1. ATP-Binding Cassette Transporter Gene Family
Plants employ ATP-binding cassette transporters, or ABC transporters, to carry out various functions. One of these functions involves upholding homeostasis when the plant is exposed to heavy metals
[1]. The most critical processes that have evolved in plants to protect them from negative effects such as Cd are the
glutathione—dependent phytochelatin, as has been mentioned and PDR8-dependent pathways
[2].
AtPDR8 is one of the fifteen genes encoding the family of ABC transporters in
Arabidopsis thaliana, showing overall altered regulation following exposure to external and internal stressors
[3].
WRKY13 has a unique role in response to exposure of Cd. Moreover, their upregulation increases resilience to stress from Cd. They affect upregulation of
PDR8 in
Arabidopsis thaliana, which decreases accumulation Cd in plants
[4]. Plants with overexpression of
MMDH2 have been detected in the regulation of the
PDR8 gene in a negative way
[5]. The fact that higher expression of the
PDR8 gene helps plants become more resilient was described. Moreover, it has been concluded that
AtPDR8 acts as a pump for draining excess Cd in
Arabidopsis thaliana [6].
2. PCR Gene Family
To date, PCRs have not been sufficiently well studied. However, it has been shown to be able to transfer Cd and zinc (Zn) in plants. They contain the CCXXXXCPC and CLXXXXCPC motifs and also the PLAC8 motif. How much, if at all, these motifs are related to Cd and Zn transfer is not yet known
[7]. Several regulated genes from this family of genes have been discovered in a variety of plants.
HvPCR2 plays an important role in the detoxification of
Hordeum vulgare L.
[8].
SaPCR2 gene is regulated in
Sedum alfredii [9]. The
OsPCR1 gene has been found to be associated with detoxification and accumulation
[10]. Subsequently, another gene was discovered in
Oryza sativa [11]. The results were obtained using two different plant species, so that the focus was on the difference in their ability to accumulate Cd. Overexpression of the gene
SIPCR6 underpinned the high resistance of
Salix linearistipularis. Its function was verified using the established transgenic
Populus [12].
3. ZIP Gene Family
Homologous genes occurring in the plant kingdom may not show the same expression after exposure to Cd. Evidence shows that expression of homologues ZIP family genes in
Oryza sativa after Cd treatment is different than in
Arabidopsis thaliana. The difference is in the location of expression. While in
Arabidopsis thaliana, there is a higher expression of ZIP genes, especially in the root, in
Oryza sativa, it is mostly in the shoot. Interestingly, quite the opposite regulation of specific genes:
AtIRT3,
AtZIP5,
AtZIP12,
OsIRT1 and
OsZIP1 was detected
[13]. An increase in the expression of the
OsIRT1 and
OsIRT2 genes has been observed in response to Fe deficiency. This overexpression, according to one study, could lead to increased uptake of Cd into the plant. The author goes on to explain that, thanks to OsIRT1, cadmium does not enter the plant through the roots, but is subsequently transported to other parts of the plant as well
[14].
AtZIP9 and
AtIRT1 genes were further detected, where expression was decreased in the roots of
Arabidopsis thaliana. If a disturbance of expression in
AtFC1 occurs, there is a change in gene transcription.. Detoxification processes in plants with high Cd exposure comprise mainly genes that are involved in GSH-dependent phytochelatin synthetic pathway
[15]. A link has been found between
WRKY33 and the expression of
ATL31, which subsequently affects expression of the
IRT1 gene. The whole mechanism works on the principle of decreasing absorption of Cd. After Cd treatment, gene encodes
WRKY33 is upregulated. In theory, it links to the promotor of the
ATL31 gene.
ATL31 causes degradation of IRT1. Although the IRT1 transporter is the primary carrier for iron, it is also involved in transporting of other metals, including Cd
[16]. It is likely that the transfer of a particular metal depends on a conserved residue in or in the immediate vicinity of the transmembrane domain
[17]. Cd displaces other elements, such as Fe, Zn and Mn, from the transport pathways
[18].
4. CDF/MTP Gene Family
The CDF gene family is also named MTPs, which means metal tolerance proteins. In
Citrus sinensis L. overexpression of eight genes was detected in
CitMTP1,
CitMTP3,
CitMTP4,
CitMTP5,
CitMTP7,
CitMTP10,
CitMTP11, and CitMTP12 in the underground section of plant. Apart from in the leaves overexpression of five genes was detected in CitMTP1, CitMTP3, CitMTP5, CitMTP8, and CitMTP12. Samples used in the research was affected by Cd for 7 days and 15 days with the form of Cd 0.038 mM CdSO
4 and 0.38 mM CdSO
4. The highest expression was analysed in
CitMTP11 gene in the root with 0.038 mM CdSO
4 and 15 days affected by Cd
[19]. Increases in PCR gene expression were also detected in the sensitive plant
Glycine max. In the leaves were upregulated genes GmaMTP1.1, GmaMTP1.2, GmaMTP3.1, GmaMTP3.2 and GmaMTP4, whereas in root were upregulated GmaMTP1.1, GmaMTP1.2, GmaMTP3.1, GmaMTP4.1, GmaMTP4.3, GmaMTP10.4, and GmaMTP11.1
[20]. In Fagopyrum tartaricum one gene FtMTP8.2 has been detected so far
[21]. In Medicago truncatula there are twelve known CDS genes, of which five respond to Cd.
MtMTP1.2 and
MtMTP4 are upregulated in the root,
MtMTP1.2 and
MtMTP4 in stems,
MtMTP4 in leaves. Based on bioinformatics analyses, it grouped all of the MTP genes in
Medicago truncatula into clusters, and within these clusters, the genes showed a high degree of concordance. Further analyses showed that these genes contain domains that can be influenced by abiotic stressors and hormones
[22].
5. NRAMP Gene Family
Many NRAMP genes have been identified in plants so far. These NRAMP genes are famous for encoding metal transporters. The study of plant genomes using modern techniques has led to new insights into the adaptive processes that have played an important role during evolution
[23]. Phylogenetic studies show that the number of introns and motif in the NRAMP genes family is changing. This information could mean that plants are adapting to stresses during evolution. A consequence of NRAMP genes being able to respond to heavy metals-induced stress is that the promoters of these genes contain many motifs or elements. In a comparative study, the MYB MYC and STRE elements were the most abundant in the observed
Spirodela polyrhiza plant. In addition, ABRE motifs linked with the hormones were present in the compared plants. Following Cd exposure, the expression of NRAMP genes in the plant is altered. The genes
SpNramp1,
SpNramp2, and
SpNramp3 of Spirodela polyrhiza are sensitive to Cd treatment. These genes showed their activity mainly in the root
[24]. In
Arabidopsis thaliana, NRAMP genes AtNRAMP2, AtNRAMP3, AtNRAMP4 and AtNRAMP5 contain up to 67–75% conserved regions in amino acid sequence. After Cd treatment overexpression, the
AtNRAMP3 gene causes changes in Fe accumulations and root growth
[25]. That this gene is involved in the maintenance of metal homeostasis is also demonstrated by Oomen
[26]. Furthermore, the function of these genes in response to heavy metals was analysed in
Thlaspi caerulescens. Expression of NRAMP3 and NRAMP4 was significantly higher in
Thlaspi caerulescens than in
Arabidopsis thaliana [26]. It has been discovered that there is a difference in gene expression, specifically
NRAMP1,
NRAMP2,
NRAMP3,
NRAMP5 and
NRAMP6 in two varieties of peppers
[27].
OsNRAMP1 has also been discovered in Oryza sativa which is associated with increased uptake and accumulation in the plant
[28].
6. ACS and ACO Multigene Family
It is established that ACO and ACS share a common role in the production of ethylene
[29] and that the overexpression of their genes by Cd leads to an increase in the production of ethylene. The further determined genes which are involved in response to heavy metals have been demonstrated in previous studies. An increase of gene expression of
ACS1,
ACS2,
ACS4,
ACS5,
ACS6,
ACS7,
ACS8 was identified in
Arabidopsis thaliana after Cd treatment. While the higher level of the expression in the root was
ACS6 after 72 h, in the leaves was after 24 h. These studies have also observed increased expression in members of the ACO multigene family. The most highly expressed genes,
ACO2 and
ACO4 were detected in the roots and in leaves after Cd treatment
[30].
7. HIPP/HPP Gene Family
The number of genes encoding HIPP/HPP in plants is very high. In some plant species, there are more than two hundred
[31]. The
OsHIPP56 gene is thought to play an important role. For testing this, the CRISPR/Cas9 technique was used to create rice in which this gene was knocked out. The mutation resulted in large amounts of Cd entering the edible parts of the plant
[32].
8. PCs Gene Family
The
PCS1 and
PCS2 genes have also been implicated in the response to Cd in plants defence mechanisms. However, the transcriptomic level is also influenced by ZAT6, which belongs to the ZIP transporter family
[33]. Strikingly, in the study by Santoro
[34] in the plant
Arundo donax L., Cd did not alter the expression of PCS genes. This is a consequence of the fact that in this experiment, the plant defence mechanism was not triggered by PCS
[34]. Following the application of 50 μM Cd in
Vicia sativa, the
VsPC1 gene was upregulated, probably leading to increased tolerance to Cd
[35]. It is suggested that
PCS1 is affected by overexpression of the
MAN3 gene.
MAN3 in
Arabidopsis thaliana is important in responding to heavy metal stress. After exposure to Cd, there is an increase in the expression of
MAN3.
MAN3 has a critical role in increasing mannose concentration and the consequent activation of PC synthesis related genes such as
PCS1 and
PCS2 [36]. This whole cascade likely contains up to four different components involved in responding to Cd. This entire complex includes the MYB4 transcription factor, which binds to the
MAN3 gene and the MAN3-mediated mannose-binding-lectin 1 (MNB1) gene. It is the MNB1-related GNA domain that influences the resistance of this complex, as it is capable of association with MAN3
[37].
9. MT Gene Family
MTs provide sites on their genes for transcription factors to bind, resulting in differential expression and response to heavy metal exposure. A differential expression of the
ZmMTs has been found in
Zea mays. In the underground part of the plant, the expression of
ZmMT3 and
ZmMT7 genes is reduced. On the one hand, a higher expression of
ZmMT3, and lower expression of
ZmMT1,
ZmMT7 and
ZmMT8 were observed in the stems. In the leaves, a different expression was observed. The
ZmMT3,
ZmMT7 and
ZmMT9 genes were more highly expressed and the
ZmMT1 and
ZmMT8 genes were less highly expressed
[38]. Additional studies identified the
Spirodela polyrhiza SpMT2a gene, which is highly likely to be involved with Cd tolerance based on expression at the transcriptome level after 24 h
[39]. The genes
MTB2,
MTB3 and
MTB15 were found to be upregulated in
Calotropis gigantea L.
[40]. In
Oryza sativa and
Triticum aestivum, there is a relatively high probability that
HsfA4a influences the MT genes, and thus enhances the defence of the plants
[41].
10. Antioxidant Genes
The presence of SOD genes in plants enhances their ability to withstand stress caused by different abiotic factors
[42]. The presence or expression of antioxidant enzymes such as SOD can enhance the plant’s ability to resist damage caused by Cd exposure. However, the expression of genes related to antioxidant enzymes such as POD, CAT, APX, FeSOD, and MnSOD varies with the concentration of Cd present. Interestingly, these genes do not exhibit similar expression patterns and respond differently to varying levels of Cd. These observations have been validated in the case of
Lolium perenne L.
[43]. Following Cd treatment in
Lolium perenne L., overexpression of SOD genes was observed. These genes encode different isoforms of SOD, including Cyt Cu/ZnSOD, MnSOD, and ChlCu/ZnSOD, each with unique expression patterns. The maximum expression of
Cyt Cu/Zn SOD gene is between 6–24 h, while the maximum expression of
MnSOD is between 4–6 h and the overexpression of
MnSOD is also between 4–6 h
[44]. In another plant, many SOD genes have been studied that are essential for the response to Cd treatment.
Kandelia obovata was also examined for the expression of a family of SOD genes. A recent study has provided insight into the
KoCSD1,
KoCSD2,
KoCSD3,
KoFSD1,
KoFSD2,
KoFSD3 and
KoMSD genes and their expression after exposure to Cd. By means of the quantitative RT-PCR method, they conclude that each of these genes is upregulated after Cd treatment. There was a significant upregulation in the roots and in the above-ground parts of
Kandelia obovata. There has been an interesting breakthrough about gene expression in medical plants
[45]. Two years later, selected
KoFSD2 and
KoCSD3 genes were published and transferred into
Nicotiana bethamiana, where they came up with the idea that it was
KoCSD3 that might have a function in plant defence, preventing Cd from reaching the roots by affecting the roots at the cellular level. Its overexpression in the root exodermis confirmed this discovery
[46]. Gene
BjCAT3 from
Brassica juncea was studied in connection with Cd exposure. The changes of expression were observed by the Northern block. To confirm the association of this gene with Cd, a transgenic plant was generated where higher expression of this gene was shown to be associated with Cd
[5].
11. HMA Gene Family
HMA genes, particularly
HMA1 and
HMA2, were overexpressed in another study
[27]. Several genes in plants such as
NcHMA4 and
NcHMA3 in
Noccaea caerulescens,
AhHMA3 Arabidopsis halleri in shoots
[47],
SpHMA7 [48] and
SpHMA3 in
Sedum plumbizincicola [49],
OsHMA3 in
Oryza sativa [50] were reported. A detailed analysis of all eight HMA-encoding genes was performed for
Sedum plumbizincicola. This led to the conclusion that these genes contain the elements DKTGT, GDGxNDxP, PxxK S/TGE, HP and CPx/SPC
[48]. Twenty-one genes encoding HMA have been identified in
Hordeum vulgare L., Hv. From this number, five HMA genes were selected that had changes in regulation by Cd
[51]. Analyses comparing HMA-encoding genes in
Arabidopsis thaliana and
Brassica rapa var.
rapa showed that the genes in
Brassica rapa var.
rapa had undergone evolutionary changes. The genes have been separated into two groups. Different heavy metals species are associated with each group. Out of a total of fourteen genes, upregulation changes were observed for four genes
BrrHMA1,
BrrHMA2.1,
BrrHMA2.2 and
BrrHMA4.1, which were observed in roots of Cd-resistant plants. The upregulation of the
BrrHMA2.2 gene in the root was observed in the Cd-sensitive variety, while over expression of the other genes was not observed here. One gene,
BrrHMA1, showed the opposite expression in the sensitive plant, where it was downregulated, and conversely upregulated in the resistant plant
[52].
Table 1 is a summary of the genes that were affected by Cd treatment. The genes are divided into groups called families. These are shown in the first column. The second column is the plant in which the gene was under regulation and the third column is the gene identification number from the NCBI database
[53]. The fourth column gives the name of the gene and the fifth column gives the gene’s function in relation to Cd treatment.
Table 1. Genes regulated by Cd treatment.