Compared to the key proteins for Cd uptake in rice and Arabidopsis, it was found that these proteins were mainly expressed in plant roots and located on the cell membrane, indicating that the free Cd in the soil solution was mainly transported into the plant through root transporters (Table 1). The NRAMP protein mainly relies on H co-transport drive to provide energy, while the transport mechanism of ZIP is currently unclear. In particular, there are only about 5 NRAMP members whose evolution tends to be conservative in plants, and there is no large-scale expansion. DPGN residues are only found in NRAMP, which may be a key and special function residue. In animals, members of the NRAMP family have been shown to play an important role in the immune response, usually in the phagocytosis and presentation of antigens by pathogens, but whether there are similar functions in plants has not been reported. In addition to the Cd stress response, it seems likely that it participates in a series of foreign invasion defenses. However, there are many members in the ZIP family and their evolution has undergone specific changes. For example, all of the ZIP proteins involved in Cd influx have a signal peptide at the N-terminus. Signal peptides can play a role in controlling protein secretion rate, determining protein folding state, affecting downstream transmembrane behavior and N-terminal glycosylation, and nuclear localization signals [50]. The changes may lead to differences in subcellular functions between them, and may produce specific stress responses to different metal ions. Between TMD3 and TMD4 of the ZIP protein, there is a histidine-rich residue (HX)n (X = any amino acid, n = 3–6) that can act as a Zn buffer bag and a cytoplasmic surface zinc level sensor, and it may be a potential metal (Zn, Co and/or Cd) binding domain (Figure S2).  This residue is also ubiquitous in the MTP family, but surprisingly, the MTP principally produces an effect in the vacuole [51]. Interestingly, OsZIP1 can also be located in the endoplasmic reticulum, while OsZIP3 can be expressed in xylem parenchyma cells. The parenchyma of the xylem of plant roots is the main “controller” of Cd loading into the xylem and its transport to the branches [52], indicating that the functions of the family members have diversified. However, these changes may benefit from the emergence of the N-terminal specific signal peptide of the ZIP family. Future research should aim to determine whether it will play a more unique role, for example, the special signal peptide may be improved to become a part of the NRAMP family.

Table 1. Summary of tissue structure-specific expression, structural characteristics and functions of key Cd influx proteins.

The Key Proteins	Tissue Specific Expression	Subcellular Localization	Structural Features	References
AtNRAMP1	Mainly expressed in the root	Cell membrane	12 TMDs, DPGN near to TMD1.	[53,54]

105].

4.4. Comparison of Core Characteristics of Key Proteins in Vacuolar Transport

Vacuoles are the largest organelles in mature plant cells. They are a reservoir of ions and metabolites and are indispensable for the detoxification process and normal cell development. By comparing the results of subcellular localization, it is found that CAX/MTP/ABCC family members are mainly located on the vacuole membrane, which will increase the content of Cd in overexpressed plants, indicating that these proteins are detoxified by transferring Cd to the vacuole storage (Table 3). H/DXXXD residues are widely distributed in these three family members (Figures S5 and S6), whether they are a specific Cd binding site remains to be considered. The difference is that CAX and ABCC family members can be expressed both in the vacuole membrane and in the vacuole. Whether they have multiple functions, such as chelating Cd in the vacuole and transporting on the vacuole membrane, remains to be studied in depth. The ABCC transporter in particular can not only transport Cd directly, but also transport the PC-Cd complex. Whether there is a specific theme to give this interesting substrate difference or not requires continued research.

Table 3. Tissue structure-specific expression, structural characteristics and function summary of key proteins in vacuolar transport.

The Key Proteins	Tissue Specific Expression	Subcellular Localization	Structural Features	References
AtCAX2	/	Tonoplast	8 TMDs; with GNXXE residue on TMD2 and TMD7.	[81]
OsNRAMP1	Mainly expressed in the mature zone of the root	Cell membrane	12 TMDs, DPGN near to TMD1.	[32,55]
AtCAX4	Mainly expressed in the root	Tonoplast	10 TMDs; a GNXXE residue on TMD7, and the DXXXD residues between TMD5-6.	[82]		OsNRAMP5	Mainly expressed in the root	Cell membrane	10 TMDs, DPGN is near to TMD1. There is a Asparticacid residue (DXXXD, X= any amino acid) at the C-terminus.
OsCAX1a	expressed at a high level in flowering spikelet	[	Vacuole	31]
10 TMDs; GNXXE residues on TMD3 and TMD8, and the DXXXD residues between TMD6-TMD7.	[		83		]		AtIRT1	Root epidermal cells	Cell membrane
OsCAX1c	8 TMDs; there is a signal peptide at the N-terminus, histidine, Asparticacid residues (HXXXD, X= any amino acid) on TMD2, and (HX)n between TMD3 and TMD4.	[	40	]
Strongly expressed in leaves	Tonoplast	10 TMDs; GNXXE residues exist on TMD2, TMD, and DXXXD residues near TMD6.	[		83]		OsIRT1	Mainly expressed in the root	Cell membrane	8 TMDs; there is a signal peptide at the N-terminus, HXXXD residues on TMD2, and the (HX)n residues between TMD3 and TMD4.	[
AtABCC1	Mainly expressed in the root	Vacuole	15 TMDs, nucleotide binding site at the C-terminus, cysteine-rich loop, Q-loop, Walker B residue.	56]
[		86		]		OsIRT2	Mainly expressed in the root	Cell membrane	There are 8 TMDs with signal peptide and DXXXD residues at the N-terminus; the (HX)n residues between TMD3-TMD4.
AtABCC2	Mainly expressed in the root	Vacuole	10 TMDs, cysteine-rich loop, Walker B residue, DXXXD residues exist between TMD4-TMD5.	[56]
OsZIP1	Mainly expressed in the root	Cell membrane	8 TMDs, with signal peptide and DXXXD residues at the N-terminus; HXXXD residues on TMD2, and the (HX)n residues between TMD3-TMD4.	[41]
OsZIP3	Xylem parenchyma cells	Cell membrane	There are 7 TMDs, there is a signal peptide at the N-terminus, and there are (HX)n residues and HXXXD conserved residues between TMD3-TMD4.	[41,57]

3. Chelating Protein

Only a small part of the metal content in plants exists in the form of free water ions, and most of the ions are combined with low molecular weight ligands or proteins. The cell wall is the first protective barrier to prevent heavy metals from invading cells, the first “living” structure, and the target of heavy metal damage. The immobilization of Cd in the cell wall participates in the tolerance mechanism of plants to Cd, and PDF exists in the cell wall and can participate in the chelation of it. However, MT mainly exists in the cytoplasm and can combine with free Cd to reduce the harm of heavy metals to plants.

3.1. A Brief Introduction to the MT Family, Key Proteins and Chelation Mechanism

MT is a low-molecular-weight metalloprotein in cells that is rich in cysteine and exists in all organisms except archaea [58]. According to the distribution pattern of cysteine, it can be divided into 4 sub-families: MT1, MT2, MT3 and MT4 [59]. The MT1-3 subfamily has two cysteine-rich regions, 6 cysteines in the C-terminal domain, and 6, 8 and 4 cysteines in the N-terminal cysteine-rich region, respectively. M4 has three cysteine-rich regions, each containing 6, 6 and 5 cysteine residues [60]. It can be combined with heavy metal Cd with high affinity, and plays a role in detoxification of heavy metals and maintaining the stable state of essential metal ions in cells. Among them, MT1, because of its unique metal bonding characteristics, can endow Cd resistance and contribute to the stable state of zinc [61]. The heterologous expression of OsMTI-1b can improve the tolerance of E. coli cells to Cd by chelating metal ions [62]. Overexpression of OsMT1e improved rice growth under Cd stress [63]. After CdCl₂ treatment, Arabidopsis lines overexpressing OsMT-3a accumulated higher levels of Cd in shoots and roots [64]. From Figure S3, wWe can see that the primary structure of MT is obviously rich in highly conserved cysteine residues such as CC, CXC and CXXC (X = any amino acid). These residues have the remarkable ability to bind a large number of monovalent or divalent metal ions, as well as maintain the steady-state of basic metals and the ability to detoxify (toxic) metals, especially copper, zinc and Cd [65]. The sulfhydryl groups on MTs can combine with heavy metal ions to form non-toxic or low-toxic compounds, thereby eliminating the toxicity of heavy metals [66]. Furthermore, the mercaptans (esters) in MTs can act as powerful antioxidants. They usually bind metal ions in metal thiol clusters to provide high thermodynamic stability and kinetic instability [67]. MT constitutes a highly complex system, even though the same peptide can show different final 3D folds, it is expected that these folds are related to different functions [68].

3.2. A Brief Introduction to the PDF Family, Key Proteins and Chelation Mechanism

PDF is a type of small molecule protein, each containing 45 to 54 amino acids (Figure S4), and 13 members have been identified in Arabidopsis [69]. PDF can be divided into two families, namely PDF1 and PDF2, which mainly play a role in fungal resistance and zinc tolerance, but it is interesting that the recent reports in the literature show that they can participate in the chelation of Cd. For instance, the PDF2 (AtPDF2.5) in Arabidopsis is involved in Cd tolerance and accumulation, and Cd binding analysis shows that AtPDF2.5 has Cd chelating activity in vitro [70]. Overexpression of AtPDF2.6 increases the tolerance of E. coli and yeast to Cd, and the in vitro Cd binding test shows that AtPDF2.6 also has Cd chelating activity [71]. The PDF1.5 is located in the cell wall and may detoxify Cd by chelating Cd to the cell wall [61]. PDF has a characteristic three-dimensional folding pattern, which is stabilized by eight disulfide bonds between cysteines [72]. Cysteine and other key residues are necessary to induce changes in the three-dimensional structure of AtPDF, which can mediate and increase yeast tolerance to Cd [73].

3.3. Comparison of the Core Features of Key Cd Chelation Proteins

Analyzing the proteins involved in Cd chelation in the MT and PDF families showed that these proteins all have a cysteine-rich domain (Table 2). Cysteine is a sulfur-containing amino acid with physiological activity, with amino and sulfhydryl groups. Among protein amino acids, cysteine may be the only amino acid that contains a thiol group on the side chain and has a binding site for heavy metals of plant cells [74]. PDF can chelate Cd in the cell wall, thereby preventing Cd from destroying the cell structure, and has a more effective detoxification ability. Nevertheless, MT forms a chelate with Cd in the cytoplasm, and is then transferred to the vacuole by other transporters for detoxification. Interestingly, there is a special signal peptide at the N-terminus of the PDF protein, which may mediate its secretion in the cell wall. Surprisingly, OsMTI-1b and OsMT1e are expressed in the nucleus, indicating that members of the MT family seem to be involved in the regulation of gene transcription. On the whole, the cell wall is a unique cell structure of plants, and it is the first protective barrier to prevent heavy metals from invading cells. The specific signal peptide at the N-terminus of PDF protein has great research value for the genetic improvement of crop Cd tolerance, and how the MT family members regulate gene expression in the nucleus remains to be explored.

Table 2. The tissue structure specific expression, structure characteristics and function summary of the key proteins of Cd chelation.

The Key Proteins	Tissue Specific Expression	Subcellular Localization	Structural Features	References
AtMT2a	/	Cytoplasm	CXCXXXCXC and DXXXD are distributed at the C end, without TMDs.	[75]
AtMT3	/	Cytoplasm	CXCXXXCXCXXXCXC is distributed at the C-terminus and has no TMDs.	[75,76]
OsMTI-1b	/	Cytoplasm, Nucleus	CXCXXXCXCXXXCXC is distributed at the C-terminus and has no TMDs.	[62]
OsMTI-2b	Highly expressed in rice stems	Cytoplasm	N-terminal has 8 cysteine residues arranged in CC, CXC and CXXC residues.	[77]
OsMT1e	Expressed in roots at all developmental stages	Nucleus	CXCXXXCXCXXCXCXCXCX is distributed at the N-terminal and has no TMDs.	[78]
OsMT-3a	/	Cytoplasm	CXCXXXCXCXXCXCXCXCX is distributed at the C-terminus and has no TMDs.	[59,64]
AtPDF1.5	Mainly expressed in knots and peels	Cell wall and cytoplasm	There is a signal peptide at the N-terminus, a cysteine-rich domain, and a DXXXD residue.	[72]
AtPDF2.5	mainly expressed in root vascular bundle	Cell wall	Cysteine-rich domain and secretion signal peptide.	[70]
AtPDF2.6	mainly expressed in root vascular bundle	Cell wall	There is a signal peptide at the N-terminus, a cysteine-rich domain.	[71]

4. Vacuolar Protein

The cellular mechanism of Cd is generally considered to include isolation in the cytoplasm by glutathione, glutathione-related plant chelator, and isolation in the vacuole by the vacuolar membrane Cd/H antiporter. In fact, most plants detoxify heavy metals by storing Cd-chelating complexes or free Cd in vacuoles, because this organelle contains enzymes involved in Cd detoxification, such as phosphatase, lipase, and protease. The main proteins involved in the transfer of Cd into vacuoles in plants are CAX, ABCC and MTP family members.

4.1. Introduction to CAX Family, Functional Proteins and Vacuolar Transfer Mechanism

CAX is a secondary charged ion transporter located in the vacuole, which relies on the secondary energy transport of metal ions in the proton transmembrane gradient [79]. The main function of CAX protein is to limit the toxicity of certain free metal ions to cells, such as Mn or Cd, provide tolerance to metal toxicity, and maintain cytosolic concentration [80]. For example, the expression of AtCAX2 in tobacco increases the transport of Cd in root vacuoles [81]. AtCAX4 compensates to a certain extent for the reduction of the vacuolar membrane H pump and the increase in proton leakage, which helps to improve the higher tolerance of plants to Cd [82]. OsCAX1a/OsCAX1c may also be involved in the process of Cd transfer to vacuole storage [83]. In plants, CAX is driven by the pH gradient produced by vacuolar H-ATPase and H-pyrophosphatase to drive metal ion transport. Studies have shown that the local primary structure determines the cation specificity of CAX binding [84]. There is a highly conserved 36 motif region between TM3 and TM4, TM8 and TM9, named c-1 loop and c-2 loop, respectively, which may form a vestibule or filter for cation selection, and there are highly conserved GNXXE residues (Figure S5)  [85]. CAX has a common motif, which is the region rich in acidic amino acid motifs between TM6 and TM7, which is believed to be related to the capture and selection of metal ions.

4.2. An Introduction to the ABCC Subfamily, Functional Proteins and Vacuolar Transfer Mechanism

The ABC transporter is one of the largest and oldest protein families; it plays a very crucial part in plant heavy metal transport and plant disease resistance. A total of 9 subfamilies (ABCA-ABCI) have been identified in eukaryotes, of which 8 subfamilies (ABCA-ABCG and ABCI) exist in the plant genome. In plants, the two subfamilies that are currently reported to be related to heavy metals are mainly ABCC and ABCG. ABCC mainly transfers Cd to vacuoles, while ABCG removes Cd from cells for detoxification. The AtABCC1 and AtABCC2 participate in the vacuolar separation of PC-Cd (II) and PC-Hg (II), thereby making plants tolerant to these toxic heavy metals [86]. AtABCC3 detoxifies by transporting the PC-Cd complex into the vacuole [87], and the OsABCC9 isolates Cd into the vacuoles of rice roots to mediate Cd tolerance and accumulation [88]. They are usually composed of two membrane domains or TMDs and two cytoplasmic nucleotide binding domains (NBD) [89]. Two TMDs provide channels for metal ions, while two NBDs combine and hydrolyze ATP to provide energy for transport reactions [90]. As the input proteins, TMD and NBD act as separate polypeptide chains, while in the bacterial export protein, a TMD and NBD are fused to form a homodimer or a heterodimer, thereby producing a complete and functional transport protein [91]. In particular, high-resolution electron microscopy has determined the structure of 8 different states and conformations, including two inward (IF), four outward (OF) and two asymmetric hydrolyzed states [92]. A single conformational transition from IF to OF conformation, triggered by ATP binding, drives unidirectional substrate translocation across the membrane. After this power stroke, ATP hydrolysis and phosphate release start to return to a static state, which promotes nucleotide exchange and a new round of substrate binding and translocation [93].

4.3. Introduction to MTP Family, Functional Proteins and Vacuolar Transfer Mechanism

CDF is also known as MTP in plants. MTP/CDF is a type of membrane-bound protein, which can maintain cell homeostasis and play a vital part in the process of plants dealing with heavy metal stress. It is a ubiquitous divalent cation transporter and is essential for the metal homeostasis and tolerance of archaea, bacteria and eukaryotes. Most of the CDF family members confer heavy metal tolerance by affecting the outflow of heavy metals from the cytoplasm. Due to the different members, outflow refers to the outflow from the cell, or into the internal compartment [94,95]. In the model plants rice and Arabidopsis, members of the MTP family mainly transfer heavy metals into vacuoles to increase plant tolerance. For instance, heterologous expression of OsMTP1 in tobacco plants increases the plant’s tolerance to Cd toxicity and allows transgenic plants to accumulate more Cd [96]. After 24 h of Cd stress, the expression levels of OsMTP6, OsMTP7, OsMTP9 and MTP11.1 in the shoots of rice increased significantly [90], but there is no research showing how they respond to Cd stress [97]. All CDF transporters consist of two domains: the TMD for cation transport and the regulatory cytoplasmic C-terminal domain (CTD) [98]. The combination of metal ions and CTD will induce its dimerization, and it will lead to a higher CTD accumulation [99]. Although CTD shows a high degree of sequence variability between different species, all available CTD structures have similar metal chaperone-like folds [100]. Metal chaperones are cytoplasmic metal carrier proteins that form a metal donor–acceptor interface with related transport mechanisms to transport metal ions to various protein targets and participate in cytoplasmic metal transport [101]. They usually have 6 TMDs and have a highly conserved characteristic sequence in TMD 2 and TMD 5, which has a potential effect on metal selectivity [102,103]. There are also HXXXD or DXXXD conserved residues on or near these 2 TMDs. If this residue is subjected to site-directed mutation, the CDF transporter will lose its ability to bind to Cd and impair Cd transport [104]. However, so far, the metal selectivity of the substrate among members of the CDF family is still unclear. We speculate that these two conserved and aspartic acid-rich residues are key targets for the binding and transport of metal ions. Furthermore, some MTP transporters have an (HX)n between TMD 4 and TMD 5 that also exists in members of the ZIP family and has similar functions. It can be used as a Zn buffer bag and a cytoplasmic surface zinc level that recognizes Sensor [

[
86
]
AtABCC3
/	Tonoplast	15 TMDs, cysteine-rich loop, Walker B residue, HXXXD residues near TMD3, and DXXXD residues between TMD9-TMD10.	[		87]
OsABCC9	Significantly induced by Cd treatment in roots	Tonoplast	8 TMDs, cysteine-rich loop, Q-loop, Walker B residue, and HXXXD and DXXXD residues at the N-terminus.	[88]
OsMTP1	Significantly induced by Cd treatment in roots	Vacuole	The characteristic residue of CDF (SLAILTDAAHLLSDVAA), 6 TMDs and histidine-rich regions, HXXXD residues exist on and/or near TMD2, TMD5.	[96]