Plant Transcription Factors: Molecular Mechanisms in Cadmium (Cd) Detoxification and Applications for Reducing Cd Accumulation in Rice Grains

Abstract

Cadmium (Cd) toxicity threatens global food security and agricultural sustainability. Transcription factors (TFs) act as master regulators of the complex molecular networks involved in Cd detoxification. This review provides a focused synthesis of the molecular mechanisms governing Cd tolerance in plants, encompassing antioxidant defense, Cd chelation and sequestration, Cd uptake and transport, signal transduction, and damage repair pathways. We highlight the pivotal roles of key TFs in these specific processes, such as OsMYB45 in antioxidant defense, OsIRO2 in regulating chelation and storage, OsNAC5 in modulating Cd transport, and OsE2F in facilitating the repair of DNA and protein damage. Furthermore, we evaluate the potential of harnessing these TF-mediated regulatory mechanisms for developing low-Cd rice varieties. By delineating precise correlations between specific TFs and detoxification pathways, this review proposes actionable molecular strategies to mitigate Cd contamination, thereby contributing to ecological and food safety.

1. Introduction

Cadmium (Cd), recognized as one of the ‘five toxic elements’, is extensively disseminated through industrial emissions, mining activities, and fertilizer application [1]. Global soil Cd contamination is intensifying due to increasing anthropogenic activities. Using an Extreme Random Tree (ERT) machine learning model that incorporates climatic, geological, pedological, topographical, and socioeconomic variables (e.g., mining intensity, irrigation ratio), Hou et al. predicted that approximately 9% of global topsoil Cd concentrations exceed national agricultural thresholds (ATs) and human health/ecological thresholds (HHETs), with hotspots concentrated in East and South Asia [2]. China’s National Soil Pollution Survey Bulletin reports a 7.0% Cd exceedance rate in arable land, particularly severe in major rice-producing regions such as the Yangtze River Delta. As a major global cereal crop, rice (Oryza sativa L.) is a primary caloric source for billions of people. Its significance is particularly pronounced in Southeast Asia, where it is a dietary staple. Compared to other cereals like wheat or maize, rice exhibits a higher propensity for Cd uptake, resulting in pronounced Cd accumulation in grains [3]. Global surveys indicate that median rice grain Cd concentrations in India (27.55 μg/kg) exceed the global median (19 μg/kg), while China shows the highest median (69.3 μg/kg) [4]. In China, 2.2–10% of rice samples exceeded the national safety limit (200 μg/kg) between 2009 and 2018. Moreover, the average monthly dietary Cd intake doubled from 1990 to 2015, reaching 15.3 μg/kg body weight (bw). This intake level surpasses the safety limits set by the Agency for Toxic Substances and Disease Registry (ATSDR, 3 μg/kg bw) and the European Food Safety Authority (EFSA, 10.8 μg/kg bw) [5].

Cd enters the human body via the food chain, accumulating over time and potentially causing severe diseases such as renal dysfunction, osteomalacia, neurodegenerative disorders, and cancer [6]. ‘Itai-Itai’ disease, first documented in Japan, resulted from consuming rice irrigated with Cd-contaminated water from the Jinzū River, manifesting as hepatic fibrosis, chronic nephropathy, bronchopneumonia, and gastrointestinal disturbances [7]. In rice, Cd stress significantly inhibits root growth, reduces lateral root formation, and triggers excessive reactive oxygen species (ROS) production, leading to oxidative stress, cellular damage, organelle disruption (e.g., chloroplasts), and impaired photosynthesis, ultimately reducing biomass and grain yield [8]. Cd also competes with essential metals (e.g., Zn, Fe) for transport proteins, inhibiting nutrient uptake, exacerbating growth inhibition, and compromising grain quality [9]. For instance, Li et al. demonstrated that Cd disrupts Ca²⁺ and K⁺ uptake/translocation, significantly reducing their content in roots and shoots, contributing to chlorosis and growth inhibition [10]. Furthermore, Xie et al. reported that Cd stress reduced yields by 17–27% in the low-grain-Cd genotype Xiushui 817 and by 20–21% in the high-grain-Cd genotype Zheda 821 [11].

To counteract Cd toxicity, plants have evolved multifaceted detoxification strategies. These include activating antioxidant systems to mitigate ROS bursts; synthesizing ligands such as phytochelatins (PCs) or metallothioneins (MTs) to chelate Cd²⁺; sequestering Cd into vacuoles via tonoplast transporters; and modulating plasma membrane transporter activity to reduce uptake or enhance efflux [12] (Figure 1). These physiological processes are primarily executed by specific functional genes. For example, ABCC1/2 transporters are crucial for the vacuolar sequestration of Cd-chelates [13], while TaNCL2-A (encoding a sodium/calcium exchanger-like protein) alleviates Cd toxicity by increasing calcium accumulation and enhancing enzymatic antioxidant activities [14]. The spatiotemporal expression of these functional genes is precisely orchestrated by a hierarchical regulatory network of transcription factors (TFs). Prominent TF families, such as WRKY, MYB, NAC, and bHLH, act as key regulators within this network. Typically, WRKY and MYB TFs are central to modulating antioxidant defense and metal uptake, whereas NAC and bHLH TFs are indispensable for directing phytochelatin synthesis and sequestration [12,15]. Key regulators such as OsNAC5 [16], AtMYB49 [17], and OsMYB45 [18] exemplify how TFs bridge initial stress perception with downstream physiological adaptations required to maintain cellular homeostasis [19].

Moving beyond traditional family-based classifications, this review uniquely provides a systems-level framework that integrates early Cd perception with terminal grain allocation. We incorporate the latest research—encompassing epigenetic regulatory hierarchies, microRNA-encoded peptides (miPEPs), and single-cell transcriptomics—into a cohesive model of the plant Cd-response network. Unlike prior work, this synthesis reorganizes transcription factors (TFs) according to the specific detoxification mechanisms they orchestrate, such as antioxidant defense, chelation, compartmentalization, and transport regulation, thereby enhancing the mechanistic clarity of the regulatory landscape. Furthermore, we emphasize the dual functionality of certain TFs that concurrently influence Cd allocation while regulating detoxification. For example, the soybean TF GmWRKY172 [20] significantly enhances Cd tolerance and yield while reducing seed Cd accumulation. By distinguishing between “direct effector engineering” and “systemic network reprogramming,” and by analyzing the molecular basis of genotypic variations and tissue-specific “barrier” effects, this review culminates in a forward-looking roadmap centered on three technological pillars—multi-omics integration, epigenetic decoding, and precision promoter engineering—to provide a robust foundation for the precision breeding of “Cd-safe” rice varieties.

2. Mechanisms and Transcription Factors Regulating Cd Tolerance and/or Accumulation in Plants

2.1. Activation of the Antioxidant Defense System

Under normal conditions, plant cells maintain ROS homeostasis through efficient antioxidant systems. Abiotic stresses such as Cd exposure disrupt this balance, triggering excessive ROS production. Elevated ROS levels cause damage to DNA and proteins, impair photosynthesis and respiration, and may induce programmed cell death. Plants employ multi-layered antioxidant defenses comprising enzymatic and non-enzymatic components. Under Cd stress, ROS also act as signaling molecules that activate stress-responsive TFs, which enhance enzymatic ROS scavenging or stimulate non-enzymatic antioxidant synthesis, maintaining cellular redox homeostasis [21].

2.1.1. Enzymatic Antioxidant System

The enzymatic antioxidant system includes peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPX), which collectively mitigate ROS via precise catalytic reactions [22]. Cd-tolerant species typically exhibit higher activities of SOD, POD, and APX, whereas Cd-sensitive plants show reduced expression of antioxidant enzyme genes [23].

Studies demonstrate that Cd stress activates TFs that regulate antioxidant enzyme expression, alleviating oxidative damage (Table 1). In Populus, PyWRKY75 overexpression enhances POD, SOD, CAT, and APX activities, increasing Cd tolerance and accumulation [24]. In Tamarix hispida, ThRAX2 upregulates SOD activity and transporter function, improving ion homeostasis and Cd tolerance while reducing Cd accumulation [25]. Conversely, ThDIV2 overexpression elevates ROS and proline levels by suppressing SOD, POD, and CAT activities, negatively regulating Cd tolerance [26]. ThbZIP1 overexpression enhances POD/SOD activities and soluble protein/sugar content, bolstering Cd tolerance [27]. In soybean, Cd-induced GmWRKY172 reduces malondialdehyde (MDA) and H₂O₂ levels while increasing flavonoids, lignin, and POD activity, enhancing tolerance and reducing shoot Cd accumulation [20].

In crops, TFs also mitigate Cd toxicity via antioxidant regulation. In maize, Cd stress induces ZmWRKY4, which activates ABA-dependent SOD/APX expression to counteract ROS bursts [28]. Heterologous expression of wheat TaWRKY70 in Arabidopsis elevates CAT activity and Cd resistance [29]. The ethylene-responsive TF TdSHN1 from durum wheat confers Cd/Cu/Zn tolerance in tobacco by enhancing SOD/CAT activities, reducing ROS accumulation, and improving chlorophyll content and biomass [30]. In rice, OsMYB45 knockout doubles H₂O₂ levels and halves CAT activity, impairing Cd tolerance; complementation restores wild-type phenotypes, confirming its role in regulating CAT-mediated detoxification [18]. Heterologous expression of OsTAZ4 in Arabidopsis improves heavy metal resistance by suppressing ROS accumulation [31].

Table 1. Transcription factors regulating the expression of antioxidant enzyme genes.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
WRKY	Poplar	Cd	PyWRKY75	Increased chlorophyll and carotenoid content; enhanced activities of POD, SOD, CAT, and APX	/	Induced	/	Enhanced	Enhanced	[24]
WRKY	Maize	Cd	ZmWRKY4	Binds promoters of ZmSOD4 and ZmcAPX to increase antioxidant enzyme activities	Leaf	Induced	ZmSOD4, ZMcAPX	Enhanced	/	[28]
WRKY	Soybean	Cd	GmWRKY172	Increased POD activity, flavonoid, and lignin content	Leaf, flower, root, stem	Induced	/	Enhanced	Reduced	[20]
WRKY	Sorghum	Cd	SbWRKY54	Enhanced SOD, POD, and CAT activities; restricted Cd transport	Root, stem	Induced	SbHKT2b	Enhanced	Reduced	[32]
MYB	Tamarix	Cd	ThRAX2	Regulates intracellular ion homeostasis, transporter activity, and enhances antioxidant enzyme activities	Root, leaf	Induced (root), Inhibited (leaf)	ThSOS1, ThCKX3, ThCAX3A, ThMYB78	Enhanced	Reduced	[25]
MYB	Tamarix	Cd	ThDIV2	Decreased AOC, SOD, POD, CAT activities and GSH content	Root, leaf	Inhibited	ThAO1, ThAO2	Reduced	/	[26]
MYB	Rice	Cd	OsMYB45	Knockdown decreased expression of OsCATA and OsCATC, and reduced CAT activity	Leaf, hull, stamen, pistil, lateral root	Induced (root), Unchanged (leaf)	/	Enhanced	/	[18]
MYB	Populus euphratica	Cd	PeRAX2	Increased H2O2 synthesis, inhibited CAT, SOD, POD activities, promoted Cd accumulation	Root, leaf	Induced	AtANN1	Reduced	Enhanced	[33]
ERF	Wheat	Cd, Cu, Zn	TdSHN1	Increased chlorophyll content, SOD, and CAT activities	/	Induced	/	Enhanced	/	[30]
bZIP	Tamarix	Cd	ThbZIP1	Enhanced POD and SOD activities, increased soluble sugar and soluble protein content	Root, leaf, stem	Induced	/	Enhanced	/	[27]
TEAD	Rice	Cd, Cr	OsTAZ4	Regulates ROS homeostasis; interacts with OsMYB34 and OsFHA9 to promote resistance to heavy metals	Whole plant	Induced	OsMYB34, OsFHA9	Enhanced	/	[31]

Table 1. Transcription factors regulating the expression of antioxidant enzyme genes.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
WRKY	Poplar	Cd	PyWRKY75	Increased chlorophyll and carotenoid content; enhanced activities of POD, SOD, CAT, and APX	/	Induced	/	Enhanced	Enhanced	[24]
WRKY	Maize	Cd	ZmWRKY4	Binds promoters of ZmSOD4 and ZmcAPX to increase antioxidant enzyme activities	Leaf	Induced	ZmSOD4, ZMcAPX	Enhanced	/	[28]
WRKY	Soybean	Cd	GmWRKY172	Increased POD activity, flavonoid, and lignin content	Leaf, flower, root, stem	Induced	/	Enhanced	Reduced	[20]
WRKY	Sorghum	Cd	SbWRKY54	Enhanced SOD, POD, and CAT activities; restricted Cd transport	Root, stem	Induced	SbHKT2b	Enhanced	Reduced	[32]
MYB	Tamarix	Cd	ThRAX2	Regulates intracellular ion homeostasis, transporter activity, and enhances antioxidant enzyme activities	Root, leaf	Induced (root), Inhibited (leaf)	ThSOS1, ThCKX3, ThCAX3A, ThMYB78	Enhanced	Reduced	[25]
MYB	Tamarix	Cd	ThDIV2	Decreased AOC, SOD, POD, CAT activities and GSH content	Root, leaf	Inhibited	ThAO1, ThAO2	Reduced	/	[26]
MYB	Rice	Cd	OsMYB45	Knockdown decreased expression of OsCATA and OsCATC, and reduced CAT activity	Leaf, hull, stamen, pistil, lateral root	Induced (root), Unchanged (leaf)	/	Enhanced	/	[18]
MYB	Populus euphratica	Cd	PeRAX2	Increased H2O2 synthesis, inhibited CAT, SOD, POD activities, promoted Cd accumulation	Root, leaf	Induced	AtANN1	Reduced	Enhanced	[33]
ERF	Wheat	Cd, Cu, Zn	TdSHN1	Increased chlorophyll content, SOD, and CAT activities	/	Induced	/	Enhanced	/	[30]
bZIP	Tamarix	Cd	ThbZIP1	Enhanced POD and SOD activities, increased soluble sugar and soluble protein content	Root, leaf, stem	Induced	/	Enhanced	/	[27]
TEAD	Rice	Cd, Cr	OsTAZ4	Regulates ROS homeostasis; interacts with OsMYB34 and OsFHA9 to promote resistance to heavy metals	Whole plant	Induced	OsMYB34, OsFHA9	Enhanced	/	[31]

2.1.2. Non-Enzymatic Antioxidants

Non-enzymatic antioxidants (e.g., ascorbate (AsA), glutathione (GSH), hydrogen sulfide (H₂S), melatonin, flavonoids) operate synergistically with enzymatic systems across cellular compartments via electron donation, radical quenching, and redox buffering [21]. TFs critically regulate genes involved in their biosynthesis and recycling pathways (Table 2).

The AsA-GSH cycle is a key antioxidant mechanism. In Populus, overexpression of SpHsfA4c activates this pathway, elevating AsA and GSH levels and optimizing GSH/GSSG ratio to enhance Cd tolerance [34]. H₂S also significantly contributes to Cd tolerance by maintaining redox homeostasis. Notably, exogenous H₂S has been shown to upregulate both non-enzymatic AsA/GSH levels and enzymatic SOD/CAT activities, thereby ameliorating Cd toxicity in rice [35,36]. At the transcriptional level, WRKY TFs (e.g., WRKY18/40/60) regulate H₂S biosynthesis genes by binding to W-box elements in their promoters, thereby modulating endogenous H₂S production and Cd resistance [37].

Melatonin further enhances heavy metal tolerance by improving antioxidant defenses, inducing metal transporters, and stimulating GSH/PC synthesis [21,38,39]. For example, tomato heat shock factor HsfA1a binds to the COMT1 (encoding Caffeic acid O-methyltransferase 1) promoter, increasing melatonin synthesis and subsequent PC/GSH production for vacuolar Cd sequestration [40]. Similarly, flavonoids mitigate Cd stress through their superior antioxidant capacity [41,42]. In rice, Cd-induced OsNAC300 activates OsCHS1 (encoding a key flavonoid biosynthesis enzyme) to enhance flavonoid-mediated antioxidant defense [43]. Correspondingly, overexpression of GmWRKY172 in soybean promotes Cd tolerance and reduces seed Cd accumulation by upregulating the flavonoid biosynthetic network [20].

In summary, TFs mitigate Cd-induced oxidative stress by modulating enzymatic and non-enzymatic antioxidants. However, ROS reduction alone is insufficient; plants employ additional strategies (chelation, compartmentalization, transporter regulation, signaling, and damage repair) for comprehensive Cd detoxification.

Table 2. Transcription factors regulating the expression of genes related to non-enzymatic antioxidant synthesis.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
WRKY	Arabidopsis	Cd	AtWRKY18, AtWRKY40, AtWRKY60	Inhibit H2S synthesis	/	Induced	LCD, DCD, DCD2, DES, NFS2	Reduced	/	[37]
NAC	Rice	Cd	OsNAC300	Increased pathogenesis-related (PR) protein and flavonoid synthesis	Root	Induced	OsPR10a, OsPR10b, OsCHS1	Enhanced	Unchanged	[43]
HSF	Sedum	Cd	SpHsfA4c	Increased GSH and AsA synthesis	/	Induced	/	Enhanced	Enhanced	[34]
HSF	Tomato	Cd	HsfA1a	Induces melatonin biosynthesis and heat shock protein (HSP) expression	/	Induced	COMT1	Enhanced	/	[40]

Table 2. Transcription factors regulating the expression of genes related to non-enzymatic antioxidant synthesis.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
WRKY	Arabidopsis	Cd	AtWRKY18, AtWRKY40, AtWRKY60	Inhibit H2S synthesis	/	Induced	LCD, DCD, DCD2, DES, NFS2	Reduced	/	[37]
NAC	Rice	Cd	OsNAC300	Increased pathogenesis-related (PR) protein and flavonoid synthesis	Root	Induced	OsPR10a, OsPR10b, OsCHS1	Enhanced	Unchanged	[43]
HSF	Sedum	Cd	SpHsfA4c	Increased GSH and AsA synthesis	/	Induced	/	Enhanced	Enhanced	[34]
HSF	Tomato	Cd	HsfA1a	Induces melatonin biosynthesis and heat shock protein (HSP) expression	/	Induced	COMT1	Enhanced	/	[40]

2.2. Chelation of Cd

Chelation of free Cd into stable complexes by molecules such as phytochelatins (PCs) and metallothioneins (MTs) significantly reduces its biological toxicity. Studies demonstrate that TF families including WRKY, MYB, bHLH, bZIP, and ZAT directly or indirectly activate genes involved in PC and MT synthesis, thereby enhancing plant tolerance and reducing Cd accumulation [44,45].

2.2.1. Regulation of Phytochelatin Synthesis

PCs are cysteine-rich small peptides that bind metals via thiol groups. Their synthesis is induced by various metals, primarily facilitating detoxification of metals with high affinity for -SH groups, such as Cd, As (III), Zn, Hg, Pb, and Cu [46]. The glutathione-dependent PC synthesis pathway is critical for Cd tolerance in plant [47]. Reduced glutathione (GSH) serves as the precursor for PCs. Phytochelatin synthase 1 (PCS1) and PCS2 play key roles in PC synthesis, while γ-glutamylcysteine synthetase (GSH1) and glutathione synthase (GSH2) are essential for GSH production [45,48].

Under Cd stress, TFs enhance plant Cd chelation capacity by regulating key genes involved in GSH and PC synthesis, significantly improving Cd tolerance (

Table 3. Transcription factors regulating the expression of genes related to phytochelatin synthesis.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
WRKY	Arabidopsis	Cd	AtWRKY12	Directly targets GSH1, inhibits expression of genes related to PC synthesis	Root, leaf, seed	Inhibited	GSH1	Reduced	Reduced	[49]
WRKY	Arabidopsis	Cd	AtWRKY45	Promotes expression of PC synthesis-related genes PCS1 and PCS2	Root, leaf	Induced	PCS1, PCS2	Enhanced	Enhanced	[45]
WRKY	Rape	Cd	BnWRKY11, BnWRKY28, BnWRKY33, BnWRKY75	Upregulates genes encoding GST and PCS, promoting formation of low molecular weight complexes (PC-Cd)	/	Induced	/	/	/	[51]
MYB	Arabidopsis	Cd	AtMYB75	Increases GSH and PC content; binds promoters of ACBP2 and ABCC2, promoting chelation and compartmentalization	Leaf	Induced	ACBP2, ABCC2	Enhanced	Enhanced	[44]
bZIP	Arabidopsis	Cd	AtbZIP30, AtERF2	Regulates expression of glutathione S-transferase genes (AtGST)	/	/	AtGST11	/	/	[50]
ZAT	Arabidopsis	Cd	ZAT6	Positively regulates genes involved in GSH1 and PC biosynthesis	/	Induced	ZAT	Enhanced	Enhanced	[47]

). For instance, in Arabidopsis, Cd-induced AtWRKY45 upregulates PCS1 and PCS2 expression, increasing PC content, enhancing Cd tolerance, and promoting Cd accumulation. Additionally, AtWRKY45 facilitates Fe uptake, further mitigating Cd toxicity [45]. In contrast, AtWRKY12 negatively regulates Cd tolerance by binding to the GSH1 promoter and suppressing its expression, indirectly inhibiting PC synthesis [49]. The zinc-finger TF ZAT6 positively regulates transcription of GSH1, GSH2, PCS1, and PCS2; its overexpression enhances Cd tolerance and accumulation in Arabidopsis, while loss-of-function mutants exhibit the opposite phenotype [47]. Both AtbZIP30 and AtERF2 bind to the promoter of glutathione S-transferase gene AtGST1, positively regulating its expression, improving ROS scavenging capacity and Cd chelation, and thereby enhancing Cd tolerance [50]. In rapeseed, TFs BnWRKY11, BnWRKY28, BnWRKY33, and BnWRKY75 are upregulated under Cd stress. Transcriptome analysis revealed significant enrichment of differentially expressed genes in glutathione metabolism pathways, particularly BnPCS1, BnGSTU12, and BnGSTU5, suggesting these TFs enhance Cd chelation and tolerance by promoting GSH and PC synthesis [51].

2.2.2. Regulation of Metallothionein Synthesis

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that play a vital role in heavy metal homeostasis and detoxification in plants [52]. They confer Cd tolerance through two primary mechanisms: high-affinity chelation of Cd²⁺ via their abundant thiol groups, and efficient scavenging of reactive oxygen species (ROS) to alleviate oxidative stress [53].

Functional studies in rice have provided direct evidence for these roles. For example, the expression of OsMT1e is strongly induced by Cd. Compared to wild-type controls, OsMT1e-overexpressing lines exhibit enhanced Cd tolerance, whereas OsMT1e-RNAi lines show inhibited growth, reduced chlorophyll content, and lower biomass under Cd stress. Furthermore, heterologous expression of OsMT1e in yeast confers a robust growth phenotype under Cd stress, consistent with its role in detoxification [54]. Similarly, the rice MT gene OsMT2b is co-induced by drought and Cd stress. Under drought, OsMT2b enhances tolerance by modulating the expression of ROS-scavenging genes. Its role in Cd tolerance is likely attributed to the direct binding of OsMT2b protein to Cd, thereby reducing the concentration of active Cd²⁺ [55].

Currently, systematic studies on TFs that regulate MT genes remain limited (Table 4), highlighting this as an emerging and promising research frontier. Known examples include the wheat A4-class heat shock TF gene TaHsfA4a and its rice ortholog OsHsfA4a, both of which are upregulated under Cd stress. Overexpression of TaHsfA4a enhances Cd tolerance in yeast and rice, while knockout of OsHsfA4a reduces tolerance. These TFs function by activating the expression of MT genes [56]. In Arabidopsis, AtMYB4 binds to the promoters of phytochelatin synthase (PCS) and metallothionein 1C (MT1C), positively regulating their expression to promote Cd chelation and increase tolerance [57]. Furthermore, in Iris lactea var. chinensis, the AP2/ERF transcription factor IlAP2 mitigates Cd toxicity through a non-canonical mechanism. Instead of activating transcription, IlAP2 physically interacts with the metallothionein IlMT2a. This interaction is proposed to reduce Cd accumulation and enhance tolerance by interfering with metal transport and modulating stress-responsive signaling pathways [58].

To systematically expand this regulatory network and advance explorations towards precision molecular breeding, future research should focus on the following interconnected directions: First, it is essential to deeply analyze the regulatory logic and key cis-regulatory elements (CREs)—such as metal response elements (MREs) and copper response elements (CuREs)—within MT gene promoters. Studies on promoters like OsMT-I-4b have already revealed their precise tissue specificity and responsiveness to multiple metals [59], laying the foundation for discovering upstream transcription factors and designing synthetic inducible systems [60]. Second, it is necessary to elucidate how transcription factors coordinate MT synthesis to balance the nutritional allocation of essential elements such as zinc with cadmium detoxification. Notably, OsMT2b and OsMT2c, which are highly expressed in the phloem of rice nodes, are crucial for zinc allocation to grains [61]. This suggests that transcriptional engineering of nodal MT expression may become a key strategy for maintaining mineral nutrition while reducing grain cadmium accumulation. Finally, cutting-edge technologies such as ATAC-seq should be integrated to map genome-wide chromatin accessibility and identify regulatory regions that become “open” under cadmium stress. Meanwhile, single-cell RNA sequencing (scRNA-seq) can help uncover endogenous, tissue-specific promoters with practical potential. Furthermore, CRISPR-mediated promoter engineering to drive root-specific expression of genes such as MTs could effectively “intercept” cadmium at critical sites, thereby minimizing its translocation to grains [60].

Table 4. Transcription factors regulating the expression of genes related to metallothionein synthesis.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
MYB	Arabidopsis	Cd	AtMYB4	Improves oxidative defense; increases AtPCS1 and AtMT1C expression	Root, leaf	Induced	PCS1, MT1C	Enhanced	Enhanced	[57]
HSF	Wheat	Cd	TaHsfA4a	Enhances Cd tolerance in rice and yeast	/	Induced	CUP1	Enhanced	/	[56]
HSF	Rice	Cd	OsHsfa4a	Upregulates MT gene expression to enhance Cd tolerance	/	Induced	OsMT-I-1a	Enhanced	/	[56]
ZAT	Arabidopsis	Cd	ZAT10	Inhibits Cd uptake genes; positively regulates Cd chelation gene expression	/	Induced	NAS1, NAS2, IRT2, MTP3	Enhanced	Reduced	[62]
ERF	Iris lactea var. chinensis	Cd	IlAP2	Enhances Cd tolerance	/	Induced	IlMT2a	Enhanced	/	[58]

Table 4. Transcription factors regulating the expression of genes related to metallothionein synthesis.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
MYB	Arabidopsis	Cd	AtMYB4	Improves oxidative defense; increases AtPCS1 and AtMT1C expression	Root, leaf	Induced	PCS1, MT1C	Enhanced	Enhanced	[57]
HSF	Wheat	Cd	TaHsfA4a	Enhances Cd tolerance in rice and yeast	/	Induced	CUP1	Enhanced	/	[56]
HSF	Rice	Cd	OsHsfa4a	Upregulates MT gene expression to enhance Cd tolerance	/	Induced	OsMT-I-1a	Enhanced	/	[56]
ZAT	Arabidopsis	Cd	ZAT10	Inhibits Cd uptake genes; positively regulates Cd chelation gene expression	/	Induced	NAS1, NAS2, IRT2, MTP3	Enhanced	Reduced	[62]
ERF	Iris lactea var. chinensis	Cd	IlAP2	Enhances Cd tolerance	/	Induced	IlMT2a	Enhanced	/	[58]

2.3. Compartmentalization (Sequestration)

Compartmentalization, the regulated transport and redistribution of Cd within plants, is a decisive process governing the final accumulation of Cd in grains. This process is orchestrated by diverse transcription factors that coordinate the sequestration of Cd into immobile compartments such as cell walls and vacuoles. Beyond cellular detoxification, this sequestration critically modulates the pool of free Cd ions available for xylem loading and subsequent translocation. As indicated by Yue et al., the efficacy of these sequestration mechanisms—which are governed by specific transcriptional networks in roots, nodes, and leaves—serves as a major checkpoint for grain safety. By transcriptionally enhancing tissue-specific “barrier” effects, plants can effectively restrict the flux of Cd destined for the grain during the reproductive stage [63]. Therefore, elucidating how transcription factors regulate these sequestration pathways presents a practical strategy for minimizing grain Cd accumulation without disrupting essential micronutrient homeostasis.

2.3.1. Cell Wall Binding

Plant cell walls, composed primarily of cellulose, hemicellulose, pectin, and lignin, exhibit strong adsorption capacity for Cd. The root cell wall serves as the primary physical barrier against heavy metal stress (e.g., Cd) [64]. Following Cd entry into roots, a significant fraction binds to cell wall components, limiting cytoplasmic influx and reducing phytotoxicity [65]. Hemicellulose constitutes a major Cd-binding site [66]. Notably, Cd-inducible OsCDT1 in rice encodes a cysteine-rich protein localized to the plasma membrane and cell wall, which chelates Cd at the cell surface, restricting intracellular entry and enhancing Cd tolerance while reducing accumulation [67].

Transcription factors induce genes involved in cell wall biosynthesis, strengthening Cd sequestration capacity and minimizing cellular damage (

Table 5. Transcription factors regulating the expression of cell wall synthesis-related genes.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
bHLH	Maize	Cd	ZmbHLH105	Binds promoters of ZmNCED1/2 to regulate ABA biosynthesis for ROS scavenging; confers Cd tolerance via ABA-mediated lignin deposition and root cell wall thickening	Root, leaf	Induced	ZmNCED1/2	Enhanced	Reduced	[71]
bZIP	Arabidopsis	Cd	AtbZIP44	Regulates mannanase MAN7 activity, enhancing cell wall Cd fixation capacity	Root, stem	Induced	AtMAN7	Enhanced	Reduced	[69]
NAC	Arabidopsis	Cd	AtNAC102	Regulates cell wall pectin metabolism and Cd binding, conferring Cd tolerance in Arabidopsis	Root	Induced (root), unchanged (shoot)	WAKL11	Enhanced	Reduced	[72]
NAC	Arabidopsis	Cd	ANAC004	Enhances Cd cell wall fixation; induces Cd chelation and compartmentalization gene expression; downregulates Cd translocation genes, reducing root-to-shoot Cd transfer	Root, flower, silique	Induced	CS1/2, NAS4, ABCC1/2/3, MTP1/3, IREG2, NRAMP3/4	Enhanced	Reduced	[68]

). In Arabidopsis, ANAC004 knockout reduced cell wall-bound Cd by ≥19.6% and hemicellulose-bound Cd by ≥28.3%, suggesting compromised Cd tolerance due to reduced sequestration [68]. AtbZIP44 positively regulates MAN7 (encoding the endo-β-mannanase), modulating mannose content in the cell wall and influencing Cd binding, thereby reducing Cd uptake and root-to-shoot translocation [69]. Lignin mitigates Cd toxicity through functional group interactions [70]. In maize, ZmbHLH105 overexpression enhances activities of lignin biosynthesis enzymes (PAL, CAD, LAC, POD), promoting cell wall thickening and Cd binding capacity, ultimately increasing Cd tolerance [71].

2.3.2. Vacuolar Sequestration

Vacuolar sequestration is a pivotal Cd detoxification mechanism. Proton pumps establish a transmembrane proton gradient that drives transporters to actively transport cytosolic Cd or Cd-complexes into vacuoles, reducing cytoplasmic toxicity [73]. For instance, ABCC1/2 transporters in Arabidopsis sequester Cd-PCs complexes into vacuoles, significantly enhancing tolerance [13]. In rice, the tonoplast transporter OsHMA3 sequesters Cd into root vacuoles, limiting translocation to shoots and reducing grain Cd accumulation [74]. CRISPR/Cas9-generated OsHMA3 knockout mutants exhibit heightened Cd sensitivity, reduced root Cd accumulation, and increased shoot Cd content, confirming OsHMA3-mediated root vacuolar sequestration enhances tolerance by restricting Cd translocation [74].

Specific TFs activated by Cd stress regulate downstream vacuolar transporter genes, enhancing Cd sequestration into vacuoles (Table 6). This process effectively lowers cytosolic Cd concentration and alleviates toxicity. In Arabidopsis, AtMYB75 overexpression increases GSH and PC levels and upregulates chelation/sequestration genes, enhancing tolerance and accumulation [44]. Co-overexpression of bHLH TFs (FIT, AtbHLH38, AtbHLH39) activates HMA3, MTP3, IRT2, and IREG2, alleviating Cd toxicity by maintaining stem Fe levels and increasing vacuolar Cd storage [75]. AtbHLH104 positively regulates four Cd detoxification genes (IREG2, MTP3, HMA3, NAS4); its knockout mutants are Cd-sensitive, while overexpression lines exhibit high root Cd accumulation, reduced shoot Cd, and enhanced tolerance [76]. Conversely, AtMYB43 negatively regulates tolerance by suppressing key Cd transporter genes (HMA2, HMA4), hindering xylem transport and vacuolar sequestration [77].

In rice, OsCS1 (allelic to OsMTP11) critically regulates the translocation of Cd from leaves to grains [73]. It is predominantly expressed in leaf vascular parenchyma cells, where it sequesters Cd into vacuoles via the OsVSR2-mediated TGN–PVC–vacuole trafficking pathway (OsVSR2 is a vacuole-sorting receptor protein). By actively “trapping” Cd within the leaf endomembrane system during vegetative growth, OsCS1 effectively prevents its phloem loading and subsequent translocation to grains after heading. The high efficiency of this sequestration mechanism in indica rice stems from a natural variation in the OsCS1 promoter: a duplication of a G-box-like motif. This structural variation strengthens the binding of the transcription factor OsIRO2, leading to intensified transcriptional activation and upregulated OsCS1 expression. Consequently, Cd sequestration in leaf vacuoles is maximized, which provides the genetic basis for the superior low-Cd accumulation trait characteristic of indica rice varieties [78].

Table 6. Transcription factors regulating the expression of vacuolar sequestration-related genes.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
MYB	Arabidopsis	Cd	AtMYB43	Downregulates HMA2, HMA3, and HMA4 gene expression	Whole plant	Induced	PRL1	Reduced	Enhanced (root), Reduced (shoot)	[77]
bHLH	Arabidopsis	Cd, Fe	AtbHLH38, AtbHLH39	Maintains high iron levels via iron transport and nicotianamine accumulation	Root, stem	Induced	HMA3, MTP3, IREG2, IRTN2, NAS1, NAS2	Enhanced	/	[75]
bHLH	Arabidopsis	Cd, Fe	AtbHLH104	Positively regulates genes for heavy metal detoxification; inhibits root-to-shoot Cd translocation	/	/	IREG2, MTP3, HMA3, NAS4	Enhanced	Enhanced (root), Reduced (shoot)	[76]
bHLH	Rice	Cd, Mn	OsIRO2	Upregulates OsCS1	/	/	OsCS1	Enhanced	Reduced	[78]

Table 6. Transcription factors regulating the expression of vacuolar sequestration-related genes.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
MYB	Arabidopsis	Cd	AtMYB43	Downregulates HMA2, HMA3, and HMA4 gene expression	Whole plant	Induced	PRL1	Reduced	Enhanced (root), Reduced (shoot)	[77]
bHLH	Arabidopsis	Cd, Fe	AtbHLH38, AtbHLH39	Maintains high iron levels via iron transport and nicotianamine accumulation	Root, stem	Induced	HMA3, MTP3, IREG2, IRTN2, NAS1, NAS2	Enhanced	/	[75]
bHLH	Arabidopsis	Cd, Fe	AtbHLH104	Positively regulates genes for heavy metal detoxification; inhibits root-to-shoot Cd translocation	/	/	IREG2, MTP3, HMA3, NAS4	Enhanced	Enhanced (root), Reduced (shoot)	[76]
bHLH	Rice	Cd, Mn	OsIRO2	Upregulates OsCS1	/	/	OsCS1	Enhanced	Reduced	[78]

2.4. Regulation of Transporter Activity

A variety of metal transporters are involved in Cd uptake from roots and its translocation to grains in rice. These include: Zinc/Iron-regulated transporter-like Proteins (ZIPs), Natural Resistance-Associated Macrophage Proteins (NRAMPs), Heavy Metal-transporting ATPases (HMAs), Metal Tolerance Proteins (MTPs), ATP-Binding Cassette transporters (ABCs), Cation Exchangers (CAXs), Yellow Stripe-Like proteins (YSLs), and Iron-Regulated Transporters (IRTs) [79,80,81,82]. Upon Cd stress, plants activate specific transcription factors (TFs) to modulate the activity of these transporters (Table 7). Based on their primary regulatory roles, these TFs can be categorized into three groups: those enhancing Cd efflux, those suppressing Cd influx, and those coordinating long-distance transport.

2.4.1. Enhancing Cd Efflux and Cellular Exclusion

A primary plant defense strategy is to activate efflux transporters that export Cd from the cytoplasm. For example, in Arabidopsis, AtWRKY13 upregulates PDR8 expression, leading to enhanced Cd extrusion and improved tolerance [83]. Similarly, PyWRKY48 in poplar promotes Cd exclusion by inducing heavy metal transporters and antioxidant enzymes [84]. In pepper, Cd stress-induced CaMYB TFs increase the expression of transporter genes involved in maintaining cellular homeostasis [85]. By reducing intracellular Cd levels, these TFs constitute a first line of defense against Cd toxicity.

2.4.2. Suppressing Cd Influx and Root Uptake

Limiting Cd uptake at the root level is crucial for reducing overall plant accumulation. This is achieved mainly through transcriptional repression or post-translational regulation of influx transporters:

(1) Transcriptional repression: In rice, OsNAC15 binds to the ZDRE cis-element in the promoters of OsZIP7 and OsZIP10, suppressing their transcription and thereby decreasing Cd accumulation in shoots [86]. In wheat, TaWRKY70, TaNAC22, and TaWRKY74 repress several Cd transporter genes while enhancing antioxidant defense systems [29,87,88]. Moreover, AemNAC2/3 from Aegilops markgrafii significantly lowers Cd accumulation in wheat by downregulating TaNRAMP5 and TaHMA2 expression [89].

(2) Post-translational regulation: In Arabidopsis, AtWRKY33 employs a distinct mechanism by activating ATL31, which encodes an E3 ubiquitin ligase. ATL31 promotes ubiquitination and degradation of the influx transporter IRT1, thereby blocking Cd entry into cells [90].

2.4.3. Regulating Long-Distance Transport and Grain Allocation

Transcription factors that control Cd translocation from roots to grains are particularly important for food safety. In wheat, TaWRKY22 activates the TaCOPT3D promoter, enhancing Cd fixation in roots and limiting the metal pool available for grain translocation [91]. In maize, ZmWRKY64 acts as a broad regulator that coordinates multiple genes—such as ZmABCC4, ZmNRAMP5, and ZmHMA3—to balance Cd translocation with reactive oxygen species scavenging [92].

In rice, OsNAC5 functions as a key regulatory switch for grain Cd accumulation: it activates OsNRAMP1 to promote Cd uptake, and its knockout significantly reduces grain Cd levels [16]. Conversely, in cotton, the GhbHLH121-GhRCD-GhMYB44 module fine-tunes the transcription of HMA genes to regulate transport efficiency [93]. In potato, StWRKY6 enhances Cd transport and chelation, thereby reducing overall accumulation [94].

Table 7. Transcription factors regulating the expression of Cd transporter genes.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
WRKY	Arabidopsis	Cd	AtWRKY13	Binds promoter of transporter PDR8 to pump out Cd	Whole plant	Induced	PRD1, PRD2, PRD8	Enhanced	Reduced	[83]
WRKY	Arabidopsis	Cd	AtWRKY33	Interacts with ATL31, ubiquitinates and promotes IRT1 degradation	Root, leaf, seed	Induced	ATL31, IRT1	Enhanced	Reduced	[90]
WRKY	Wheat	Cd	TaWRKY22	Binds TaCOPT3D, enhances HMA3 expression	/	Induced	TaCOPT3D	Enhanced	Enhanced (root), Reduced (grain)	[91]
WRKY	Wheat	Cd	TaWRKY70	Downregulates AtHMA3, AtNRAMP5, AtYSL3, AtIRT1	Root, stem	Induced	TaCAT5	Enhanced	Enhanced (root), Reduced (shoot)	[29]
WRKY	Wheat	Cd	TaWRKY74	Affects expression of AsA-GSH synthesis genes and inhibits expression of Cd transporter genes	Root, leaf, stem	Induced	/	Enhanced	Reduced	[88]
WRKY	Poplar	Cd	PyWRKY48, PyWRKY71, PyWRKY45	Upregulates transporter, heavy metal-binding protein, and xylem protein gene activity	Root, leaf, stem	Induced	PaABC, PaHIP, PaNFP, PaBSP	Enhanced	Enhanced	[84]
WRKY	Maize	Cd	ZmWRKY64	Regulates ROS homeostasis; positively regulates transporters	Root, leaf	Induced	ZMSRG7	Enhanced	Reduced	[92]
WRKY	Potato	Cd	StWRKY6	Upregulates genes for Cd chelation, plant defense, transporters, photomorphogenesis, and auxin signaling	Root, leaf, stem	Induced	ERF013, BBX20, BAM5, VSP2, ABCG1, PDF1.4	Enhanced	Reduced	[94]
MYB	Pepper	Cd, Cu, Zn	CaMYB	MYB protein interacts with Cd transporters (e.g., HMA) to improve plant tolerance to heavy metals	/	Induced	/	/	/	[85]
bHLH	Cotton	Cd	GhBHLH12	Interacts with GhRCD1, binds to and relieves transcriptional repression of GhMYB44; GhMYB44 further interacts with GhPYL8 and activates transcription of heavy metal transporter GhHMA1, affecting Cd transport efficiency and tolerance	/	/	GhRCD1, GhMYB44, GhHMA1	Reduced	Reduced	[93]
NAC	Rice	Cd	OsNAC15	Binds promoters of OsZIP7 and OsZIP10, inhibiting their transcription	Root	Induced	OsZIP7, OsZIP10	Enhanced	Enhanced (root), Reduced (shoot)	[86]
NAC	Rice	Cd	OsNAC5	Upregulates OsNRAMP1 and OsLEA3	Root	Induced	OsNRAMP1	Enhanced	Enhanced	[16]
NAC	Wheat	Cd	AemNAC2, AemNAC3	Inhibit expression of TaNRAMP5	Root, shoot	Induced	TaNRAMP5	Enhanced	Reduced	[88]
NAC	Wheat	Cd	TaNAC22	Reduces Cd transporter levels and enhances antioxidant enzyme system	Leaf	Induced	TaHMA2, TaIRt2, TaLCT1, TaHMA3, TaNRAMP1, TaNRAMP5	Enhanced	Reduced	[87]

Table 7. Transcription factors regulating the expression of Cd transporter genes.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
WRKY	Arabidopsis	Cd	AtWRKY13	Binds promoter of transporter PDR8 to pump out Cd	Whole plant	Induced	PRD1, PRD2, PRD8	Enhanced	Reduced	[83]
WRKY	Arabidopsis	Cd	AtWRKY33	Interacts with ATL31, ubiquitinates and promotes IRT1 degradation	Root, leaf, seed	Induced	ATL31, IRT1	Enhanced	Reduced	[90]
WRKY	Wheat	Cd	TaWRKY22	Binds TaCOPT3D, enhances HMA3 expression	/	Induced	TaCOPT3D	Enhanced	Enhanced (root), Reduced (grain)	[91]
WRKY	Wheat	Cd	TaWRKY70	Downregulates AtHMA3, AtNRAMP5, AtYSL3, AtIRT1	Root, stem	Induced	TaCAT5	Enhanced	Enhanced (root), Reduced (shoot)	[29]
WRKY	Wheat	Cd	TaWRKY74	Affects expression of AsA-GSH synthesis genes and inhibits expression of Cd transporter genes	Root, leaf, stem	Induced	/	Enhanced	Reduced	[88]
WRKY	Poplar	Cd	PyWRKY48, PyWRKY71, PyWRKY45	Upregulates transporter, heavy metal-binding protein, and xylem protein gene activity	Root, leaf, stem	Induced	PaABC, PaHIP, PaNFP, PaBSP	Enhanced	Enhanced	[84]
WRKY	Maize	Cd	ZmWRKY64	Regulates ROS homeostasis; positively regulates transporters	Root, leaf	Induced	ZMSRG7	Enhanced	Reduced	[92]
WRKY	Potato	Cd	StWRKY6	Upregulates genes for Cd chelation, plant defense, transporters, photomorphogenesis, and auxin signaling	Root, leaf, stem	Induced	ERF013, BBX20, BAM5, VSP2, ABCG1, PDF1.4	Enhanced	Reduced	[94]
MYB	Pepper	Cd, Cu, Zn	CaMYB	MYB protein interacts with Cd transporters (e.g., HMA) to improve plant tolerance to heavy metals	/	Induced	/	/	/	[85]
bHLH	Cotton	Cd	GhBHLH12	Interacts with GhRCD1, binds to and relieves transcriptional repression of GhMYB44; GhMYB44 further interacts with GhPYL8 and activates transcription of heavy metal transporter GhHMA1, affecting Cd transport efficiency and tolerance	/	/	GhRCD1, GhMYB44, GhHMA1	Reduced	Reduced	[93]
NAC	Rice	Cd	OsNAC15	Binds promoters of OsZIP7 and OsZIP10, inhibiting their transcription	Root	Induced	OsZIP7, OsZIP10	Enhanced	Enhanced (root), Reduced (shoot)	[86]
NAC	Rice	Cd	OsNAC5	Upregulates OsNRAMP1 and OsLEA3	Root	Induced	OsNRAMP1	Enhanced	Enhanced	[16]
NAC	Wheat	Cd	AemNAC2, AemNAC3	Inhibit expression of TaNRAMP5	Root, shoot	Induced	TaNRAMP5	Enhanced	Reduced	[88]
NAC	Wheat	Cd	TaNAC22	Reduces Cd transporter levels and enhances antioxidant enzyme system	Leaf	Induced	TaHMA2, TaIRt2, TaLCT1, TaHMA3, TaNRAMP1, TaNRAMP5	Enhanced	Reduced	[87]

2.5. Regulation of Signal Transduction Pathways and Integration of Stress Signaling

Transcription factors (TFs) act as pivotal downstream effectors in signal transduction pathways, playing a central role in coordinating Cd detoxification mechanisms and enhancing plant tolerance [15,95]. The regulatory process begins with the early perception of Cd stress. Cd signals are detected by membrane-localized sensor modules, such as G protein-coupled receptors (GPCRs), calcium channels, and glutamate receptor-like (GLR) channels.

These sensors rapidly trigger the production of secondary messengers, including Ca²⁺, calmodulin (CaM), ROS, and phytohormones [19,95]. These messengers are then decoded by specialized signaling cascades, such as calcium-dependent protein kinases (CDPKs), CBL-interacting protein kinases (CIPKs), and calcineurin B-like proteins (CBLs) [15]. Specifically, certain CDPKs or mitogen-activated protein kinases (MAPKs) modulate the DNA-binding affinity or stability of downstream TFs through phosphorylation, thereby initiating precise transcriptional responses to Cd [19].

Notably, MAPK and SnRK2 (sucrose non-fermenting-1-related protein kinase 2) pathways serve as common “signaling hubs” that mediate crosstalk between Cd stress and other biotic or abiotic stresses [15,19]. Given the substantial overlap between Cd-induced ROS/Ca²⁺ signals and those triggered by drought, salinity, or pathogen infection, TFs can integrate multiple environmental cues to optimize defense strategies [19]. For instance, under Cd-induced oxidative stress, the MAPK cascade coordinates synergistic defense against heavy metal toxicity and biological invasion by interacting with jasmonic acid (JA) and salicylic acid (SA) pathways, highlighting plant capacity for signal integration under combined stresses [19].

This signal integration mechanism has been demonstrated in several studies (Table 8). In Arabidopsis, AtMYB59 functions as a key molecular switch that negatively regulates growth and Cd tolerance by maintaining Ca²⁺ homeostasis and modulating Ca²⁺ signaling [95]. In soybean, MYBZ2 is regulated by NADPH oxidase-derived ROS, implicating it in early oxidative stress signaling [96]. In rice, SNAC1 enhances Cd tolerance by coordinating MAPK signaling with ABA-dependent pathways, linking heavy metal response to osmotic stress adaptation [97].

TFs also participate in phytohormone-mediated signaling. In Arabidopsis, AtMYB49 interacts with ABI5 to participate in ABA-mediated inhibition of Cd accumulation [17], whereas in wheat, TabHLH094 interacts with TaMYC8 and limits ethylene synthesis to suppress Cd uptake and reduce grain Cd content [98,99]. Collectively, the extensive interplay between TFs and multiple hormone pathways (e.g., ABA, ethylene, and JA) indicates that the Cd-responsive network is deeply integrated with broader plant growth and defense systems, enabling robust adaption to complex environmental fluctuations [19].

Table 8. Transcription factors regulating the expression of signal transduction-related genes.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
MYB	Arabidopsis	Cd	AtMYB59	Negatively regulates Ca2+ homeostasis and signaling	Root, leaf	Induced	CMLs, KIC, CAX1, ACA1	Enhanced	/	[95]
MYB	Soybean	Cd	MYBZ2	/	/	Induced	/	Enhanced	/	[96]
MYB	Arabidopsis	Cd	AtMYB49	Interacts with ABI5, participates in ABA synthesis; activates IRT1, HIPP22, HIPP44	Root, flower, stem, bud	Induced	ABI5, bHLH38, bHLH101, HIPP22, HIPP44	Enhanced	Enhanced	[17]
bHLH	Wheat	Cd	TabHLH094	Regulates transcriptional activity of TaMYBC8 to reduce ethylene production	Root, stem	Induced	TaMYC8, ACF, ACO	Enhanced	Reduced	[98]
ERF	Wheat	Cd	TaMYC8	Inhibits ethylene biosynthesis pathway	Root, stem	Inhibited	TaERF6	Reduced	Enhanced	[99]
NAC	Rice	Cd	SNAC1	Regulates mitogen-activated protein kinase (MAPK) signaling cascade	/	/	/	Enhanced	/	[97]

Table 8. Transcription factors regulating the expression of signal transduction-related genes.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
MYB	Arabidopsis	Cd	AtMYB59	Negatively regulates Ca2+ homeostasis and signaling	Root, leaf	Induced	CMLs, KIC, CAX1, ACA1	Enhanced	/	[95]
MYB	Soybean	Cd	MYBZ2	/	/	Induced	/	Enhanced	/	[96]
MYB	Arabidopsis	Cd	AtMYB49	Interacts with ABI5, participates in ABA synthesis; activates IRT1, HIPP22, HIPP44	Root, flower, stem, bud	Induced	ABI5, bHLH38, bHLH101, HIPP22, HIPP44	Enhanced	Enhanced	[17]
bHLH	Wheat	Cd	TabHLH094	Regulates transcriptional activity of TaMYBC8 to reduce ethylene production	Root, stem	Induced	TaMYC8, ACF, ACO	Enhanced	Reduced	[98]
ERF	Wheat	Cd	TaMYC8	Inhibits ethylene biosynthesis pathway	Root, stem	Inhibited	TaERF6	Reduced	Enhanced	[99]
NAC	Rice	Cd	SNAC1	Regulates mitogen-activated protein kinase (MAPK) signaling cascade	/	/	/	Enhanced	/	[97]

2.6. Regulation of DNA and Protein Damage Repair

Cd induces DNA and protein damage and endoplasmic reticulum (ER) stress. Low Cd concentrations can directly bind to DNA, causing damage such as base mismatches, insertions, deletions, and breaks [100]. Cd also disrupts protein stability, causing misfolding and loss of function [101]. The MutS protein complex (encoded by MSH genes) recognizes DNA mismatches and initiates mismatch repair (MMR), implicating it in plant Cd tolerance. In rice, msh mutants exhibit more severe growth inhibition under Cd stress than wild-type plants [102]. Cao et al. found that MSH2 and MSH6 perceive Cd-induced DNA damage, activating cell cycle checkpoint genes and triggering G2/M phase arrest to allow time for DNA repair. In msh2 and msh6 mutants, upregulation of cell cycle-related genes is attenuated, G2/M arrest is reduced, and root growth is more severely inhibited [103]. Under Cd stress, misfolded protein accumulation triggers the unfolded protein response (UPR), activating TFs and upregulating chaperone to restore proteostasis. Prolonged stress may lead UPR to induce programmed cell death (PCD), inhibiting growth [104]. In soybean, Cd stress activates the developmental cell death (DCD)/asparagine-rich protein (NRP) signaling cascade, inducing GmNAC81 and GmNAC30, which activate vacuolar processing enzyme (VPE) to trigger PCD. Overexpression of the ER chaperone BIP alleviates ER stress and cell death by suppressing UPR and DCD/NRP pathway [101].

TFs repair Cd-induced DNA and protein damage by regulating relevant gene expression (Table 9). In rice, OsE2F activates MSH target genes, promoting DNA damage repair [102]. OsREX1-S enhances Cd tolerance by improving DNA damage repair capacity [105]. In Arabidopsis, AtbZIP60 upregulates ER chaperone genes, promoting folding and degradation of misfolded proteins to restore ER homeostasis. Under severe Cd stress, UPR activates NAC089, initiating PCD. In atbzip60 mutants, expression of ER stress and PCD-related genes is reduced, suppressing UPR-mediated PCD and enhancing Cd tolerance [104]. NtbZIP60 is involved in ER stress and UPRs, regulating Cd tolerance in tobacco [106].

In summary, TFs counter Cd stress by orchestrating a range of detoxification mechanisms, thereby enhancing plant Cd tolerance and modulating Cd allocation and accumulation. Beyond these predominant pathways, a subset of TFs employ alternative strategies to confer Cd tolerance, as exemplified in Table 10. For example, in Arabidopsis, AtERF1B and AtERF104 activate NRT1.8, promoting nitrate allocation to roots and subsequently enhancing Cd tolerance [107]. AtZAT17 interacts with MAC spliceosome components, affecting alternative splicing of Cd stress-responsive genes and negatively regulating Cd tolerance [108]. In common bean, PvMTF-1 binds to the ASA2 promoter, increasing free tryptophan levels, which contributes to reduced Cd accumulation, and enhanced Cd tolerance [109]. In rice, OsMYB36 promotes Casparian strip formation, thereby restricting apoplastic Cd transport and reducing Cd accumulation [110]. These specialized mechanisms likely reflect evolutionary adaptations that enable different species to thrive in specific Cd-stressed environments. They function synergistically with the primary detoxification pathways, collectively fine-tuning the plant’s response to enhance environmental adaptability.

Table 9. Transcription factors regulating the expression of DNA and protein damage repair-related genes.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
bZIP	Arabidopsis	Cd	AtbZIP28, AtbZIP60	Reduces UPR and PCD, thereby increasing Cd tolerance	/	Induced	BIP3	Enhanced	/	[104]
bZIP	Tobacco	Cd	NtbZIP60	Associated with ER stress	/	Induced	/	/	/	[106]
bZIP	Rice	Cd	OsbZIP39	Activates defense protein OsCAL2 to increase Cd uptake; upregulates UPR	/	Induced	OsCAL2	Reduced	Enhanced	[111]
E2F	Rice	Cd	OsE2F	Interacts with OsMSHs, promotes recognition and correction of mismatched bases; inhibits DNA damage	/	Induced	OsMSHs	Enhanced	/	[102]
TFIIH	Rice	Cd	OsREX1-S	Enhances DNA excision repair, conferring tolerance to Cd and UV-induced damage	/	Induced	/	Enhanced	/	[105]

Table 9. Transcription factors regulating the expression of DNA and protein damage repair-related genes.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
bZIP	Arabidopsis	Cd	AtbZIP28, AtbZIP60	Reduces UPR and PCD, thereby increasing Cd tolerance	/	Induced	BIP3	Enhanced	/	[104]
bZIP	Tobacco	Cd	NtbZIP60	Associated with ER stress	/	Induced	/	/	/	[106]
bZIP	Rice	Cd	OsbZIP39	Activates defense protein OsCAL2 to increase Cd uptake; upregulates UPR	/	Induced	OsCAL2	Reduced	Enhanced	[111]
E2F	Rice	Cd	OsE2F	Interacts with OsMSHs, promotes recognition and correction of mismatched bases; inhibits DNA damage	/	Induced	OsMSHs	Enhanced	/	[102]
TFIIH	Rice	Cd	OsREX1-S	Enhances DNA excision repair, conferring tolerance to Cd and UV-induced damage	/	Induced	/	Enhanced	/	[105]

Table 10. Transcription factors involved in other regulatory mechanisms.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
WRKY	Rice	Cd	OsWRKY71, OsTRAB-1	Acts as nodal components in Cd signaling pathways	Root, leaf, stem, seed	Induced	/	/	/	[112]
MYB	Rice	Cd	OsMYB36	Regulates Casparian strip formation in plants	Root, leaf, stem, lemma, leaf sheath	/	OsCASP, OsESB1	/	Reduced	[110]
bHLH	Soybean	Cd, Fe	ORG3	Increases Fe transport rate but reduces Cd translocation from root to stem	Root	Induced	/	Enhanced	Enhanced (root), Reduced (shoot)	[113]
ERF	Arabidopsis	Cd	AtERF1b, AtERF104	Promotes nitrate assimilation and photosynthesis	/	/	NRT1.8, NRT1.5	Enhanced	/	[107]
HSF	Sedum	Cd	SaHSFA4c	Upregulates antioxidant enzymes and heat shock proteins (HSPs), reduces ROS accumulation	Root, leaf, stem	Induced	/	Enhanced	/	[114]
ZAT	Arabidopsis	Cd	ZAT17	Induces IRT1 and NRT2.1 expression; interacts with MAC splicing complex (PRL1, CDC5), affecting alternative splicing (AS) of multiple Cd response genes	Root, leaf	Induced	IRT1, NRT2.1, HMA3, PCR1	Reduced	/	[108]
ERF	Common bean	Cd	PVMTF-1	Activates tryptophan biosynthesis	/	Induced	AsA2	Enhanced	/	[109]

Table 10. Transcription factors involved in other regulatory mechanisms.

Family	Plant	Heavy Metal (Primarily Cd)	Gene Name	Gene Function	Expression Site	Response to Cd (Inhibition/Induction)	Up/Downstream Genes	Role in Cd Tolerance	Role in Cd Accumulation	Reference
WRKY	Rice	Cd	OsWRKY71, OsTRAB-1	Acts as nodal components in Cd signaling pathways	Root, leaf, stem, seed	Induced	/	/	/	[112]
MYB	Rice	Cd	OsMYB36	Regulates Casparian strip formation in plants	Root, leaf, stem, lemma, leaf sheath	/	OsCASP, OsESB1	/	Reduced	[110]
bHLH	Soybean	Cd, Fe	ORG3	Increases Fe transport rate but reduces Cd translocation from root to stem	Root	Induced	/	Enhanced	Enhanced (root), Reduced (shoot)	[113]
ERF	Arabidopsis	Cd	AtERF1b, AtERF104	Promotes nitrate assimilation and photosynthesis	/	/	NRT1.8, NRT1.5	Enhanced	/	[107]
HSF	Sedum	Cd	SaHSFA4c	Upregulates antioxidant enzymes and heat shock proteins (HSPs), reduces ROS accumulation	Root, leaf, stem	Induced	/	Enhanced	/	[114]
ZAT	Arabidopsis	Cd	ZAT17	Induces IRT1 and NRT2.1 expression; interacts with MAC splicing complex (PRL1, CDC5), affecting alternative splicing (AS) of multiple Cd response genes	Root, leaf	Induced	IRT1, NRT2.1, HMA3, PCR1	Reduced	/	[108]
ERF	Common bean	Cd	PVMTF-1	Activates tryptophan biosynthesis	/	Induced	AsA2	Enhanced	/	[109]

3. Practical Applications of Transcription Factors for Reducing Cd Accumulation in Rice

3.1. Genetic Engineering Strategies: From Direct Effectors to Network Regulators

Genetic engineering offers a dual-track strategy for developing low-Cd rice cultivars: the precise modification of downstream functional genes, or the systemic reconfiguration of master-regulatory networks [60,115].

3.1.1. Precision Engineering of Structural and Functional Genes

The most direct approach involves manipulating functional genes—primarily encoding transport proteins and chelators—to alter specific physiological processes. For example, OsNRAMP5 knockout reduces Cd uptake while promoting root-to-shoot translocation. The impact on shoot Cd accumulation depends on environmental Cd concentration: at low Cd (0–5 μM), knockout significantly reduces uptake and accumulation; at high Cd (10–15 μM), reduced uptake is less pronounced but translocation persists, increasing shoot Cd. Regardless, OsNRAMP5 knockout enhances Cd tolerance [116]. OsLCT1 transports various cations; RNAi-mediated OsLCT1 knockdown reduces phloem-mediated Cd transport to grains, decreasing grain Cd by 50% without compromising growth or essential metal content [117]. Defensins enhance metal tolerance; rice CAL1 encodes a defensin-like protein that chelates Cd via cysteine residues and secretes it extracellularly, reducing cytosolic Cd while facilitating Cd loading into xylem sap. CAL1-Cd complexes cannot enter phloem, resulting in preferential Cd accumulation in stems/leaves and low grain Cd [118]. OsTHi9 overexpression enhances cell wall Cd binding, reduces root-to-shoot translocation and grain Cd accumulation without compromising grain quality [118].

3.1.2. Systemic Reprogramming via Transcription Factors (TFs)

Unlike single transporters, TFs can reconfigure entire defense networks simultaneously. Recent studies show that TFs coordinately modulate Cd allocation and accumulation. For example, OsNAC5 positively regulates Cd tolerance; osnac5 mutants show significantly reduced Cd in grains and husks, while complementation restores near-WT levels [16]. OsWRKY72 negatively regulates OsGLP8-7, reducing lignin synthesis and weakening cell wall Cd binding, increasing cytosolic Cd accumulation. oswrky72 mutants exhibit lower whole-plant Cd concentrations and reduced root Cd uptake, whereas overexpression lines show higher uptake [119]. Cd-inducible OsHB4 overexpression increases Cd translocation factor, elevates xylem sap Cd, and enhances shoot/grain Cd accumulation. Conversely, OsHB4 RNAi lines accumulate more Cd in roots and less in shoots, with lower grain Cd [120]. Uclacyanin (UCL23) negatively regulates Cd tolerance by elevating ROS. OsWRKY51 activates the UCL23 promoter while suppressing its post-transcriptional repressor miR528, increasing ROS and reducing Cd tolerance. OsWRKY51 overexpression elevates Cd in all tissues, whereas knockout reduces grain Cd by 36% without affecting essential elements or agronomic traits [121].

3.2. Advantages of TFs as Master Regulators: The “Added Value” for Rice Improvement

While transgenic approaches targeting specific genes successfully reduce grain Cd accumulation, single-gene manipulations often fail to address the complexity of plant Cd detoxification networks and may cause unintended effects. TFs function as master regulators of gene expression networks, offering distinct advantages:

(1) Coordinated multi-gene regulation: A single TF can activate or suppress multiple functional genes, forming synergistic networks that comprehensively regulate Cd uptake, transport, detoxification, and accumulation. This “one-to-many” characteristic makes TFs pivotal for deciphering complex stress adaptation mechanisms [122]. For example, ANAC004 overexpression enhances root hemicellulose-mediated Cd binding while regulating multiple detoxification genes, reducing Cd influx, promoting chelation/vacuolar sequestration, elevating ABA levels, and boosting antioxidant enzyme activity [68]. Similarly, ZmbHLH105 enhances ABA biosynthesis, lignin deposition, and cell wall thickening while inhibiting root Cd uptake [71].

(2) Key signaling nodes: TFs reside at critical junctions between stress signal perception and physiological responses. Studying them illuminates early Cd signaling pathways. For instance, AtMYB59 negatively regulates growth and tolerance via Ca² homeostasis and signaling pathways [95]. Researching such “molecular switches” elucidates the complete “Cd perception-signaling-response” pathway.

(3) Cross-stress and developmental coordination: TFs optimize resource allocation and coordinate multi-stress responses. Many Cd-responsive TFs also regulate salinity/drought pathways. For example, OsNAC5 enhances Cd tolerance and activates OsCCR10, increasing root lignin and improving drought tolerance [123]. OsNAC15 enhances Cd tolerance while interacting with ABA biosynthesis genes, conferring drought and salinity tolerance [124]. This multifunctionality enables plants to integrate internal and external signals for maximal adaptive fitness.

3.3. TF-Driven Genotypic Variations in Cd Distribution

The genetic potential of TFs is naturally reflected in the genotypic variations among rice cultivars. Significant diversity in Cd accumulation and translocation provides a natural template for identifying key regulatory nodes. Comprehensive field and pot experiments show that Cd concentrations in rice organs (roots, stems, leaves, grains) can vary more than eightfold across cultivars [125]. Physiologically, resistant genotypes (e.g., ‘Xiushui 817’) exhibit a superior capacity to restrict Cd root-to-shoot mobility compared to susceptible ones (e.g., ‘Zheda 821’), especially under combined heavy metal stress [11]. These studies highlight that grain Cd is determined not only by total uptake but, more critically, by the root-to-shoot translocation factor and the stem-to-grain redistribution ratio during different growth stages [11,126].

Molecularly, these phenotypic variations are orchestrated by differential TF expression or activity. In resistant genotypes, Cd-responsive TFs (e.g., NAC, WRKY, MYB) are often induced more rapidly or exhibit higher baseline expression, leading to robust activation of antioxidant systems and transporter genes. For instance, in high-Cd-accumulating cultivars, TFs may more effectively upregulate OsHMA3, OsCCX2, OsNRAMP5, and OsHMA9 to facilitate Cd uptake and translocation. Conversely, in low-Cd accumulators, TFs likely upregulate genes like OsIRT1, OsPCR1, and OsMTP1 to hinder Cd absorption [126]. Furthermore, TF interactions with multiple metal ions (Cd, Pb, Cr, Cu) appear genotype-specific, suggesting that some varieties possess more sophisticated TF-mediated networks capable of integrating diverse stress signals to maintain mineral homeostasis [11]. Understanding these natural variations is essential for identifying superior alleles and devising precision breeding strategies to develop “safe” rice varieties for contaminated soils.

4. Summary and Future Perspectives

This review comprehensively explores plant Cd detoxification mechanisms and the pivotal regulatory roles of TFs (Figure 2). Upon root exposure, Cd is initially bound by negatively charged groups in the cell wall. The residual Cd that enters cells via non-selective ion channels triggers cytotoxicity. Elevated Cd and reactive oxygen species (ROS) then activate diverse TFs, which mitigate toxicity by coordinately regulating: (1) Restriction of root uptake through enhanced Cd binding in the cell wall. (2) Modulation of transporters to inhibit Cd influx, promote efflux, and reduce root-to-shoot translocation. (3) Chelation and sequestration via the upregulation of phytochelatin/metallothionein synthesis and vacuolar compartmentalization. (4) ROS scavenging by enhancing enzymatic and non-enzymatic antioxidant systems. (5) Damage repair and systemic coordination by facilitating DNA/protein repair, nitrate assimilation, tryptophan/hormone synthesis, and integrating stress signaling pathways.

These interconnected mechanisms constitute a dynamic, TF-coordinated network. However, to bridge the gap between mechanistic discovery and practical crop improvement, future research must evolve from characterizing isolated regulatory modules toward a systems-level roadmap. This holistic vision is supported by three emerging technological pillars:

(1) Unveiling Precision Breeding Targets via Multi-omics Integration

The deep integration of multi-omics technologies—including phenomics, genomics, transcriptomics, proteomics, and metabolomics—is advancing Cd resistance research from single-gene characterization toward a systems-level “safe-by-design” breeding framework [127]. This approach enables the identification of “hub” TFs that orchestrate Cd-response networks, effectively overcoming functional redundancy within TF families. For example, multi-omics-based co-expression networks have revealed that mitostasis (mitochondrial homeostasis) is a conserved core hub in cross-species Cd responses, with bHLH family members acting as central regulators that coordinate ROS scavenging and energy metabolism to maintain cell viability [128]. High-resolution multi-omics further delineates tissue- and genotype-specific strategies: rice root tips exhibit higher Cd accumulation and greater abundance of transporters (e.g., OsNramp1, OsNramp5) compared to mature zones, reflecting TF-mediated dynamic regulation in specific tissues [129]. Comparative proteomics between cultivars (e.g., Indica and Japonica) shows that tolerant varieties maintain homeostasis by enhancing antioxidant defense and energy metabolism proteins [130]—differences ultimately orchestrated by variations in TF expression or binding affinity. In summary, the key TFs and metabolic modules revealed through multi-omics integration provide high-confidence targets for precisely screening low-Cd germplasm and designing ideal varieties with combined tolerance and low accumulation via genome editing.

(2) Decoding the Multidimensional Regulatory Hierarchy via Epigenetics

Plant responses to Cd are profoundly shaped by epigenetic modifications—chemical alterations to DNA and associated proteins (e.g., histones) that regulate gene expression without changing the DNA sequence. These modifications, including DNA methylation, histone modifications, non-coding RNAs (ncRNAs), and microRNA-encoded peptides (miPEPs), cooperatively form a multidimensional network that fine-tunes Cd accumulation and tolerance. For instance, the Microrchidia protein OsMORC6 dynamically regulates Cd uptake and tolerance by mediating DNA methylation; its knockout alters differentially methylated regions (DMRs) and affects cell-wall and redox-related genes, thereby reducing rice Cd tolerance [131]. ncRNAs and their derived peptides extend this regulation post-transcriptionally, often using TFs as functional hubs. Cd-induced miR535 negatively regulates accumulation by targeting the TF OsSPL7, which derepresses transporters such as OsNRAMP5 [132]. Conversely, the peptide miPEP156e, encoded by a primary miRNA, enhances miR156 expression to inhibit SPL TFs, reducing Cd influx transporters (e.g., OsNRAMP5, OsZIP1/3/9) while improving ROS scavenging [133]. In rice roots, lncRNAs respond to Cd stress by activating MAPK signaling, diterpenoid biosynthesis, or phenylpropanoid pathways, and interact with TFs to modulate hormone signaling [134]. This hierarchical framework—integrating epigenetic marks, ncRNAs, peptides, and core TFs—provides a novel theoretical basis for designing multidimensional molecular modules to develop high-performance, low-Cd rice varieties.

(3) Precise Spatiotemporal Control of Cd Responses via Promoter Engineering

Future research must shift from constitutive overexpression to precise spatiotemporal control. A key strategy is developing inducible promoters that drive gene expression only under specific stimuli (e.g., hormones, chemicals, or Cd stress). These promoters contain cis-regulatory elements (CREs)—such as metal response elements (MREs), copper response elements (CuREs), or G-boxes—that recruit trans-acting factors under defined conditions. Such “molecular switches” maintain low baseline expression to minimize metabolic cost, while enabling strong, Cd-specific induction to precisely activate detoxification pathways [60].

Complementing inducible systems, tissue-specific promoters can sequester Cd in “gatekeeper” tissues to block grain translocation. A definitive example is expressing the vacuolar transporter OsHMA3 under the root-, node-, and stem-enriched OsHMA2 promoter. This targeted expression intercepts Cd at critical entry and redistribution checkpoints, reducing brown rice Cd to <10% of wild-type levels without compromising yield or mineral nutrition [135].

Moving forward, research should integrate these spatiotemporal strategies—e.g., using root- or phloem-specific promoters—to ensure precise localization of gene expression. Moreover, leveraging RNA-seq to identify endogenous rice promoters that respond robustly and specifically to Cd stress, and using them to drive functional genes, is a highly promising direction. This approach not only optimizes detoxification efficiency but also avoids the instability and ethical concerns associated with exogenous strong promoters, offering a sustainable and socially acceptable molecular toolkit for developing high-yielding, “Cd-safe” rice varieties.

Figure 2. Schematic Diagram of Cd Uptake, Detoxification, and Regulatory Network in Plant Cells. Under Cd exposure, plants activate specific transcription factors (TFs) via signal transduction pathways, which precisely regulate the expression of downstream functional genes, forming a coordinated multi-modular core defense network. Key components of this network include: the antioxidant system, which alleviates oxidative damage by enhancing the activities of enzymes such as SOD, POD, and CAT, along with the synthesis of non-enzymatic antioxidants; chelation and compartmentalization, involving the production of phytochelatins (PCs) and metallothioneins (MTs), and the sequestration of Cd into vacuoles or its immobilization in the cell wall; regulation of Cd uptake and transport, through inhibition of Cd absorption and promotion of its efflux; as well as signaling cascades and repair of DNA and protein damage. Through the synergistic action of these finely tuned processes, plants achieve effective Cd detoxification and accumulation control, thereby enhancing tolerance while ensuring agricultural product safety. Created in BioGDP. com. Jiang, S. (2024) https://base-img.rjmart.cn/image/diagram/cacgbc/42b020ba-f26c-4cc5-8bf2-6ee5761d29f1.png (accessed on 12 January 2026) [136].

Figure 2. Schematic Diagram of Cd Uptake, Detoxification, and Regulatory Network in Plant Cells. Under Cd exposure, plants activate specific transcription factors (TFs) via signal transduction pathways, which precisely regulate the expression of downstream functional genes, forming a coordinated multi-modular core defense network. Key components of this network include: the antioxidant system, which alleviates oxidative damage by enhancing the activities of enzymes such as SOD, POD, and CAT, along with the synthesis of non-enzymatic antioxidants; chelation and compartmentalization, involving the production of phytochelatins (PCs) and metallothioneins (MTs), and the sequestration of Cd into vacuoles or its immobilization in the cell wall; regulation of Cd uptake and transport, through inhibition of Cd absorption and promotion of its efflux; as well as signaling cascades and repair of DNA and protein damage. Through the synergistic action of these finely tuned processes, plants achieve effective Cd detoxification and accumulation control, thereby enhancing tolerance while ensuring agricultural product safety. Created in BioGDP. com. Jiang, S. (2024) https://base-img.rjmart.cn/image/diagram/cacgbc/42b020ba-f26c-4cc5-8bf2-6ee5761d29f1.png (accessed on 12 January 2026) [136].