the inward flow of Cd and enhancing its outward flow (Han et al., 2023; Kanwal et al., 2024). Numerous heavy metal transporter families are responsible for the absorption, accumulation, and transportation of Cd in plants (Yang et al., 2022; Bari et al., 2021). The cation diffusion facilitator (CDF) family, members of which act as integral membrane transporters for divalent cations (Mn²⁺, Zn²⁺, Fe²⁺, Co²⁺, Cd²⁺, and Ni²⁺), is involved in the efflux of these ions, either transporting them from the cytoplasm into vacuoles or to the extracellular space (Gustin et al., 2011). Due to their evolutionary conservation, members of the CDF family have been comprehensively identified in prokaryotes, archaea, and eukaryotes (including plants) (Montanini et al., 2007). Based on substrate specificity for divalent metal ions, the CDF family is classified into three major subfamilies: Mn-, Zn/Fe-, and Zn-CDFs. Most CDF proteins are characterised by the presence of six transmembrane domains (TMDs) and a conserved C-terminal cation efflux domain that extends into the cytoplasm (Montanini et al., 2007).

In plants, CDF transporters are typically referred to as metal tolerance proteins (MTPs). Plant MTPs function as H⁺/metal-ion antiporters driven by proton gradients (Yang et al., 2022). These transporters confer metal tolerance by compartmentalising excess or toxic metal ions (e.g., Cd²⁺) into vacuolar compartments. In addition, MTPs mediate the transport of indispensable metal ions, such as Zn²⁺, Mn²⁺, Fe²⁺, and Co²⁺, which is crucial for promoting plant growth and development (Haney et al., 2005). The identification of Arabidopsis thaliana MTP1 (AtMTP1) marked the first report of an MTP protein in plants (van der Zaal et al., 1999). Transgenic plants overexpressing AtMTP1 exhibited enhanced resistance to Zn²⁺-induced stress, along with significantly elevated zinc ion concentrations in root tissues (Desbrosses-Fonrouge et al., 2005). MTP family members play essential roles in various plant species by mediating the transport of diverse divalent metal ions. For instance, in response to heavy metal stress from elevated Zn²⁺ and Cd²⁺ levels, the transcript levels of MTP1, MTP3, and MTP4 in Citrus sinensis were markedly induced in root or foliar tissues. AtMTP11 enhances Mn²⁺ tolerance in both yeast and A. thaliana (Delhaize et al., 2007). Heterologous expression of Populus trichocarpa MTP8.1, MTP9, and MTP10.4 in yeast cells demonstrated their specific roles in Mn²⁺ translocation, whereas MTP6 showed broad substrate specificity for Co²⁺, Fe²⁺, and Mn²⁺ ions (Gao et al., 2020). Collectively, these findings underscore the critical functions of the MTP gene family in mediating plant responses to heavy metal stress.

With the increasing availability of genome sequences for a wide range of plant species, numerous MTP proteins have been identified and verified in species including A. thaliana (van der Zaal et al., 1999), Triticum aestivum (Vatansever et al., 2017), sweet orange (Fu et al., 2017), grape (Shirazi et al., 2019), tobacco (Liu et al., 2019), rice (Ram et al., 2019), P. trichocarpa (Gao et al., 2020), potato (Li et al., 2021), tomato (El-Sappah et al, 2021), Quercus dentata (Jiang et al., 2024), and Nelumbo nucifera (Hu et al., 2025). Coptis chinensis Franch. is a well-known traditional medicinal plant in China. Its dried rhizome is noted for its strong antibacterial and antifungal activities and is used in traditional medicine for clearing heat, eliminating dampness, and detoxifying (Liu et al., 2022). Due to the high levels of alkaloids in its rhizomes, C. chinensis is often referred to as the ‘Chinese antibiotic’. However, as a perennial herb, the rhizome of C. chinensis has been found to accumulate at more than 1.64 µM Cd in numerous batches of commercial samples (Huang et al., 2019). The presence of such elevated Cd levels notably impacts the quality of the rhizome and poses a severe threat to the safety and efficacy of C. chinensis. Therefore, identifying and characterising MTP family genes in C. chinensis is important for understanding their roles in heavy metal detoxification in this species. With the availability of high-quality genome sequencing and assembly for C. chinensis, a systematic analysis of the MTP gene family is now feasible (Liu et al., 2021).

A comprehensive functional elucidation and systematic investigation of CcMTPs in C. chinensis remain unconducted. In this study, we aimed to identify CcMTPs in the C. chinensis genome and comprehensively investigate the MTP gene family, including phylogenetic relationships, gene structure, conserved motifs, cis-regulatory elements, protein-protein interaction network, and expression patterns in different tissues, in response to Cd toxicity. The rationale was to elucidate novel perspectives on the functions of CcMTP genes in response to Cd stress in C. chinensis.

Materials and methods

Plant materials and Cd treatments

Four-year-old C. chinensis samples were collected from Gaochuan Township, Anzhou District, Mianyang City, China (31°37′49.8″N, 104°12′5.7″E). Seedlings of C. chinensis were cultivated in a greenhouse maintained at 22°C under 16-h day/8-h night cycles with 70% relative humidity at the Key Laboratory of Plant Genetics and Molecular Breeding for 7d. Based on previous research, the seedlings were washed and then transferred into aerated containers filled with half-strength Hoagland’s solution supplemented with 100μM CdCl₂ for Cd treatment (Huang et al., 2019). After exposure to Cd for 0h, 6h, 12h, 24h, and 48h, the roots and leaves of C. chinensis seedlings were harvested for analysis of the expression patterns of the CcMTP gene family. Samples were immediately frozen in liquid nitrogen and stored at –80°C.

Identification of CcMTP genes

The genome sequence of C. chinensis was retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_015680905.1/). To identify all MTP genes in C. chinensis, the HMM profile of the cation efflux domain (PF01545) was downloaded from the Pfam protein family database (http://pfam.xfam.org) and used to screen for potential MTP genes within the C. chinensis genome using HMMER 3.2.1 (http://hmmer.janelia.org/) (Prakash et al., 2017). After removing redundant sequences, the putative CcMTP protein sequences were submitted to the NCBI Conserved Domains Database (https://www.ncbi.nlm.nih.gov/cdd/), SMART database (http://smart.embl.de/) and Pfam for additional confirmation of conserved domains (Marchler-Bauer et al., 2017; Letunic et al., 2021). To further validate the completeness of our identification, we conducted a BLASTP search against the C. chinensis proteome using experimentally characterised MTP protein sequences from A. thaliana and Oryza sativa as queries. This independent analysis confirmed that all candidates identified via the HMM-based approach were consistently recovered, with no additional CcMTP homologs detected.

Chromosomal localisation and duplication analysis of CcMTPs in C. chinensis

The chromosomal location data of CcMTPs were retrieved from the assembled C. chinensis genomic repository. These spatial arrangements and gene duplication events were subsequently subjected to graphical representation through TBtools software (Chen et al., 2020). The rates of synonymous (Ks) and non-synonymous (Ka) substitutions per site for duplicated gene pairs were also calculated using TBtools (Chen et al., 2020).

Phylogenetic analysis of CcMTPs

Twenty-five CcMTP protein sequences were obtained from NCBI. Subsequently, protein sequences of the MTP gene family from Arabidopsis and rice were used to construct a phylogenetic tree using MEGA version 10.0, based on the maximum likelihood method with bootstrap values calculated from 1000 replicates (Tamura et al., 2011). The phylogenetic tree was annotated and visualised using EvolView 2.0 server (https://www.evolgenius.info/evolview-v2/).

Gene structure, conserved motifs, cis-regulatory elements, and protein modelling of CcMTPs

The physicochemical properties of the confirmed CcMTP proteins, including molecular weight (MW) and theoretical isoelectric point (pI), were calculated using the ExPASy server (https://web.expasy.org/protparam/). TMHMM and WoLF PSORT were used to predict transmembrane domains and subcellular localisation, respectively (https://wolfpsort.hgc.jp/) (Krogh et al., 2001; Yu et al., 2006). Conserved motifs within CcMTP proteins were identified using the MEME server (http://meme-suite.org/tools/meme) with default parameters, specifying up to 10 motifs per gene and motif widths between 15 and 40 amino acids. Conserved domains of CcMTP sequences were further confirmed using InterPro (https://www.ebi.ac.uk/interpro/). Exon-intron structures of CcMTPs were analysed using the GSDS 2.0 online tool (http://gsds.gao-lab.org/). The 2000 bp upstream regions of the 25 identified CcMTPs were extracted as promoter sequences, and related cis-regulatory elements were identified using the PLACE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Computational structural modelling of CcMTP proteins was performed using the intensive mode of the Phyre2 webserver (http://www.sbg.bio.ic.ac.uk/phyre2/) (Kelley et al., 2015).

Sequence similarity network (SSN) analysis

Protein sequences of MTPs from A. thaliana, O. sativa, and C. chinensis were subjected to pairwise alignment using the NCBI BLASTP algorithm with default parameters (e-value ≤ 1e–5). Gene pairs with sequence similarity greater than 46% were retained. An SSN was constructed by EFI-EST (Oberg et al., 2023) and visualised using Cytoscape software (v3.10.3). In the network, nodes represent individual protein sequences, and edges represent significant BLASTP alignments meeting a significance threshold (e-value ≤1e-5), and topological organisation was optimised via Cytoscape force-directed layout (Shannon et al., 2003).

Expression patterns of CcMTPs in C. chinensis

Gene-specific oligonucleotide primers were designed and optimised using Primer3 software (Supplementary Table S2) for RT-qPCR analysis. Total RNA was extracted from the 12 samples described previously using the HiPure Total RNA Plus Kit (Magen, R411103). First-strand cDNA synthesis was performed using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, RR047A), following the manufacturer’s protocol. The synthesised cDNA served as a template for qPCR, which was performed using the CFX96™ Real-Time PCR Detection System (Bio-Rad, USA) with SYBR Premix Ex Taq™ (Tli RNaseH Plus, Takara Bio Inc.). The amplification protocol was as follows: initial denaturation at 95°C for 3min, followed by 40 cycles of 95°C for 10s and 60°C for 30s. All reactions were performed in triplicate under identical conditions. The fold changes in RNA transcripts were computed using the 2^−ΔΔCt method (Livak and Schmittgen, 2001), with the 18S rRNA-encoding gene (DQ406855) in C. chinensis used as the internal reference (Liu et al., 2022) (Supplementary Table S2).

Yeast complementation assay

To validate the functional roles of CcMTPs, the full-length coding sequences (CDS) of CcMTPs were amplified using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China). Following sequencing confirmation, PCR amplicons of three target genes (CcMTP11, CcMTP16, and CcMTP24) were directionally cloned into Hind III/Sac I restriction sites of the pYES2 yeast expression vector, generating three recombinant plasmids (pYES2-CcMTP11, pYES2-CcMTP16, and pYES2-CcMTP24) along with an empty vector control. These plasmids were transformed into the Cd -hypersensitive Saccharomyces cerevisiae mutant strain Δycf1 via lithium acetate-mediated transformation for heterologous complementation analysis. Primary transformants were selected on uracil dropout (SD-Ura) agar medium. Cultures were grown at 28°C with orbital shaking (225 rpm) at 28°C until reaching mid-exponential phase (OD₆₀₀ = 1.0 ± 0.05) and serially diluted 10-fold (10^-1, 10^-2, 10^-3). A 50-μL aliquot from each dilution was spotted onto SGR–Ura plates supplemented with various heavy metals: Cd²⁺ (0, 10, 30, 55μM), Mn²⁺ (0, 20, 30, 40mM), Fe²⁺ (0, 40, 60, 65mM), and Zn²⁺ (0, 8, 10, 12mM). Plates were incubated at 30°C for 3–5 d before imaging.

To assess Cd -induced growth inhibition, yeast cells were cultured in 20mL SGR–Ura medium to an OD₆₀₀ = 0.2 and treated with 35μM CdCl₂. OD₆₀₀ was measured at 6-h intervals to generate a growth curve of the transformants. To analyse Cd accumulation within yeast cells, yeast cells were propagated in 20 mL of SGR-Ura medium until the OD₆₀₀ reached 0.2, followed by a 48-h exposure to 35 μM CdCl₂ under standard culture conditions. The yeast cells were then harvested and washed three times with distilled water. Subsequently, they were lyophilised for 48h at 85°C to constant weight, and digested in 5mL nitric acid. Intracellular metal content was determined using inductively coupled plasma-optical emission spectrometry (ICP-OES 7000, Thermo Fisher Scientific, New York, USA).

Statistical analysis

Statistical analyses of numerical data were performed using one-way analysis of variance (ANOVA) and independent-sample t-tests, followed by post-hoc Duncan tests (p < 0.01) and least significant difference (LSD) comparisons. All computations were executed via the SPSS, version 23.

Results

Sequence characteristics of CcMTP proteins

Using 12 AtMTP and 10 OsMTP protein sequences as the queries, a total of 25 MTP proteins were identified in the C. chinensis genome. According to the sequence analysis, multiple distinct characteristics of the CcMTPs were elucidated (Table 1; Supplementary Figure S1): (1) ORF lengths of CcMTPs were determined to span 300–1572 base pairs (CcMTP1 to CcMTP25), encoding 99–523 amino acid residues. Predicted molecular weights (MWs) ranged from 11.4 to 58.6 kDa, and isoelectric points (pI) ranged from 4.72 to 9.05 across the CcMTP family. (2) Most of CcMTPs were predicted to contain two to six transmembrane domains (TMDs), except for CcMTP1, CcMTP18, and CcMTP21, which harboured ten, seven, and eight TMDs, respectively. (3) Subcellular localisation by Wolfpsort predicted that 24 of the 25 proteins localised in both the plasma membrane and tonoplast, with only CcMTP11 predicted to be located in the Golgi apparatus. (4) Multiple sequence alignment revealed that three distinct subfamilies (Zn-, Zn/Fe-, Mn-MTPs) exhibited low sequence homology (Supplementary Figure S1). The CcMTP members classified within the Zn-MTP and Zn/Fe-MTP clades exhibited a conserved HXXXD (X = variable residue) signature sequence spanning both TMD-I and TMD-V. Conversely, the Mn-MTP subgroup was characterised by a divergent DXXXD motif localised in these equivalent transmembrane regions, suggesting evolutionary adaptation for differential metal ion selectivity (Supplementary Figure S1).

Table 1

Table 1. Physicochemical characterisation and subcellular localisation profiling of 25 metal tolerance proteins (MTPs) identified in Coptis chinensis.

Chromosomal localisation and synteny analysis of CcMTPs

To characterise the chromosomal distribution of C. chinensis MTP genes, we mapped the genomic locations of the 25 CcMTP genes across the C. chinensis genome (Figure 1A). This study demonstrated a heterogeneous distribution pattern across the nine chromosomes. Chr2, Chr5, and Chr7 harboured the highest number of CcMTP genes (4) CcMTP genes, with Chr1 and Chr6 following closely behind, each containing three CcMTP genes. Chr4, Chr8, and Chr9 each harboured two CcMTP genes, whereas the lowest number (one CcMTP gene) was identified on Chr3.

Figure 1

Figure 1. Chromosomal locations and synteny analysis of CcMTP genes. (A) Chromosomal locations of CcMTP genes in Coptis chinensis. (B) The chromosomal relationships of CcMTPs in C. chinensis were identified using multiple collinear scanning toolkits (specifically the one Step MCScanX super Fast module in TBtools 2.0) and visualised with TBtools. Gray lines denote collinear modules within the C. chinensis genome, whereas blue lines highlight MTP collinear genes.

In this study, we employed the TBtools software to investigate gene duplication events within the CcMTP gene family. Genomic analyses revealed that the CcMTP gene family generated a single segmental duplication event involving the CcMTP12 and CcMTP20 gene pair, as evidenced by phylogenetic and syntenic conservation patterns (Figure 1B). This isolated chromosomal duplication event likely represents an evolutionary mechanism driving the functional diversification of metal transport systems in C. chinensis. To investigate the evolutionary dynamics and selective pressures influencing the CcMTP gene family, the values of Ka, Ks, and the Ka/Ks ratio were calculated in the C. chinensis genome. The duplicated CcMTP gene pairs exhibited a Ka/Ks ratio of 0.69 (Supplementary Table S3), suggesting that MTP family genes in C. chinensis may have undergone selective pressure during their evolutionary history.

Phylogenetic relationships and classification of MTP genes

To investigate the evolutionary relationships of the MTP gene family, phylogenetic analyses were conducted using the maximum likelihood (ML) method (Figure 2). A phylogenetic tree was constructed using MEGA X following multiple sequence alignment of 25 C. chinensis MTP proteins (CcMTPs), 12 A. thaliana MTP proteins (AtMTPs), and 10 rice MTP proteins (OsMTPs). CcMTP protein sequences are listed in Supplementary Table S4. Based on the topology of the ML phylogenetic tree, MTP genes from C. chinensis, A. thaliana, and rice were divided into three major clades: Zn-MTP, Zn/Fe-MTP, and Mn-MTP. The three subfamilies were resolved to comprise 19, 13, and 15 MTP proteins, respectively. Multiple sequence alignment analysis of CcMTPs and AtMTPs revealed significant sequence homology, highlighting conserved structural features among these proteins (Supplementary Figure S1).

Figure 2

Figure 2. Phylogenetic analysis of MTP proteins from different species. Species abbreviations: At, Arabidopsis thaliana; Os, Oryza sativa; Cc, Coptis chinensis.

Modelling and SSN analysis of CcMTP proteins

All twenty-five CcMTPs were modelled using Phyre2 with >98% confidence in normal mode to elucidate their molecular mechanisms in C. chinensis. Four known structural templates, c7kzxA and c8f6fA (Cd and zinc efflux pump FieF), c8j7tF and c8j7yA (zinc transporter 7), c7y5gA and c6xpdA (zinc transporter 8), and c8f6fD (another FieF structure), were used for modelling CcMTP1–9, CcMTP11–14, CcMTP16–22, and CcMTP24, respectively. Models of c3h90D (ferrous-iron efflux pump FieF), c7ttiA (solute carrier family 12 member 4), and c8xmaB and c8zsbA (proton-coupled zinc antiporter SLC30A1) were used for the remaining five CcMTPs (Supplementary Figure S2; Supplementary Table S1).

The SSN was defined as a graph structure composed of nodes and edges, where each node represents a non-redundant protein sequence (90% identity threshold), and edges connect node pairs with >40% pairwise sequence identity (BLASTP, e-value <1e-10). The MTP family SSN comprised 47 nodes (non-redundant at 90% identity) and 520 edges (≥46% sequence identity). A total of five clusters were obtained from the SSN, with clusters 1, 2, and 3 accounting for 89% of the total nodes (Supplementary Figure S3). The results showed that 11 CcMTP members within the Zn-MTP subfamily clustered in cluster 2, 9 transporters classified as Zn/Fe-MTPs in cluster 3, and 5 CcMTPs within the Mn-MTP clade in cluster 1. In cluster 1, six CcMTPs and AtMTP8 exhibited sequence homology ranging from 47% to 56%, whereas CcMTP1 shared as high as 67% sequence homology with AtMTP12 at the amino acid level. Seven CcMTP genes, CcMTP3, CcMTP6, CcMTP7, CcMTP8, CcMTP15, CcMTP17, and CcMTP19, showing significant homology with AtMTP1 and AtMTP3 were co-clustered in cluster 2 (Supplementary Figure S3).

Conserved motifs, domains, and structure of CcMTPs

To acquire a more in-depth understanding of the structural properties of CcMTPs in C. chinensis, conserved motif analysis was performed using the MEME server to identify 10 evolutionarily conserved protein motifs (Supplementary Figure S4). The 25 CcMTP genes exhibited motif counts ranging from 1 to 5, demonstrating quantitative heterogeneity in conserved domain architectures (Figure 3A). Motif composition analysis showed that most genes (n = 20) harboured 2–4 motifs, whereas two genes (CcMTP10 and CcMTP23) harboured only a single motif. Three genes (CcMTP14, CcMTP20, and CcMTP21) possessed five motifs. Notably, tandem duplication of motif 1 was detected in 64% of the analysed CcMTP proteins, whereas duplication was absent in CcMTP3, CcMTP12, CcMTP13, CcMTP14, CcMTP20, CcMTP21, CcMTP22, CcMTP24, and CcMTP25. Additionally, CcMTP10 and CcMTP23 exclusively contained motif 1, suggesting that motif 1 may serve as a core functional element, potentially mediating key substrate recognition activities involved in metal transport.

Figure 3

Figure 3. Annotation of the CcMTP gene family in C. chinensis including comprehensive analysis of conserved motifs, exon–intron organisation, and transmembrane helix (TM) architecture. (A) Phylogenetic analysis and motif characterisation of CcMTP gene family. Conserved motifs are distinguished by distinct colour coding across the phylogenetic tree. (B) Gene structures of CcMTPs. Exons, untranslated regions (UTRs), and introns are represented by yellow boxes, green boxes, and connecting lines, respectively. The dimensions of boxes and lines are proportional to the corresponding gene lengths. (C) Conserved motif arrangement across transmembrane helices (TMs). Transmembrane structures are labelled with black numbers, whereas distinct colour coding shows differently conserved motifs.

Notably, phylogenetic subgrouping revealed that members within the same clade demonstrated significantly similar conserved motif distribution patterns, whereas members from different clades displayed marked divergence in motif architecture (Figure 3A). This observation supports the hypothesis that phylogenetic clustering within the same clade correlates with functional homology, as reflected by the conserved motif arrangements among closely related members. A systematic investigation was conducted to examine the topological distribution of conserved structural motifs within transmembrane α-helices in six representative MTP family members (CcMTP1, CcMTP7, CcMTP8, CcMTP19, CcMTP21, and CcMTP22), with particular emphasis on their spatial arrangement across helical domains (Figure 3C).

To assess this further, domain architecture analysis of the CcMTP proteins was performed. The results showed that all CcMTP proteins contained the cation efflux domain. In contrast, the zinc transporter domain was exclusively identified in CcMTP5, CcMTP14, CcMTP20, and CcMTP21 (Supplementary Figure S5).

To further investigate the genomic organisation of CcMTPs, a comprehensive analysis of exon-intron structure was systematically performed via comparison of genomic DNA and CDS sequences (Figure 3B). Genomic characterisation of the CcMTP gene family revealed a stable exon number distribution from 1 to 12 across all members. The CcMTP gene family exhibited a distinct distribution pattern, with 10 members (the majority) harbouring 3–4 exons, whereas five genes (CcMTP22, CcMTP9, CcMTP21, CcMTP18, and CcMTP5) represented the extremes with 2, 6, 9, 10, and 12 exons, respectively. In contrast to the conserved motif patterns, no significant congruence was observed between phylogenetic grouping and exon-intron architecture among CcMTP genes within the same clade.

Characterisation of cis-acting elements in promoters of CcMTP genes

To characterise the cis-regulatory elements within the promoters of CcMTP genes, 2000 bp upstream sequences from the translation start site were computationally analysed for all family members. A total of 16 major cis-acting elements were identified across the 25 CcMTP gene promoters (Figure 4). These elements were functionally categorised into four major regulatory pathways: growth and development, biotic/abiotic stress response, light responsiveness, and phytohormone responsiveness. Promoter analysis revealed that CcMTP gene promoters were particularly enriched in elements associated with light and biotic/abiotic stress responses, including motifs for light responsiveness, drought inducibility, low temperature responsiveness, and general defence and stress response. In addition to the major categories, CcMTPs gene regulatory regions comprised a small subset of elements related to phytohormone response and growth and development responsiveness, such as those responsive to abscisic acid, MeJA, and salicylic acid, along with circadian control elements.

Figure 4

Figure 4. Cis-acting elements analysis of CcMTPs family promoters. (A) Distribution of cis-acting elements. (B) The statistics of cis-acting elements of each CcMTP gene.

qRT-PCR analysis of CcMTP genes in different tissues under Cd²⁺ stress

The relative expression patterns of 24 CcMTP genes under Cd²⁺ stress at 0, 6, 12, 24, and 48 h were assessed using qRT-PCR (Figure 5). CcMTP3 was excluded because of unsuccessful primer design. The CcMTP genes exhibited diverse expression responses to Cd²⁺ exposure across roots, stems, and leaves.

Figure 5

Figure 5. Relative expression levels of CcMTP genes under Cd stress in different tissues. Asterisks indicate significant differences from the control by one-way ANOVA and t-test followed by an independent-samples Dunnett’s post hoc test (*p ≤ 0.05; **p ≤ 0.01).

In roots, Cd²⁺ increased the expression of 11 CcMTPs (CcMTP2, CcMTP5, CcMTP6, CcMTP9, CcMTP10, CcMTP11, CcMTP14, CcMTP16, CcMTP19, CcMTP24, and CcMTP25); however, it repressed the expression of five CcMTPs (CcMTP7, CcMTP8, CcMTP12, CcMTP13, and CcMTP17). In stems, Cd²⁺ markedly enhanced the expression of 62.5% of CcMTP genes, except for CcMTP6, CcMTP10, CcMTP20, CcMTP21, and CcMTP23. In contrast, it remarkably suppressed the expression of CcMTP12, CcMTP13, CcMTP14, and CcMTP22. Notably, the transcript level of most of CcMTPs reached the peak at 48 h after Cd treatment. For instance, the expression levels of CcMTP4 in stems reached their highest at 48 h, which were 40.5-fold higher than those in the control. In leaves, 58.3% of the CcMTP genes were remarkably upregulated under Cd treatment, except for CcMTP1, CcMTP6, CcMTP8, CcMTP18, CcMTP20, CcMTP23, and CcMTP25, whereas the expression levels of CcMTP12, CcMTP13, and CcMTP14 were downregulated. Excess Cd significantly upregulated the expression levels of CcMTP2, CcMTP5, CcMTP9, CcMTP11, CcMTP16, CcMTP19, and CcMTP24. In contrast, it substantially downregulated the expression levels of CcMTP12 and CcMTP13 in three tissues. Given that smaller gene size facilitates PCR amplification, efficient transformation into yeast for functional characterisation, as well as proper expression and folding, CcMTP11, CcMTP16, and CcMTP24 were selected for subsequent functional validation in this study. CcMTP11, CcMTP16, and CcMTP24 were selected for subsequent functional validation in this study.

Functional analysis of CcMTP proteins in yeast

Based on the expression patterns of CcMTP genes in Cd-treated C. chinensis plants, CcMTP11, CcMTP16, and CcMTP24, which exhibited high expression levels in roots, stems, and leaves, were selected as the candidate genes for functional analysis in yeast. The yeast mutant Δycf1, deficient in yeast Cd factor 1, was transformed with the yeast expression vector pYES2 carrying CcMTP11, CcMTP16, or CcMTP24, as well as with the empty vector as a control. When cultured on standard SGR-Ura medium without Cd, or with 10 µM and 30 µM Cd, the growth phenotypes of yeast strains overexpressing CcMTPs were indistinguishable from those harbouring the empty vector. However, on medium supplemented with 55 μM Cd, CcMTP24 significantly enhanced the Cd tolerance of the Δycf1 mutant, whereas CcMTP11 and CcMTP16 marginally improved Cd tolerance (Figure 6A).

Figure 6

Figure 6. Functional analysis of CcMTP11, CcMTP16, and CcMTP24 in the yeast cells. (A) △ycf1 mutant harbouring empty vectors or recombinant plasmids encoding CcMTP11/CcMTP16/CcMTP24 were cultured on SGR-Ura solid medium supplemented with 0, 10, 30 or 55 µM Cd for 3 days. (B, C) Growth dynamics of yeast cells at different time intervals in the absence (B) or presence of CdCl₂ (C). Yeast cultures were adjusted to an OD₆₀₀ of 0.2, followed by the addition of CdCl₂ to achieve a final concentration of 35 µM in the medium. Samples were harvested and OD₆₀₀ was measured at 6-hour intervals thereafter. (D) The content of Cd in yeast cells. ycf1, CcMTP11, CcMTP16, and CcMTP24 represented △ycf1 mutant harbouring empty vectors or recombinant plasmids encoding CcMTP11, CcMTP16, and CcMTP24. Asterisks indicate significant differences from the control by one-way ANOVA and t-test followed by an independent-samples Dunnett’s post hoc test (*, p ≤ 0.05).

Growth curve analysis further confirmed that yeast strains transformed with CcMTP11, CcMTP16, and CcMTP24 exhibited significantly higher growth rates under Cd stress compared with the Δycf1-pYES2 control (Figure 6C). In contrast, no significant growth differences were observed under normal conditions (Figure 6B). Notably, after 48 h of exposure to 35 µM Cd, all three transformants accumulated lower Cd levels than the Δycf1 control, with CcMTP16 and CcMTP24 showing especially reduced Cd accumulation (Figure 6D). These results suggest that CcMTP24 may mediate Cd detoxification by facilitating its efflux into the extracellular medium.

Additionally, we investigated whether CcMTP11, CcMTP16, and CcMTP24 conferred tolerance to other heavy metals, such as Mn, Zn, and Fe. When expressed in yeast, CcMTP11 and CcMTP24 conferred tolerance to Mn, Zn, and Fe, whereas CcMTP16 enhanced tolerance only to Mn and Zn (Figure 7). These findings demonstrate that the heterologous expression of CcMTP11, CcMTP16, and CcMTP24 in yeast enhances cellular tolerance to various heavy metals.

Figure 7

Figure 7. Heterologous expression-mediated tolerance of yeast to Mn, Zn, and Fe conferred by CcMTP11, CcMTP16, and CcMTP24. Yeast cells were subjected to transformation with either the empty vector pYES2 or CcMTP11, CcMTP16, and CcMTP24, then inoculated onto SGR-Ura solid medium amended with 0, 20, 30, 40 mM MnCl₂ (A), 0, 40, 60, 65 mM FeSO₄ (B); 0, 8, 10–12 mM ZnSO₄ (C).

Discussion

MTP genes exhibit ubiquitous distribution across the plant kingdom and encode membrane-localised divalent cation transporters that function in mediating tolerance to and transmembrane transport of diverse heavy metals, potentially playing critical roles in maintaining plant mineral nutrition homeostasis and enhancing resistance to metal-induced stresses (Liu et al., 2024; Li et al., 2024; Hu et al., 2025).

In this study, 25 CcMTP genes were identified and classified into four groups in conjunction with orthologs from A. thaliana and rice and further assigned to three major substrate-specific clades: Zn-MTPs, Zn/Fe-MTPs, and Mn-MTPs (Figure 2). These findings are consistent with the results reported in peanut (Wang et al., 2022), tulip (Lu et al., 2024), Q. dentata (Jiang et al., 2024), T. aestivum (Vatansever et al., 2017), Brassica napus (Xie et al., 2022), and Nicotiana tabacum (Liu et al., 2019), indicating that CcMTPs may exhibit functional similarities to their orthologs in these species. Among plant species with characterised MTP gene families, C. chinensis harbours fewer MTP genes than B. napus (33 MTPs), T. aestivum (33 MTPs), and N. tabacum (26 MTPs), whereas most other species contain 10–12 MTP members (Xie et al., 2022; Vatansever et al., 2017; Liu et al., 2019; Zhao et al., 2024).

Phylogenetic analysis revealed that the CcMTPs cluster into three primary groups (Figure 2), a classification that aligns with the corresponding groupings of A. thaliana and rice. Most plants (e.g., tomato, potato, soybean, Q. dentata, and B. napus) contain more Mn-MTP members than Zn-MTPs (El-Sappah et al., 2021, 2023; Xie et al., 2022; Jiang et al., 2024; Li et al., 2021); however, the contrary pattern was observed in C. chinensis and A. thaliana (van der Zaal et al., 1999), where Zn-MTP members outnumber Mn-MTP members. Phylogenetic analysis further revealed that the six Mn-MTP subfamily members in C. chinensis cluster closely with AtMTP8, AtMTP11, OsMTP8, and OsMTP11, whereas 10 Zn-MTP members and nine additional MTPs formed a clade with MTPs from A. thaliana and rice (Figure 2). Notably, homologous genes within the same phylogenetic clade are likely to exhibit shared or analogous biological functions (Liu et al., 2023).

As previously described, while the cation efflux domain represents a characteristic feature of MTP transporters (Liu et al., 2019), to date, the biological roles of CcMTPs remain largely unelucidated. Nonetheless, the well-characterised functions of MTPs in other species (e.g., A. thaliana and rice) may facilitate the prediction of gene functions in C. chinensis via ortholog-based analysis. For instance, AtMTP8 and AtMTP11 are two Mn-MTP proteins, the functions of which, have been clearly defined. AtMTP8, a Golgi or vacuolar Mn transporter, not only contributes to the protection of plants from Mn toxicity but also is involved in the distribution of Fe and Mn in seeds (Eroglu et al., 2016, 2017; Chu et al., 2017). AtMTP11, localised in the prevacuolar compartment, exhibits Mn²⁺-specific transport activity and plays an essential role in Mn transport and tolerance (Delhaize et al., 2007). In rice, OsMTP8.1 and OsMTP8.2 are tonoplast-localised Mn transporters involved in Mn transport (Tsunemitsu et al., 2018). Compared with AtMTP11, OsMTP11 not only confers Mn tolerance but also actively sequesters Cd into leaf vascular parenchyma cells, preventing its translocation to grains (Farthing et al., 2017; Liu et al., 2024). These findings suggest a potential role for CcMTP proteins in regulating metal ion efflux and maintaining metal homeostasis, highlighting their utility as valuable genetic resources for breeding C. chinensis with low Cd.

Notably, three Zn-MTP genes (CcMTP1, CcMTP8, and CcMTP19) exhibit coding sequences composed of a single exon devoid of introns, classifying them as single-exon genes (SEGs) (Sakharkar et al., 2004). The presence of SEGs in multicellular eukaryotic genomes is of particular interest, because such genes are characteristically archetypal of prokaryotic systems (Sakharkar et al., 2004). SEGs are categorised into two distinct groups: (i) untranslated region (UTR) intron-containing SEGs (uiSEGs), which harbour introns within their UTRs; and (ii) intronless genes (IGs), which lack introns throughout the gene structure (Jorquera et al., 2018). In this study, CcMTP1, CcMTP8, and CcMTP19 were identified as IGs. SEGs have been documented within the MTP family of peanut (Wang et al., 2022), tomato (El-Sappah et al., 2021), and tobacco (Liu et al., 2019). These results suggest that during the evolutionary history of MTP genes in C. chinensis, episodes of intron gain and loss occurred. Certain genes lack introns and consist of a single exon, exhibiting reduced rates of exon gain or loss due to stronger selective pressure acting on exon sequences (Harrow et al., 2006).

Notably, CcMTP1 contains 10 TMDs, more than any other CcMTPs, and was found to possess the longest protein sequence (523 amino acids) and the highest MW (58.64 kDa). Based on a phylogenetic analysis, CcMTP1 was the only MTP member in C. chinensis that clustered closely with AtMTP12 and OsMTP12 in a single clade (Figure 2), suggesting that CcMTP1 is the homologous gene of AtMTP12. These findings are consistent with the characteristics of MTP12 observed in other plant species, which similarly harbour more TMDs, longer protein sequences, and higher MWs, for example, AhMTP12 (16 TMDs, 867 amino acids, and 97.04 kDa), PtrMTP12 (12 TMDs, 869 amino acids, and 97.5 kDa), and VvMTP12 (12 TMDs, 1092 amino acids, and 123.68 kDa) (Wang et al., 2022; Gao et al., 2020; Shirazi et al., 2019). These results suggest that CcMTP1 may possess unique physiological functions in C. chinensis.

Herein, we employed qRT-PCR to analyse the expression patterns of MTP genes across different tissues under Cd stress. The transcriptomic responses of MTP genes to the presence of their potential metal substrates exhibited notable diversity and complexity. AtMTP1, which encodes a tonoplast-localised zinc transporter, maintained stable expression at both transcriptional and translational levels under conditions of zinc excess (Kobae et al., 2004; Dräger et al., 2004). Similarly, the expression level of CsMTP1 was unaffected under Zn excess, although the encoded protein abundance was significantly upregulated in cucumber under metal treatment (Migocka et al., 2015). Varying concentrations of Mn²⁺ exert minimal effects on the expression of Mn-MTP family members (AtMTP8, AtMTP9, AtMTP10, and AtMTP11), a response pattern analogous to that observed in tobacco (Delhaize et al., 2007; Liu et al., 2019). Similarly, the expression levels of Mn subfamily members in C. chinensis exhibited relatively minor changes compared to those in the other two subfamilies, consistent with results in tobacco and alfalfa (Liu et al., 2019; El-Sappah et al., 2021). Notably, six of nine CcMTPs (CcMTP2, CcMTP5, CcMTP9, CcMTP11, CcMTP16, and CcMTP24), which were simultaneously upregulated in roots, stems, and leaves of C. chinensis, belong to the Fe/Zn-MTP class. In contrast to other species such as Q. dentata (Jiang et al., 2024), tomato (El-Sappah et al., 2021), soybean (El-Sappah et al., 2023), and P. trichocarpa (Gao et al., 2020), where the MTP genes significantly responsive to cadmium stress across different tissues predominantly belong to the Zn-MTP and Mn-MTP subfamilies, it is noteworthy that in tobacco, genes from the Zn-MTP, Mn-MTP, and Fe/Zn-MTP subfamilies exhibit responses to cadmium stress in various tissues. It highlights the specific involvement of the Fe/Zn-MTP members in C. chinensis under Cd stress. Conversely, excessive Cd²⁺ exposure induced minimal changes in the mRNA expression levels of CcMTP6, CcMTP20, and CcMTP23 in this study. Thus, further investigation is needed to characterise the protein-level responses of CcMTPs to metal ions in future research.

Currently, yeast mutants are frequently used to characterise the basic functions of genes under heavy metal stress conditions (Khan et al., 2019). CcMTP11, CcMTP16, and CcMTP24, members of the Fe/Zn subfamily with two or three TMDs located in the plasma membrane or Golgi (Table 1), were highly upregulated in roots, stems, and leaves under Cd stress (Figure 5). However, heterologous expression of CcMTP24 in yeast revealed tolerance to four heavy metals, including Cd, Mn, Fe, and Zn (Figures 6, 7). Comparatively, yeast expressing CcMTP11 exhibited tolerance to Mn, Fe, and Zn, whereas CcMTP16 conferred tolerance only to Mn and Zn (Figures 6, 7). Consistent with our results, QdMTP10.7 and PtrMTP6 also conferred tolerance to various heavy metal stresses in yeast cells (Jiang et al., 2024; Gao et al., 2020). Notably, CcMTP24 significantly reduced Cd accumulation in yeast (Figure 6), identifying it as a candidate gene for further functional analysis.

Despite the insights gained from our study, several limitations should be acknowledged. First, the Cd tolerance phenotypes observed in yeast may not fully translate to the native functions of CcMTP genes in planta. Future studies employing transgenic or CRISPR/Cas9-mediated approaches in C. chinensis would help clarify the precise mechanisms by which these MTP proteins confer heavy metal tolerance. Additionally, our functional analysis was performed specifically under Cd stress conditions; the responsiveness of CcMTP genes to other heavy metals, such as Mn, Fe, Cu, Co, and Zn, remains to be investigated in subsequent work.

Conclusion

Our study provides a comprehensive identification and characterisation of the MTP gene family in C. chinensis, a species for which this gene family had remained unexplored. 25 CcMTP genes exhibited uneven chromosomal distribution and were classified into three major substrate-specific groups: Zn-MTPs, Zn/Fe-MTPs, and Mn-MTPs. These genes appear to have undergone gene loss events and experienced selective pressure. All CcMTPs contained conserved modified signature sequences and the cation efflux domain. Expression profiles of CcMTP genes across different tissues and under Cd²⁺ stress indicated their conserved and critical roles in the growth and development of C. chinensis. Moreover, overexpression of CcMTP24 in the yeast mutant Δycf1 enhanced Cd tolerance and reduced Cd accumulation, providing functional evidence that CcMTP24 contributes to Cd stress resistance. In addition, CcMTP11, CcMTP16, and CcMTP24 improved tolerance to multiple heavy metals, including Mn, Fe, and Zn. These findings suggest that the identified CcMTPs are promising molecular targets for developing Cd -tolerant C. chinensis cultivars, offering the dual benefit of mitigating ecological risks from heavy metal toxicity and enhancing agricultural sustainability. Further characterisation of CcMTP-mediated Cd transport and detoxification pathways will improve our understanding of the genetic architecture underlying metal accumulation in C. chinensis, thereby informing strategies for regulating metal homeostasis in medicinal plants.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Author contributions

YZ: Conceptualization, Writing – original draft, Formal analysis. LW: Writing – original draft, Data curation, Formal analysis. JZ: Writing – original draft, Formal analysis, Data curation. CW: Writing – original draft, Formal analysis, Data curation. YF: Formal analysis, Data curation, Writing – original draft. HC: Writing – original draft, Formal analysis. HZ: Investigation, Writing – original draft. YM: Writing – original draft, Investigation. HW: Writing – original draft, Investigation. JK: Software, Writing – original draft. YJ: Software, Writing – original draft. XS: Investigation, Writing – original draft. XL: Writing – review & editing. KX: Funding acquisition, Writing – review & editing.

Funding

The author(s) declare financial support was received for the research and/or publication of this article. This work was supported by the National Natural Science Foundation of China (32102487), Department of Science and Technology Planning Project of Henan Province (252102111164, 242102110299, 252102110299, 252102110322).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declare that no Generative AI was used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2025.1658134/full#supplementary-material

Supplementary Figure 1 | Multiple sequence alignment of AtMTP and CcMTP proteins. The signature sequences and the consensus motifs HXXXD/DXXXD (where X denotes any amino acid) are highlighted by red and green open boxes, respectively.

Supplementary Figure 2 | Predicted 3D models of CcMTP proteins. The structural models were computationally generated via the intensive mode of the Phyre 2 server. The resultant 3D structures were visualised using a rainbow colour scheme to depict the protein backbone from the N- to C-terminus and systematically organised according to the CcMTP gene family members (CcMTP1–CcMTP25).

Supplementary Figure 3 | Sequence similarity network (SSN) for MTP proteins from C. chinensis, A. thaliana, and O. sativa. Nodes in the network represent 47 cation efflux domain-containing protein sequences from C. chinensis, A. thaliana and O. sativa. The SSN was constructed using EFI-EST and visualised with Cytoscape. Edges between pairs of nodes in the network correspond to two sequences ≥46% similarity.

Supplementary Figure 4 | Sequence logos for 10 conserved motifs in Cation efflux domains were generated using the MEME algorithm. MEME-derived motifs are visualised as stacked amino acid letters, where the total height of each stack denotes the information content (in bits) of the corresponding site within the motif. The height of each letter within a stack reflects the product of its positional probability and the stack’s total information content. The X-axis represents motif length, whereas the Y-axis indicates the information content (in bits) for each residue.

Supplementary Figure 5 | Structural distribution of conserved domains in CcMTP proteins. Cation efflux domains are denoted by green boxes, whereas zinc transporter domain and xylanase inhibitor N-terminal dimerisation domain are represented by pink and yellow boxes, respectively.

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