Jin-Song Luo ,Zhenhua Zhang ,*

a College of Resources and Environmental Sciences,Hunan Agricultural University,Changsha 410128,Hunan,China

b Hunan Provincial Key Laboratory of Farmland Pollution Control and Agricultural Resources Use,Hunan Provincial Key Laboratory of Nutrition in Common University,National Engineering Laboratory on Soil and Fertilizer Resources Efficient Utilization,Changsha 410128,Hunan,China

Keywords:Uptake Translocation Vacuolar sequestration Chelation

ABSTRACT As a consequence of industrial development,soil Cd pollution leads to crop contamination by Cd,posing a threat to food safety and human health.Excessive accumulation of Cd in plants also inhibits their growth via oxidative stress damage to their photosynthetic systems.Through evolutionary selection,plants have developed a set of efficient strategies to respond to Cd in their environments.These include the accumulation and detoxification of heavy metals.Cd is absorbed by plant roots through the apoplastic and symplastic pathways and then translocated to plant shoots via xylem loading,long-distance transport,and phloem redistribution.Simultaneously,plants initiate a series of mechanisms to reduce Cd toxicity,including cell wall adsorption,cytoplasmic chelation,and vacuolar sequestration.This review summarizes current knowledge of Cd accumulation and detoxification in plants.

1.Introduction

Soil is an integral part of the ecological environment and is necessary for human survival.With the rapid development of modern industrialization,the use of industrial wastewater,extensive agricultural fertilizer use,and accumulation of household waste leads to pollution.Cadmium(Cd)pollution in soil is a prominent example.Remediation of Cd-contaminated soil is critical to the sustainable development of agriculture,especially in parts of China.

Cd is a toxic element with no biological function in most living organisms,except in the marine diatom Thalassiosira weissflogii,in which it acts as a cofactor for carbonic anhydrase[1].In humans,Cd toxicity caused by contaminated water and food causes various physical problems,such as cancer,heart disease,vascular problems,and kidney and liver poisoning,and affects the human male reproductive system,especially sperm motility and hormone synthesis and release[2,3].In plants,Cd toxicity results in leaf chlorosis, reduced growth rates, inhibition of respiration and photosynthesis,increased oxidative damage,and reduction in nutrient uptake ability[4-6].

Over the two last decades,using various methods of plant physiology and biochemistry,plant molecular genetics,including forward and reverse genetics,and omics technologies including genomics, transcriptomics, proteomics, and metabolomics,numerous studies have investigated the physiological and molecular mechanisms of Cd accumulation and detoxification in plants,with particular focus on identifying genes responsible for Cd uptake,translocation,sequestration,and tolerance in plant tissues[7].One potential application of these studies is in breeding crop cultivars with low Cd accumulation in their edible parts[8].Another application is in cultivating plants with high Cd accumulation ability and tolerance for effective and economical remediation of contaminated land.In this review,the molecular mechanisms of Cd accumulation and tolerance in Arabidopsis thaliana and rice are summarized.

2.Cd remediation strategies

Physicochemical remediation and bioremediation represent the two main types of current remediation methods for Cdcontaminated soil.Physicochemical remediation employs mainly physical principles and specific chemical agents to remove Cd from soil.Various redox,adsorption,and complexation reactions of Cd,reduce its bioavailability and thus its toxic effects.Physicochemical remediation is simple;however,because of its high cost or inability to remove Cd completely from soil,its application is limited.Bioremediation involves biologically enriching Cd-contaminated ecosystems to remove contaminants from the soil.Bioremediation methods include phytoremediation,in which metal pollutants of soil such as Cd can be absorbed by natural or deliberately bred special plants through their roots to remove or reduce theirbioavailability[9,10].Such methods are economically feasible,environmentally friendly,and highly applicable.Phytoremediation includes plant extraction,plant stabilization,plant filtration,and plant stimulation[11,12].

Plant extraction is the transfer of heavy metal elements from the soil to the shoot of hyperaccumulating plants,thus reducing soil Cd[10,13].Plant stabilization refers to the use of plant roots through collateral binding,adsorption,and other actions to fix heavy metals in the soil,thus preventing their diffusion in the environment[14].Plant filtration includes rhizome filtration,such as embryo filtration (using seedlings)and caulofiltration (using excised plants)[15].Finally,plant stimulation promotes the phytoremediation process by stimulating roots to release compounds that enhance microbial activity.These exudates promote microbial growth by meeting the nutrient requirements of the organism.This process is used as a rhizosphere repair technique,a low-cost method for removing Cd and other organic compounds[16].

Cd-hyperaccumulator plants have application prospects in phytoremediation.To date,five Cd hyperaccumulator plant species with high-efficiency Cd detoxification ability have been found.Cd concentration in their foliar tissue is generally greater than 300μg g-1dry weight.In a recent review[17],in hyperaccumulating plants,the constitutively high expression of genes responsible for Cd uptake,long-distance transport,chelation,and vacuolar sequestration,or natural variation,determines its hyperaccumulation characteristics and supertolerance to Cd.

3.Accumulation of Cd in plants

Cd is present in soil mainly in insoluble form and has no bioavailability for plants.However,plants can increase Cd solubility by releasing root exudates that change the pH of the rhizosphere[18].Cd uptake occurs mainly through the apoplastic and symplastic pathways.The apoplastic pathway is a process of passive diffusion.The symplastic pathway is an active-transport and energy-consuming plasma-membrane transport process that requires electrochemical potential gradients and concentrations[19].When Cd enters the root,it forms a complex with various chelating agents.These complexes are immobilized in the cell wall,cytoplasm,or vacuoles,thereby losing toxicity[10].Cd storage in root vacuoles reduces its toxicity and long-distance transport to the shoot[20].Cd is sequestered in shoots,and detoxification occurs in cell walls or plant vacuoles[21].

Cd has no known biological activity in most living organisms,although its accumulation,like that of Zn and Ni,is thought to be a defense against herbivores in hyperaccumulating plants[22].Accordingly,during evolution,it is unlikely that transport systems specific to non-essential or even highly toxic metals such as Cd would be selected.Cd may be absorbed by the plasma membrane transporters of essential elements in roots,which have low substrate selectivity[23].Fig.1A and B show schematic diagrams of Cd transport in A.thaliana and rice roots,respectively.

3.1.Cd uptake transporters in plants

The Cd transporters identified to date have been divided into several families based on their sequence similarity.They include zinc-regulated transporters,iron-regulated transporter proteins(ZIP),natural resistance-associated macrophage proteins(NRAMP),and metal tolerance proteins(MTP).

Transporters from the plant ZIP family are involved in the accumulation of heavy metals,including the absorption and transport of many cations from the root to the shoot[24].For example,AtIRT1 is expressed mainly in the root epidermis,with lower expression in the root cortex.Under iron-deficient conditions,Cd accumulation in irt1 knockout mutants is greatly reduced[25].OsIRT1 and OsIRT2 were induced in roots with iron deficiency and showed Cd transport activity when expressed in yeast.Thus,iron deficiency increases the expression of these transporters and promotes Cd absorption and transport[26,27].IRT1 too may be involved in Cd absorption in the hyperaccumulator plant Noccaea caerulescens[28,29].Recently,Tan et al.[30]showed that in rice,double mutations of two zinc transporter genes,OsZIP5 and OsZIP9,expressed mainly in the cortex and located on the cell membrane,reduced plant uptake of Zn and Cd.These results suggest that these genes synergistically regulate the uptake of Zn and Cd.

The transporters of the NRAMP family are also involved in plant Cd uptake and transport.For instance,AtNRAMP3/4 is responsible for Fe,Mn,and Cd efflux from vacuoles in A.thaliana[31-33].In rice,the Fe transporter OsNRAMP1 is involved in uptake and transport of Cd and Mn[34,35].In contrast,OsNRAMP5 is a transporter that is responsible mainly for the uptake of Mn and Cd in rice roots,and the content of Mn and Cd in nramp5 mutant roots and shoots was significantly lower than that in wild-type rice[36,37].OsMTP8 and OsNRAMP5 synergistically control the Mn uptake and translocation process.However,OsMTP8 has no effect on Cd transport[38].The expression of wheat TpNRAMP5 in Arabidopsis increased Cd,Co,and Mn accumulation but had no effect on Zn and Fe accumulation[39].MicroRNA166 and RING E3 ligase OsHIR1 also regulate Cd uptake and accumulation in rice[40,41].

3.2.Cd translocation and reallocation

Heavy-metal ATPase(HMA)transporters are r esponsible for long-distance transport of Cd from root to shoot.They hydrolyze ATP and expel Cd from the cytoplasm to the apoplast along an electrochemical gradient.For instance,three plasma membranelocalized proteins:AtHMA4,AtHMA2,and OsHMA2,are responsible for mediating the influx of Cd into the stele to facilitate rootto-shoot transport of Zn and Cd in Arabidopsis and rice[42-47].Accordingly,the increased expression and the triplication of HMA4 are responsible for the supertolerance and hyperaccumulation of Cd in A.halleri and N.caerulescens[48-50].

CAL1 and CAL2,which are defensin-like proteins in rice,can chelate cytoplasmic Cd to form complexes and excrete them to the xylem sap via long-distance transport,thereby regulating the accumulation of Cd in the shoot[51,52].Recently,it was reported[53]that a major facilitator superfamily transporter OsCd1,which is expressed mainly in the root cortex and located on the cell membrane,mediates the differential Cd accumulation observed in the kernels of indica and japonica rice.Cd in the rice shoot was distributed to the grain mainly through OsHMA2,an Oryza sativa low-affinity cation transporter 1(OsLCT1),OsZIP7,PCR1,LCD,OsPCS1,OsPCS2,and OsCCX2 proteins[54-61].OsLCT1,expressed mainly at stem nodes,was responsible for phloem Cd transport into grain in a standard japonica cultivar[55].Fig.1C shows a schematic diagram of transporter participation in Cd distribution in the shoot.

3.3.Vacuolar sequestration of Cd

Vacuoles are storage organelles for many ions.Several transporters can transport free Cd and phytochelatin(PC)-Cd complexes to the vacuoles.They include ATP-binding cassette transporters(ABCCs),NRAMPs,H+/cation exchangers(CAXs),and HMAs,which have previously[31,62,63]been investigated.Among these,three ABCC vacuolar membrane transporters,ABCC1,ABCC2,and ABCC3,play an important role in PC-Cd complex sequestration[62,63].Two NRAMP tonoplast transporters,NRAMP3 and NRAMP4,play a key role in the efflux of free Cd from the vacuole to the cytosol[31].Among CAXs,AtCAX2 and AtCAX4 are tonoplast-localizedtransporters,which not only are specific to Ca but can transport other metals,including Cd,by using the proton gradient to control Ca and Cd storage in the vacuole[64].The Cd hyperaccumulator A.halleri has shown increased Cd tolerance with increased expression of AhCAX1[4].As for HMAs,AtHMA3 is responsible for the vacuolar storage of Cd and has been found by Genome wide association study(GWAS)[65,66]to be a primary determinant of leaf Cd natural variation in A.thaliana.In rice,OsHMA3 can also sequester Cd into vacuoles;natural variation of a single amino acid mutation of OsHMA3 can explain the difference in Cd accumulation between the two rice cultivars[67,68].Higher NcHMA3 and SaHMA3 expression is associated with Cd hyperaccumulation in N.caerulescens and Sedum alfredii[69,70].Recently,Liu et al.[71]reported that SpHMA3 is responsible for Cd vacuolar sequestration and detoxification in young leaf cells of Sedum plumbizincicola plants.

4.Cd detoxification

Under the stress of Cd accumulation,plants initiate a series of mechanisms to reduce Cd toxicity,including vacuolar separation,cytoplasmic chelation,and cell-wall detoxification.Fig.2 presents a schematic diagram of Cd detoxification in plant cells.

Fig.2.Schematic overview of Cd detoxification in plant cells.PC,phytochelatin;MT,metallothioneins;PDF,plant defensins;ROS,reactive oxygen species.The detoxification mechanism of Cd in plant cells includes cell wall adsorption to prevent it from entering the cell,cytoplasmic chelation mediated by defensin and metallothionein,vacuolar compartmentation and cytosolic Cd efflux to the apoplast,and activate reactive oxygen signal pathway to eliminate oxidative stress.

4.1.Glutathione and phytochelatin

Glutathione(GSH)and phytochelatin(PC)are non-protein peptides involved in the detoxification of Cd and maintenance of cell redox balance[72].PCSare small cysteine-rich peptides with basic structure(γ-Glu-Cys)nGly;with n ranging from 2 to 11.They are synthesized by GSH as a substrate under the action of phytochelatin synthase (PCS)[73-75].It is currently believed[62,76,77]that the main mechanism of PC-mediated Cd detoxification is chelation by PCs to form a complex,which is then transported into the vacuoles by ABC transporters.In rice,ABCG36,ABCG43,and OZT1 have been reported[78-80]to mediate Cd tolerance,but the molecular mechanism is unknown.

4.2.Metallothionein

Metallothionein(MT)is a small peptide with a cysteine residues content of 15%-30%.MT has metal-binding activity and is widely found in eukaryotes and cyanobacteria[81].It is involved in the balance of and tolerance to Zn,Cu,and Cd in organisms[82-85].Lately,Peng et al.[86]suggested that the elevated transcript levels and natural variation in protein sequences of MT-like protein SpMTL,which acts as a cytoplasmic Cd chelation protein,is responsible for Cd hyperaccumulation and hypertolerance in the hyperaccumulator S.plumbizincicola.The MT-like gene DcCDT1 in Digitaria ciliaris and its rice homolog,OsCDT1,when heterologously expressed in yeast cells and A.thaliana,increases Cd tolerance by reducing cytoplasmic Cd content[87].HsfA4a of wheat and rice increased Cd tolerance by upregulating MT gene expression[88].

4.3.Plant defensins

Investigations over the past few decades have revealed that another class of cysteine-rich molecule plant defensins play an important role in Cd allocation and detoxification.In vitro experiments showed that human defensin 5(HD5)had Zn and Cd chelating activity[89].Heterologous expression of plant defensin gene type 1(PDF1)increased Zn tolerance,but not Cd tolerance,in yeast and plants[90-93].Recently,Luo et al.[94]identified the function of the Arabidopsis defensin AtPDF2.5,which is located in the cell wall of xylem vascular bundles,mediates cytoplasmic Cd chelation and excretes the AtPDF2.5-Cd complex to the apoplast.Loss of AtPDF2.5 function reduced Cd tolerance and accumulation in Arabidopsis.Subsequent reports[95,96]suggested that functional disruption of AtPDF2.2,AtPDF2.3,and AtPDF2.6 increased Cd sensitivity in Arabidopsis.Xylem sap proteomic studies identified a plant defensin-like protein BnPDFL,which is present uniquely in Cd-treated samples and may increase the Cd tolerance of rapeseed(Brassica napus)[96].

4.4.Cell wall

The cell wall is composed mainly of cellulose,hemicellulose,and pectin.Current theory[97,98]holds that electronegative sites on cell wall components enable cations to be adsorbed to the cell wall.Previous studies[99-101]have shown that the cell wall increases plant Cd tolerance by preventing Cd from entering root cells.Pectin is the main component of the cell wall that binds to heavy metals[101-103].Transcriptomic and physiological analyses[100]have also indicated the importance of the role of cell walls in Cd hyperaccumulation and detoxification in S.plumbizincicola.Recently[104,105],our research group has found that boron alleviates Cd toxicity to rapeseed by increasing Cd chelation onto cell walls in shoots and roots.A multi-omics approach[106]revealed the pivotal role of the cell wall in determining rapeseed resistance to Cd toxicity.Other physiological experiments have shown[107]that cell-wall polysaccharide sorption accounts for Cd-tolerance differences between two A.thaliana ecotypes.

5.Interaction of Cd with nitrates and silicon

Cd uptake and detoxification by plants are affected by nitrates and silicon(Si).Nitrogen speciation affects Cd root uptake.Indeed,under hydroponic conditions,nitrates promoted Cd uptake and accumulation in N.caerulescens,Solanum lycopersicum,andS.alfredii more than supplied NH+4[108-110].Cd stress induced the expression of the low-affinity nitrate transporter AtNRT1.8,which removes nitrate from the xylem sap,regulating nitrate distribution to the root and increasing Cd tolerance in Arabidopsis[111].In contrast,Chen et al.[112]reported that a loss of function of the longdistance nitrate transporter AtNRT1.5 increases Cd tolerance.Our latest study[113]has revealed that the double-affinity nitrate transporter NRT1.1 regulates nitrate allocation to increase plant Cd stress tolerance.Liao et al.[114]suggested that plants with high nitrogen use efficiency and Cd accumulation capacity can be achieved by inhibiting the activity of vacuolar transporter CLCa and increasing the activity of CAX.Cd inhibits NRT1.1-mediated NO-3uptake and promotes Cd detoxification by reducing Cd entry into the root system [115].These results indicate crosstalk between Cd and nitrate uptake and translocation.

Si reduces the toxicity of Cd to plants[116].It reduces the availability of Cd in soil and reduces the absorption and/or translocation of Cd to plants[117,118].Si promoted the adsorption and complexation of Cd,restored the redox balance,and increased the antioxidant capacity of plants[119].

6.Strategies to reduce Cd pollution

6.1.Phytoremediation

Phytoremediation of Cd-contaminated soil is a common method of reducing Cd.Hyperaccumulating plants usually have welldeveloped roots with the capacity to adsorb high levels of heavy metals present in the soil and transfer them to aboveground parts[86,100].Sedum alfredii,Phytolacca americana,Arabis gemmifera,and Prosopis laevigata can hyperaccumulate Cd to above 2000 mg kg-1in shoots[120-124].Such plants could be used to remediate contaminated soils.

Maintaining a balance between food production and environmental remediation has become a global challenge in the face of soil contamination with toxic heavy metals.In China,food security is an overriding issue,and moderately polluted land needs to be used to produce food instead of being remediated.In this case,an effective and economical solution is to grow remediation crops that extract heavy metals from the soil and accumulate them in straw rather than grain.Such remediation crops can remediate the surrounding habitat while producing safe,nutritious grain[51].Recent research[51]shows that CAL1 can specifically regulate Cd accumulation in rice straw without affecting Cd accumulation in grain,providing a theoretical basis for the development of remediation rice.

6.2.Breeding low Cd-accumulating rice

There is wide variation in Cd content among rice germplasm resources.Usually,Cd accumulation in indica is higher than that in japonica rice cultivars,determined mainly by differences in xylem Cd long-distance transport from root to shoot[125-129].In some high-yielding inbred lines and hybrid rice,differences in Cd content in rice grain were determined mainly by genetic diversity.Cultivars with low Cd accumulation in grain include Shenyou 957,Longping 602,Weiyou 402,Weiyou 463,Zhuliangyou 168,TYou 535,Jiefengyou 1,and I-You 899,providing valuable material for planting in Cd-contaminated farmland or for breeding[125,130,131].Many quantitative trait loci(QTL)controlling Cd concentration in grain have been identified by map-based cloning or GWAS[132-139].These QTL can be used to select low Cdaccumulating indica-hybrid lines via molecular-marker-assisted breeding[140],and provide valuable information for cloning functional genes.In the last decade,three major genes(HMA3/OsCD1/CAL1)that control Cd-accumulation QTL in rice have been identified[51,53,67].Table 1 summarizes the currently identified genes that regulate Cd accumulation and tolerance in rice and provide genetic resources for breeding low-Cd or remediation rice cultivars.By regulation of the expression of known genes using genetic engineering methods,the uptake or translocation of Cd to cereal crops can be reduced.In one study[36],three low-cadmium mutants of non-transgenic rice were generated by heavy-ion beam irradiation,the target gene OsNRAMP5 was identified,and a DNAmarker was developed for further breeding.Functional disruption of the OsNRAMP5 gene by the CRISPR/Cas9 system in both parental lines of a rice hybrid significantly reduced Cd concentrations in the grain[143].Overexpression of a functional allele of OsHMA3 dramatically reduced Cd concentration in grain with minimal side effects[67,144,145].In summary,conventional hybridization,mutation breeding,genetic engineering,and molecular markerassisted selection can be used for breeding low Cd-accumulating rice.

Table 1Summary of identified genes involved in Cd accumulation and detoxification in rice.

7.Conclusions and perspectives

In recent decades,knowledge of Cd accumulation and detoxification mechanisms in plants has improved considerably.Numerous studies have focused on the distribution of Cd in plant tissues and organs and on transporters that control plant uptake and long-distance transport from root to shoot.The molecular mechanisms of Cd transport to the fruit are not well understood.Future work may reveal the mechanism by which Cd is distributed in the aboveground parts of plants,especially the fruits and seeds.

At present,studies of Cd accumulation and tolerance mechanisms focus mainly on the model plants A.thaliana and rice.It is desirable to broaden their scope to other food crops such as maize,wheat,and barley.Because Cd accumulation in plants is mediated by transporters of essential elements,it is difficult to increase or decrease the content of heavy metal Cd in crops simply by manipulating transporters,without impairing the plant’s ability to absorb essential elements.Targets for future research include the crystal structure of plant Fe/Zn/Mn transporters,identification of desirable allelic variants,and design of a protein specific for substrate transport.

CRediT authorship contribution statement

Jin-Song Luocontributed to the conception of the review and drafted the manuscript.Zhenhua Zhangrevised the manuscript.All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was financially supported by the Province Key R&D Program of Hunan(2018NK1010),the National Natural Science Foundation of China(31800202),the National Oilseed Rape Production Technology System of China,and the Natural Science Foundation of Hunan Agricultural University(19QN38).