Transcriptional Regulatory Network of Plant Cadmium Stress Response

Cadmium (Cd) is a non-essential heavy metal with high toxicity to plants. Plants have acquired specialized mechanisms to sense, transport, and detoxify Cd. Recent studies have identified many transporters involved in Cd uptake, transport, and detoxification. However, the complex transcriptional regulatory networks involved in Cd response remain to be elucidated. Here, we provide an overview of current knowledge regarding transcriptional regulatory networks and post-translational regulation of the transcription factors involved in Cd response. An increasing number of reports indicate that epigenetic regulation and long non-coding and small RNAs are important in Cd-induced transcriptional responses. Several kinases play important roles in Cd signaling that activate transcriptional cascades. We also discuss the perspectives to reduce grain Cd content and improve crop tolerance to Cd stress, which provides a theoretical reference for food safety and the future research of plant varieties with low Cd accumulation.


Introduction
Cadmium (Cd) is one of the naturally occurring heavy metals, which is extremely toxic to plants and humans [1]. In recent years, the increase in Cd content in soils has caused serious and widespread pollution to farmland. The accumulation of Cd in plants has toxic effects on the normal growth of plants. For example, Cd affects enzyme activity and the absorption and consumption of essential elements, generates reactive oxygen species (ROS), and impairs photosynthesis, respiration, and membrane systems. All these effects ultimately result in plant tissue necrosis, chlorosis, and eventual death [2,3]. Cd is also a threat for human health. The bone itai-itai disease in Japan in the 1950s was caused by long-term consumption of rice (Oryza sativa L.) produced in Cd-contaminated soils [4]. Cd enters the human body through the food chain and mainly accumulates in the kidneys, causing a series of diseases, such as anaemia, cancer, heart failure, steoporosis, emphysema, and renal function diseases [5][6][7][8]. Therefore, it is necessary to limit Cd in the food chain from soils to reduce health risks to humans.
Recent studies have made important progress in elucidating the physiological and molecular mechanisms of Cd transport and tolerance in plants. According to the relation between the metal content in the soil and metal in the plants, plants are divided into three groups: excluder, indicator, and hyperaccumulator plants [9]. So far, many transporters related to Cd uptake, transport, sequestration, and detoxification in plants have been identified [10][11][12][13] (Table 1, Figure 1). Metal transporters and ROS-scavenging enzymes are major functional proteins that are induced by Cd stress. Heavy metal accumulation and tolerance in plants are associated with a highly complex regulatory network system involving a large number of genes. Recent studies in rice, Arabidopsis thaliana, and other plants have revealed multi-layered transcriptional networks comprising many transcriptional factors (TFs), long The pathways of Cd absorption by roots include Cd absorption and efflux by transporters, Cd fixation by the cell wall, and Cd chelation by vacuoles. Cd is transported to shoots by loading into the xylem. Then, Cd will be redistributed through stems and nodes and further translocated to grains through the phloem. Then, Cd will be redistributed through stems and nodes and further translocated to grains through the phloem.

Cd Absorption by Transporters
At present, there are no transporters in plant roots that specifically absorb Cd. Cd can be absorbed mainly through synergistic action by other essential mineral elements, such as Zn, Fe, and manganese (Mn) ions. Several metal transporters, like OsIRT1, OsNRAMP1, and OsNRAMP5 have been reported to be responsible for Cd entry into rice roots [33,54]. OsIRT1 and OsIRT2 are located on the plasma membrane. After 10 days of 100 µM CdSO 4 treatment, the expression of OsIRT1 and OsIRT2 was highly increased in rice roots [59]. OsIRT1 and OsIRT2 both display Cd, Fe, and Zn influx activities in yeast, and overexpression of OsIRT1 increases these metals in different plant tissues [20]. These results indicate that both OsIRT1 and OsIRT2 play important roles in rice by uptaking Cd from soil to roots. OsCd1, a major facilitator superfamily (MFS) protein, has been demonstrated to be involved in Cd uptake in root cells. OsCd1 resides in the plasma membrane of roots and contributes to Cd accumulation in rice grains [48]. In addition, OsNRAMP5 is located in the plasma membrane and is mainly responsible for the transport of Cd, Fe, and Mn in the rice root system. The osnramp5 knockout mutants significantly reduced Cd concentration in roots and buds and increased Cd tolerance [11,60,61]. OsNRAMP1 is highly homologous to OsNRAMP5 and is also involved in Cd uptake and transport by root cells. Knockout of OsNRAMP1 resulted in a significant decrease in the uptake of Cd and Mn by rice roots [33]. These results have important implications for the application of OsNRAMP1 and OsNRAMP5 mutations in mitigating Cd toxicity and reducing the risk of Cd contamination in rice grains. Under 10 µM CdCl 2 stress for three days, compared with the yeast transformed with an empty vector, the growth of yeast expressing AtNRAMP1, AtNRAMP3, and AtNRAMP4 was seriously impaired. In the meantime, after 3 µM CdCl 2 treatment for 24 h, these three kinds of yeasts contained more Cd in yeast cells than the yeast transformed with empty vector [29]. Under 2 µM CdSO 4 stress for 14 days, the growth of the nramp1 Arabidopsis mutant roots was little affected compared to the wild type (WT) [28]. Under both 1 and 10 µM CdCl 2 stress for 10 days, the growth of AtNRAMP3 overexpression in Arabidopsis roots was significantly reduced compared to WT [29]. Under 500 nM CdCl 2 treatment for 14 days, the AtNRAMP4 overexpression in Arabidopsis roots accumulated more Cd than WT [31]. These results indicate that these genes are related to Cd transport in Arabidopsis roots. HvNRAMP5, located in the plasma membrane, is also a major transporter for the uptake of Cd and Mn in barley [32].

Cd Efflux by Transporters
OsZIP1 functioned as a metal-detoxified transporter through preventing excess Cd and Zn accumulation in rice [22]. OsZIP1 is located at the endoplasmic reticulum and plasma membrane [62]. OsZIP1 overexpression in rice grew better under 5 µM CdCl 2 stress for six days, but accumulated less Cd in plants. By contrast, the oszip1 mutants and RNA interference (RNAi) lines accumulated more Cd in roots and displayed Cdhypersensitive phenotypes. Tian et al. (2019) [23] found that both roots and shoots of OsZIP3-overexpressed transgenic rice plants were longer than those of WT plants under 10 µM CdSO 4 for seven days. OsZIP3 overexpression also reduced the Cd content in the roots and shoots. In addition, OsABCG36 localized at the plasma membrane was also involved in Cd efflux in rice roots. Knockout of OsABCG36 increased Cd accumulation in root cell sap and enhanced Cd sensitivity [49]. SaPCR2 is localized at the plasma membrane and plays an important role in Cd detoxification. Under 15 and 30 µM CdCl 2 stress for seven days, the Cd content in the roots of SaPCR2-overexpressed transgenic Arabidopsis plants were decreased compared to WT. Under 10 µM CdCl 2 stress for seven days, the Cd content in the roots of SaPCR2-overexpressed transgenic Sedum alfredii plants were also decreased compared to WT plants. That means SaPCR2 provided a route for Cd efflux in both Arabidopsis and non-hyperaccumulating ecotype (NHE) S. alfredii [52]. AtPDR8 is localized at the plasma membrane and was expressed in Arabidopsis roots and leaves. Kim et al. [50] found that under 5, 10, 20, and 30 µM CdCl 2 for two to three weeks, atpdr8 knockout plants and atpdr8 RNAi plants were more sensitive to Cd than WT, while AtPDR8overexpressed plants were resistant to Cd. That means AtPDR8 acts as an efflux pump of Cd 2+ in plants.

Cd Transport to Shoots by Loading into the Xylem
Cd is transported to shoots by loading into the xylem vessel. Xylem-mediated rootto-shoot translocation is shown as a major determinant for shoot Cd accumulation in many plants including rice [63,64]. CAL1 was a major quantitative trait locus (QTL) for Cd accumulation in rice leaves. CAL1 protein reduced Cd accumulation in rice leaves by specifically chelating Cd in the cytosol and promoting Cd secretion to extracellular spaces. CAL1 also regulated Cd root-to-shoot translocation through the xylem, and cal1 knockout mutants significantly reduced Cd concentration in rice leaves after 10 µM CdCl 2 treatment for seven days [51]. OsHMA2 is mainly expressed in rice roots and enriched in the vascular tissues, facilitating root-to-shoot Cd translocation. Knockout of OsHMA2 significantly reduced Cd accumulation in shoots and grains [57]. The expression of OsHMA2 was prominent in rice, which accumulated more Cd in its grains [65]. These results mean Cd can be transported from shoots to the xylem and, finally, to grains through OsHMA2. OsHMA3, a close homolog of OsHMA2, is a tonoplast-localized transporter for Cd in rice roots and is responsible for sequestering Cd in vacuoles [43]. Overexpression of OsHMA3 significantly reduced Cd transport from roots to shoots and Cd content in grains (≥90%) [58,66]. Even in seriously Cd-contaminated soils, overexpression of OsHMA3 alone produced rice grains with Cd concentration below the Chinese limit (Cd, 0.2 mg kg −1 ) [67], representing an ideal target for breeding low grain Cd rice. In Arabidopsis, the P1B-type ATPases, AtHMA2 and AtHMA4, both regulate root-to-shoot translocation of Cd and Zn and were mainly expressed in the vascular tissues of roots, stems, and leaves [39]. Overexpression of AtHMA4 led to an increased tolerance to Zn, Cd, and Co and accumulated more metals in stems than WT [41]. Another P-type ATPase family member, AtHMA3, located at the vacuolar membrane, also participates in the vacuolar storage of Cd. Under 30 µM CdCl 2 stress for 11 days, the roots and shoots of AtHMA3-overexpressed transgenic Arabidopsis plants accumulated more Cd than WT [40]. These results suggest that AtHMA3 plays a role in the detoxification of Cd through the vacuolar sequestration.

Cd Transport through the Phloem to Grains
Cd transported from the xylem to the shoots in rice is stored in nodes, transferred to the phloem, and then transported to rice grains through leaves, especially flag leaf phloem [48,68]. Phloem mediates nearly 100% of Cd deposition into grains in rice [69]. Cd can also be transferred to the grains through phloem in other plants, such as peanut (Arachis hypogaea L.), linseed (Linum usitatissimum L.), and potato (Solanum tuberosum L.) [70,71]. Cd mediated by phloem in S. alfredii participated in Cd remobilization from the older to younger leaves [72]. This reallocation could avoid excessive accumulation of Cd in leaves and stems. OsLCT1 is the first identified transporter for phloem Cd transport in plants [46]. OsLCT1 is mainly expressed in leaf blades and nodes during the reproductive stage. The Oslct1 knockdown mutant significantly reduced Cd content in rice grains as well as in phloem sap [46]. The expression of OsLCT1 was significantly enhanced in rice, which over-accumulated Cd in grains, indicating possible translocation of Cd from shoots to grains [65]. These results suggest that OsLCT1 in leaf blades functions in Cd remobilization by the phloem. In addition, OsCCX2, a putative Ca exchanger, is a node-expressed transporter involved in Cd accumulation in the grains of rice. OsCCX2 is mainly expressed in the xylem region of vascular tissues at the nodes and plays a crucial role in mediating Cd translocation and distribution. Knockout of OsCCX2 resulted in a significant decrease in Cd accumulation in rice grains when planted in 3.89 mg kg −1 Cd-contaminated paddy soils [38]. More recently, Gu et al. (2023) [69] identified a defensinlike gene, DEFENSIN 8 (DEF8), as the phloem Cd unloading transporter. DEF8 is mainly expressed in rice grains. The DEF8 mutant significantly decreased Cd accumulation in rice grains, offering an effective strategy to reduce the risk of Cd contamination without affecting important agronomic traits or the concentration of essential micronutrients. OPT3 is located at the plasma membrane and preferentially expressed in the Arabidopsis phloem. After 50 µM CdCl 2 stress for two weeks, the OPT3-overexpressed transgenic Arabidopsis plants reduced the accumulation of Cd in grains and the opt3 mutant Arabidopsis plants accumulated more Cd in grains and roots [53]. These results suggest that OPT3 plays an important role in the transport of Cd from phloem to grains.

The Transcriptional Regulation of Cd Response by TFs
Recent studies have identified the complex transcriptional networks of plant Cd stress responses ( Figure 2). TFs are major regulators of plant growth and development, as well as in abiotic and biotic stress responses. TFs belong to different families, such as WRKY, myeloblastosis protein (MYB), basic leucine zipper (bZIP), and heat shock transcription factor (HSF) [73][74][75]. They play important roles in signal transduction of Cd stress response by activating or repressing a series of genes involved in Cd uptake, transport, and tolerance in rice. The sensing of heavy metals by plants generates responses such as modulation of molecular and biochemical mechanisms of cells [58,76]. The ultimate plant Cd stress responses include altered synthesis of metal transporter proteins and metal binding proteins to counteract excessive metal stress in plants [74,77].

WRKY
The WRKY family is a unique plant TF family and plays an important regulatory role in plant development and response to various environmental stresses [75]. Under Cd stress, 35 WRKY genes were differentially expressed in rice, of which 25 were up-regulated and 10 were down-regulated. Under Cd treatment, the expression of OsWRKY15 was induced in both leaves and roots, which may participate in Cd response via NO and ABA signaling pathways. The expression of WRKY104 increased more than 90-fold after 24 h of Cd treatment [78]. Under Cd stress, WRKY12 negatively regulated Cd tolerance via the glutathione (GSH)-dependent PC synthesis pathway in Arabidopsis. WRKY12 directly targeted GSH1 by binding to its promoter and indirectly inhibited the expression of other PC synthesis-related genes (GSH1, GSH2, PCS1, and PCS2), thereby negatively regulating Cd accumulation and tolerance in Arabidopsis [79]. The expression levels of TaWRKY74 were significantly induced by Cd stress in wheat. TaWRKY74 alleviated Cd toxicity in wheat by regulating the expression of Ascorbic Acid (ASA)-GSH synthesis genes [16]. In addition, Cd stress induced the expression of WRKY13. Overexpression of WRKY13 decreased Cd accumulation and enhanced Cd tolerance, while the loss of function of WRKY13 led to Cd accumulation and increased Cd sensitivity. WRKY13 can bind the promoter of the Cd extrusion pump gene PDR8 and activate its expression to positively regulate Cd tolerance in Arabidopsis [80].

MYB
The MYB TF family is a large and functionally important class of proteins involved in the regulation of diverse biological processes. MYB proteins are divided into four classes according to the number and position of MYB repeats: 1R-MYB/MYB-related, R2R3-MYB, R1R2R3-MYB, and 4R-MYB [81]. BnMYB2, encoding a 1R-MYB protein from Boehmeria nivea (ramie), was significantly up-regulated in roots and leaves under Cd stress. The overexpression of BnMYB2 in Arabidopsis resulted a significant increase in Cd tolerance and accumulation [82]. In addition, Tiwari et al. (2020) [83] identified another member of the rice 1R-MYB family involved in heavy metal tolerance. OsMYB-R1-overexpressed rice plants exhibited a higher auxin accumulation and a significant increase in lateral roots, which resulted in the increased tolerance under 150 µM and 300 µM Chromium (Cr) (VI) exposure for 21 days. RNA-seq analysis revealed over-representation of salicylic acid (SA)-regulated genes in OsMYB-R1-overexpressed rice plants [83]. These results imply that OsMYB-R1 is part of a complex network of TFs controlling the cross-talk of auxin and SA signaling, which regulates heavy metal response.
The R2R3-MYB genes are more prevalent in plants and involved in regulating responses to environmental stresses [82,84]. Recent reports have established the role of OsMYB45 in rice tolerance to Cd stress (Figure 3). The expression of OsMYB45 was induced by Cd stress and highly expressed in the leaves, husks, stamens, pistils, and lateral roots of rice. Under 5 µM CdCl 2 treatment for three days, the Osmyb45 mutant was hypersensitive to Cd, which is associated with increased accumulation of hydrogen peroxide (H 2 O 2 ) and reduced expression of antioxidative enzymes compared with WT. Catalase (CAT) is the main antioxidant enzyme and is encoded by three genes in the rice genome (OsCATA, OsCATB, and OsCATC). OsCATA and OsCATC expression was inhibited in Osmyb45 mutations, which may be associated with Cd-sensitive phenotypes. The overexpression of OsMYB45 in the mutant complemented the mutant phenotype [85]. In addition, another R2R3-type MYB member, MYB49a, was reported to be involved in the regulation of Cd accumulation in plants by physically interacting with the central ABA signaling molecule ABI5 [14]. MYB49 was induced under Cd stress. Overexpression of MYB49 in Arabidopsis significantly increased Cd accumulation, whereas myb49 knockout plants reduced Cd accumulation. Further investigations revealed that MYB49 positively regulated the expression of basic helix-loop-helix (bHLH) TFs, bHLH38 and bHLH101, by directly binding to their promoters and indirectly up-regulating expression of the IRT1 transporter gene. MYB49 also binds to the promoter regions of the heavy metal-associated isoprenylated plant proteins, HIPP22 and HIPP44, leading to the activation of their expression and subsequent Cd uptake and accumulation [14].

bZIP
The bZIP family is one of the largest TF families in plants with important regulatory roles in various biological processes, including plant defense and responses to environmental challenges [86][87][88]. RNA-Seq results indicated that three differentially expressed genes encoding bZIP6, bZIP19, and bZIP43 were involved in Cd stress in bentgrass [89]. After 400 μM CdCl2 treatment for four days in Sedum plumbizincicola, the expression levels

bZIP
The bZIP family is one of the largest TF families in plants with important regulatory roles in various biological processes, including plant defense and responses to environmental challenges [86][87][88]. RNA-Seq results indicated that three differentially expressed genes encoding bZIP6, bZIP19, and bZIP43 were involved in Cd stress in bentgrass [89]. After 400 µM CdCl 2 treatment for four days in Sedum plumbizincicola, the expression levels of 32 SpbZIP genes changed and most of their expression levels peaked earlier in roots than in stems and leaves [88]. These results suggest that SpbZIP may play a major role in the initial response to Cd stress in the roots. In addition, TGACGTCA cis-element-binding protein (TGA) factors in Arabidopsis represent a subfamily of bZIP TFs. In Arabidopsis, TGA3 transcription was induced by Cd [90]. Compared with WT plants, the tga3 mutant accumulated higher amounts of Cd in the roots and lower amounts in the shoots [91]. Fusco et al. (2005) [92] found that under Cd treatment, BjCdR15, acting as orthologue of TGA3 in Arabidopsis, regulated the expression of several metal transporters in Brassica juncea, such as PDR8, HMA4, and NRAMP3, thus mediating long-distance root-to-shoot transport of Cd. Overexpression of BjCdR15 in Arabidopsis and Nicotiana tabacum (tobacco) enhanced Cd tolerance and accumulation in shoots [91]. These results indicate that bZIP TFs play crucial roles in the regulation of Cd accumulation, which provide useful candidates for potential biotechnological applications in the phytoextraction of Cd-contaminated soils.

HSF
The HSF family is an important member in plant stress response to several abiotic stresses by regulating the expression of stress-responsive genes, such as heat shock proteins (Hsps). In Arabidopsis, the Hsfs family is systematically divided into three classes of HsfA, B, and C [93]. In the plant response network, HsfA1 specifically interacts with HsfA2 to mediate the expression of genes encoding molecular chaperone HSPs such as HSP70 and HSP90 [94]. HSFs have been reported to play crucial roles in Cd tolerance in plants. HsfA1a conferred Cd tolerance in Solanum lycopersicum (tomato) by partially up-regulating Hsps expression [95] ( Figure 4). After 100 µM CdCl 2 stress for 15 days, Hsfa1a-silenced plants exhibited reduced melatonin levels, while HsfA1a overexpression stimulated melatonin accumulation and the expression of the melatonin biosynthetic gene caffeic acid O-methyltransferase 1 (COMT1). Exogenous melatonin promotes the modulation of GSH and PC biosynthesis which can detoxify Cd under Cd stress [96].
In S. alfredii, SaHsfA4c also played an important role in Cd tolerance. Compared with WT, the accumulation of ROS in SaHsfA4c-overexpressed Arabidopsis was reduced, and the expression of ROS-scavenging enzyme genes and Hsps was increased [97]. It has been found that the TaHsfA4a gene confers strong Cd tolerance in yeast and rice. CUP1, which encodes metallothioneins (MTs), contributes to the TaHsfA4a-induced Cd tolerance by acting as a downstream target of HsfA4a. OsHsfA4a is a rice homolog of TaHsfA4a. In rice plants expressing TaHsfA4a, Cd tolerance was enhanced, but in oshsfa4a knockdown rice plants, Cd tolerance was decreased. In addition, TaHsfA4a mediated Cd resistance in yeast by regulating MTs. The expression levels of HsfA4a and the MT gene were increased in rice roots under Cd stress. Therefore, HsfA4a in rice induced Cd tolerance by up-regulating MT gene expression in plants [98,99] (Figure 4).

Other TFs
The no apical meristem (NAM), Arabidopsis transcription activation factor (ATAF1/2), and cup-shaped cotyledon (CUC2) (NAC) family is a kind of pivotal TF in the response to various abiotic stresses [100]. They contain a conserved N-terminal DNA-binding NAC domain and a highly variable C-terminal domain. In Aegilops markgrafii, AemNAC2 was found to be associated with reducing accumulation of Cd. Overexpression of AemNAC2 could decrease accumulation of Cd in roots, shoots, and grains of transgenic wheat. In this type of transgenic wheat, AemNAC2 suppressed the expression of TaNRAMP5 and TaHMA2 [101]. HSP70 and HSP90 [94]. HSFs have been reported to play crucial roles in Cd tolerance in plants. HsfA1a conferred Cd tolerance in Solanum lycopersicum (tomato) by partially up-regulating Hsps expression [95] (Figure 4). After 100 μM CdCl2 stress for 15 days, Hsfa1a-silenced plants exhibited reduced melatonin levels, while HsfA1a overexpression stimulated melatonin accumulation and the expression of the melatonin biosynthetic gene caffeic acid O-methyltransferase 1 (COMT1). Exogenous melatonin promotes the modulation of GSH and PC biosynthesis which can detoxify Cd under Cd stress [96]. In S. alfredii, SaHsfA4c also played an important role in Cd tolerance. Compared with WT, the accumulation of ROS in SaHsfA4c-overexpressed Arabidopsis was reduced, and the expression of ROS-scavenging enzyme genes and Hsps was increased [97]. It has been found that the TaHsfA4a gene confers strong Cd tolerance in yeast and rice. CUP1, which The ethylene responsive factor (ERF) family belongs to APETALA2/ethylene responsive factor (AP2/ERF) superfamily, which is one of the largest group of TFs involved in abiotic stress response in plants [102]. In Glycyrrhiza uralensis, overexpression of lrERF061 led to maximum Cd uptake and enhanced antioxidant enzyme activities (SOD, CAT, and POD) under 10 mg L −1 Cd treatment [103]. This study contributes to the understanding of the role of LrERF061 in Cd resistance and offers a useful way to increase the phytoextraction efficiency of Cd-polluted soils.

Regulation of Cd Response by DNA Methylation, Long RNAs, and Small RNAs
As discussed above, TFs are the core regulators of transcription under Cd stress. However, increasing evidence has revealed a complex regulatory system comprising not only TFs, but also DNA methylation, long RNAs, and small RNAs with crucial roles in Cd response (Figure 2).

DNA Methylation
Heavy metal stress has an effect on DNA structure, DNA stability, DNA methylation, and the regulation of gene expression. When these effects occur in plants, changes in DNA methylation can make plants adapt to heavy metal stress, especially to Cd stress [18,104]. DNA methylation can regulate gene expression and induce corresponding phenotypic changes without altering DNA sequence [105].
Cd treatment can lead to an increase in DNA methylation levels in rice, Arabidopsis, Zostera marina, and barley, which endows plants with higher tolerance to Cd [106][107][108][109].   [110] used high-throughput single-base-resolution bisulfite sequencing (BS-Seq) and RNA-Seq to analyze DNA methylation patterns in Cd-treated rice seedlings. A group of genes encoding metal transporters, Cd-detoxified proteins, and metal-related TFs were found to be differentially methylated, implying their roles in regulating rice tolerance to Cd stress. After 80 µM CdSO 4 for four days, both GSH2 and GSHU35 upstream regions were hypermethylated. The sequence downstream of the coding region for ironrelated transcription factor 2 (OsIRO2, a bHLH TF gene) was hypermethylated, while the coding region of metal transporter OsZIP1 was hypomethylated. The expression level of OsIRO2 was repressed, while OsZIP1 was induced by Cd. These results suggest that DNA methylated modification was most likely involved in transcriptional regulation of metal transporter genes. Sun et al. (2022) [111] found that grafting significantly reduced the total sulfur and Cd accumulation in soybean, which was mediated by DNA methylation. The expression level of methyltransferase genes decreased, leading to the decreased expression of sulfur metabolism-related genes, especially S-adenosylmethionine (SAM). These results imply that DNA methylation was involved in a decrease in total sulfur and Cd content. In addition, Cd treatment can lead to a decrease in DNA methylation levels in Trifolium repens and Cannabis sativa, which reduces the tolerance of plants to Cd [112]. These results indicate that DNA methylation dynamics in response to Cd vary with species.

lncRNAs
LncRNAs are a class of non-protein coding RNAs with >200 nt, which act as 'biological regulators' to control transcriptional regulation and genome imprinting [106,113]. Many lncRNAs in plants were induced or inhibited by Cd stress, affecting plant morphology, physiology, and biochemistry, and thus producing response to stress. They were reported to play key roles in controlling the uptake of heavy metals by the plant system in order to minimize the uptake of heavy metals from soil to plants [15,114].
Chen et al. (2018) [115] used deep sequencing to study the differential expression of lncRNAs under Cd stress in rice. A total of 75 lncRNAs were down-regulated and 69 lncRNAs were up-regulated by Cd treatment. Analysis of the target gene related pathways revealed significant changes in genes associated with the cysteine (Cys) and methionine (Met) metabolic pathways, for example, Os03g0196600, which was involved in these pathways, was clearly up-regulated and might contribute to the production of Cys-rich peptides. XLOC_086307, the lncRNA targeted Os03g0196600 in cis, was also up-regulated significantly, which suggests that XLOC_086307 likely participated in Cd response in rice by regulating the Cys-rich peptide metabolism-related gene Os03g0196600. In addition,   [106] identified 301 Cd-responsive lncRNAs in Brassica napus by RNA-seq analysis, of which 67 acted as competing endogenous target mimics (eTMs) for 36 Cd-responsive miRNAs. Four lncRNAs were identified to serve as precursors of miR824, miR167d, miR156d, and miR156e in response to Cd stress. Interestingly, TCONS_00035787 was shown to target miR167d in B. napus. The target gene of miR167d encodes a NRAMP1type metal transporter, which plays an important role in Cd uptake in plants [106,116]. This is the first report of a lncRNA (TCONS_00035787)-miR167-Nramp1 pathway in plants, indicating that lncRNAs can serve as new transcripts involved in the regulation of Cd uptake and accumulation in plants.

miRNAs
MiRNAs are a new class of small non-coding RNA molecules in plants, which negatively regulate specific target mRNAs at the post-transcriptional level. They are involved in plant growth and development, organ morphogenesis, and responses to heavy metal, drought, and chilling stress [117,118]. In our lab, Ding et al. (2011) [119] used miRNA microarray to analyze miRNA expression patterns in 60 µM Cd-treated and untreated rice seedlings. In addition to the up-regulation of miR528 under Cd stress, miR166, miR171, miR159, miR390, and miR192 were significantly inhibited [119,120]. Most of these miR-NAs were reported to target TF genes, for example, miR166, miR171, and miR396 target homeodomain-leucine zipper TFs, scarecrow-like TFs, and growth regulating factor TFs, respectively. These results imply that miRNAs are key components of the transcriptional regulatory network of heavy metal stress responses in plants. The expression of miR166 was significantly repressed under 60 µM CdCl 2 exposure in rice seedlings. Overexpression of miR166 reduced both Cd translocation from roots to shoots and Cd accumulation in the grains. In 35S: miR166 plants, the expression of OsHMA2 decreased. Thus, the reduced Cd translocation in plants overexpressing miR166 may be at least partly attributable to the effect on OsHMA2 expression [121]. In addition, miR390 was found to be significantly down-regulated under Cd stress. Overexpression of miR390 increased Cd accumulation and reduced tolerance to Cd toxicity in rice [122]. Meng et al. (2017) [123] found that miR167 could cleave BnNRAMP1b (one of the NRAMP genes), thus BnNRAMP1b was a target of miR167. Huang et al. (2010) [124] validated that miR395 targeted the sulfate assimilation related genes-sulfate transporter 2; 1 (SULTR2; 1) and ATP sulfurylases (APS) by using 5 -RACE assay in B. napus. After 40 µM CdCl 2 treatment for seven days, miR395-overexpressed B. napus plants exhibited high Cd accumulation and fewer toxicity symptoms in comparison to WT, due to increased synthesis of sulfur-containing compounds used for heavy metal chelation [125]. These results demonstrate the role of miR395 in the detoxification of Cd in B. napus. MiR398 targets two closely related cuprums (Cu)/Zn-SODs (CSDs), CSD1 and CSD2, which promote defense against ROS accumulation in Arabidopsis. Transgenic Arabidopsis plants overexpressing a miR398-resistant form of CSD2 accumulated more CSD2 miRNA than plants overexpressing a regular CSD2 and were consequently much more tolerant to heavy metals and other oxidative stresses [126]. Wang et al. (2022) [127] used high-throughput sequencing to analyze miRNA expression patterns in Cd-tolerant/sensitive barley. MiR156g was identified to be Cd-induced and target nucleobase-ascorbic acid transporters 2 (HvNAT2). HvNAT2 was negatively regulated in the high-Cd-accumulating and Cd-tolerant genotype Zhenong8. Overexpression of HvNAT2 enhanced ROS enzyme activities and GSH content, thus enhancing Cd tolerance in barley. These results indicate that metal-regulated miRNAs and their target genes are involved in the diverse processes of Cd response, including metal uptake and transport, sulfate allocation, metal chelation, and ROS detoxification.

How Plants Sense and Transduce Cd Signals to Transcriptional Regulators
How plants sense and transduce Cd signals to transcriptional regulators is one of the most important open questions. Recent studies revealed that heavy metal stress activates Ca 2+ and ROS signaling that mediate signal transduction and enhance the expression of stress-responsive genes or TFs. ROS can also act downstream of the mitogen-activated protein kinase (MAPK) pathway [128]. MAPKs are among the most important and highly conserved signaling molecules that are activated by ROS production and induced upon metal stress. MAPK cascade consists of three tier components MAP kinase kinase (MAP-KKKs/MEKKs), MAP kinase kinase kinase (MAPKKs/MEKs), and MAPKs/MPKs mediating phosphorylation reactions from the upstream receptor to the downstream target [129]. It has been shown that Cd stress activates different kinase enzymes belonging to the MAPK family. The phosphorylation cascade is therefore thought to be involved in Cd signaling to the nucleus. Research confirms that transcripts for OsMSRMK2 (OsMPK3 homolog), OsM-SRMK3 (OsMPK7 homolog), OsBWMK1 (or OsMPK12), and OsWJUMK1 (OsMPK20-4 homolog) increased in response to Cd and Cu treatment in rice roots and leaves [130,131].
A connection between miRNA and MAPK signaling was deciphered by a study which showed regulation of miR398b/c by oxidative signal-inducible kinase 1 (OXI1) upon Cd and Cu treatment [132].  [134]. Apart from this, several TFs, like bZIP-, MYB-, and myelocytomatosis (MYC)-related TFs, are known to act as downstream targets of MAPKs [135,136]. In addition, Opdenakker et al. (2012) [137] reported that downstream signal transduction targets of MAPK during Cd or Cu stress included WRKY22, WRKY25, and WRKY29. MAPK cascades regulate gene transcription by activating or inhibiting TFs such as WRKY and TGA (a subfamily of bZIP TFs), thus regulating a variety of cellular responses [138,139].

Future Perspective
Cd accumulation and exposure in crops poses a serious threat to organisms and human health. Breeding of new cultivars with low Cd levels is the most cost-effective and eco-friendly strategy to reduce the risk of Cd contamination in plants. To achieve this goal, we need a comprehensive understanding of not only the mechanisms but also the regulation of Cd uptake, translocation, sequestration, and other processes important for plant Cd stress responses. Over the past decades, different families of Cd transporters have been identified in plants, and their functional analysis through molecular and genetic approaches has provided critical insights into Cd uptake and translocation mechanisms ( Figure 1). More recently, a large number of regulatory proteins including those involved in protein phosphorylation have been identified. Regulatory RNAs and DNA modifications have also been identified with roles in plant Cd accumulation and tolerance likely by affecting their expression, synthesis, activities, stability, and other properties (Figure 2). TFs are the core regulators of transcription under Cd stress. Several TFs in the transcriptional network and their functions during Cd stress have been analyzed. In addition, there is emerging evidence that epigenetic regulation through DNA methylation, lncRNAs, miR-NAs, and kinases are involved in Cd-induced transcriptional responses. These signaling and responding mechanisms at transcriptional and post-transcriptional levels will facilitate our understanding of regulatory pathways and serve as a basis for developing efficient strategies to reduce Cd in plants.
Despite the important progress, our understanding of the signaling and complex transcriptional regulatory networks in Cd stress response remains to be very limited. First, it is unclear how plants sense Cd. Do plant cells sense Cd through specific recognition of Cd itself or through indirect recognition of certain Cd-associated molecules or induced effects? Given that all identified Cd transporters also transport other metal ions, it is possible that plants sense Cd simply as a heavy metal and there are overlapping mechanisms in signaling upon exposure to different types of heavy metals. Second, upon Cd stress perception, what are the earliest signaling events? Even though MAPK cascades are implicated in plant Cd signaling and responses, there are usually other regulatory proteins that act upstream of MAPK cascades. For example, in plant immune responses, plasma membrane-localized pattern-recognition receptors can recognize specific pathogen elicitors to trigger plant immune response through activation of the MAPK cascade. Given that Cd is transported into plant cells through plasma membrane-localized transporters, it is possible that the early signaling in Cd response starts at the plasma membrane as well and could directly involve Cd transporters through coordination with other proteins such as plasma membrane-localized receptor-like proteins. Third, even though a substantial number of TFs have been identified with a role in Cd accumulation and tolerance, many lack information about their regulation and action mechanisms. For example, it is unclear how some of the identified TFs are activated or induced in response to Cd exposure. For many TFs, this remains unclear regarding direct target genes under their regulation. More importantly, there is little knowledge about the cooperation and coordination among different TFs for the effective and tight control of the transcription programs of plant Cd responses. Fourth, more recent discoveries about the role of DNA methylation and regulatory RNAs in Cd responses will expand the complex transcriptional landscape of plant Cd stress responses. It will be critical to identify the target genes that are subjected to regulation by epigenetic mechanisms and regulatory RNAs and establish the processes and pathways by which these target genes influence plant Cd accumulation and responses. Finally, most of the research on plant Cd accumulation and responses has been carried out in rice and Arabidopsis. It is very likely that there are many unknown components and mechanisms that are present in different plants with important roles in plant Cd accumulation and tolerance. There are, for example, plants that hyperaccumulate Cd and can be highly valuable research materials for discovery of novel mechanisms by which plants accumulate, sequester, and detoxify high levels of Cd from heavily contaminated soils. In the hyperaccumulator S. alfredii, some genes related to Cd uptake and hyperaccumulation have been characterized, such as SpHMA3, SaNramp6, and SaHsfA4c [97,140,141]. Isolation of new genes including those TFs and interacting factors with regulatory roles in plant Cd accumulation and tolerance will help elucidate regulatory mechanisms in response to heavy metal stress. They can also be exploited as potential targets for genetic engineering through molecular breeding and clustered regularly inter-spaced short palindromic repeat (CRISPR)-Cas9 technology to reduce grain Cd accumulation and increase Cd tolerance in crop plants. Data Availability Statement: Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Conflicts of Interest:
The authors declare no conflict of interest.