* Correspondence:
G.P. Zhang: zhanggp@zju.edu.cn
Running title: Advances in Cd accumulation by plants

ABSTRACT

Cadmium (Cd) contamination in the environment has become a hot issue worldwide, as it has posed a great risk to human health via food chain. Cd accumulation in the edible parts of crops is involved in four processes: uptake, translocation, sequestration and redistribution, which are all controlled by membrane transporters. In this review, advances in the studies on the physiological and molecular mechanisms of Cd accumulation in plants are summarized, and then the functional evolution of Cd accumulation in plants is discussed based on oneKP database. Plant Cd accumulation is a derived and polyphyletic trait that has evolved convergently several times. In the course of evolution, membrane transporter families, such as NRAMPs, HMAs, ABCCs, ZIPs, CDFs, CAXs and OPTs, have undergone the lineage specific expansion due to gene duplication. The orthologues of OsHMA2 and OsGLR3.4 in higher plants are stepwisely evolved from a common ancestor; whereas the orthologues of OsNRAMP5 evolved from a polyphyletic evolutionary lineage with several different ancestors. In addition, abundant genetic intermixing have occurred in the phylogenetic clusters of the orthologues of OsNRAMP5, indicating horizontal gene transfer from one species to another one. It may be concluded that such an evolution of Cd accumulation in plants could provide an adaptive advantage for colonization of plants to the novel habitats like metalliferous soil.
KEYWORDS: Cadmium, Evolution, Horizontal gene transfer, Xylem loading, Vacuole sequestration, Transporters

1 | INTRODUCTION

Cadmium (Cd) contamination in soil has become one of the most important environmental issues (Rai et al., 2019). It can be caused by many artificial activities, such as application of municipal sewage sludge, chemical fertilizers and pesticides containing Cd and mining (Clemens, 2019). It is estimated that about 30,000 tons of Cd pollutants are discharged into the environment annually, and 13,000 tons of them are produced from human activity (Gallego et al., 2012). Cd is easily taken up and accumulated in plant tissues, resulting in the reduced crop yield and harm to human health via food chain. (Ismael et al., 2019; Rai et al., 2019).
Cd accumulation in plants may occur through soil-plant, water-plant, and air-plant interfaces, with soil-plant interface being the most prominent (Shahid et al., 2017). There is a close association between soil Cd level and plant Cd content (Khan et al. 2015). In soil-plant system, Cd in the rhizosphere enters plant roots mainly in ionic forms, and is then translocated to above-ground tissues, including grains (Clemens and Ma, 2016). As a non-essential and toxic metal element, the excessive Cd accumulation in plants will cause various detrimental impacts on plants, such as reducing growth, inhibiting photosynthesis and respiration, interfering with biochemically- and physiologically-related signaling pathways, disturbing nutrients uptake and water relations, damaging cell membrane permeability and disrupting the cellular redox homeostasis etc., as a consequence resulting in yield reduction and even plant death (Shahid et al., 2017; Ismael et al., 2019). On the other hand, excessive intake of Cd by humans through consumption of agricultural products containing high Cd content will induce a variety of serious diseases, including kidney cancer, breast cancer anemia, heart failure, hypertension, cerebral infarction, proteinuria, eye cataract formation, osteoporosis, emphysema, and renal insufficiency (Clemens, 2019). It has been well known that crop food is the main source of Cd exposure for human, accounting for 90% of the total dietary intake (Khan et al., 2015). Therefore, minimizing Cd accumulation in plants, in particular the edible parts is crucial for alleviating its harm to human health. So a comprehensive understanding of the mechanisms underlying the accumulation in plants is imperative for developing the crop cultivars with less Cd accumulation.
In the last decade, a great deal of research has been done in revealing the physiological and molecular mechanisms of Cd uptake and accumulation (Clemens, 2019; Clemens and Ma, 2016; Manara et al., 2018). A series of genes associated with Cd transport in plants have been identified and functionally verified (Clemens and Ma, 2016; Shahid et al., 2017; Ismael et al., 2019; Shi et al., 2019). Meanwhile, the specific plant genotypes or lines with extremely low or high Cd accumulation have been developed by genetic transformation and gene editing (Fasani et al., 2018). Moreover, there is increasing evidences indicating that Cd accumulation and tolerance in some plant species might have evolved under the selection pressure for their colonization in metalliferous soils or protecting them against herbivores or pathogens (Cappa and Pilon-Smits, 2014). However, there is no report on the relationship between physiological/molecular mechanisms of Cd accumulation and evolutionary adaptation. In this review, the advances in the studies on the physiological and molecular mechanisms of Cd accumulation in plants is summarized and the functional evolution of Cd accumulation in plants is also discussed in terms of the most important membrane transporter families mediating Cd accumulation.

2 | CADMIUM UPTAKE AND TRANSPORT BY PLANTS

The mechanisms of Cd accumulation in plants have been extensively investigated (Clemens, 2019; Clemens and Ma, 2016; Shahid et al., 2017; Ismael et al., 2019; Shi et al., 2019). In general, Cd accumulation in plants can be divided into 4 processes, including (1) root Cd uptake from soil, (2) root-to-shoot translocation via xylem, (3) Cd sequestration and (4) Cd accumulation in above-ground tissues or edible parts through phloem.

2.1 | Cd uptake by roots

Plants take up Cd from soil mainly by their roots, and the rate and amount of Cd uptake in roots are dependent on Cd bio-availability or concentration in soil (Clemens, 2006), and also controlled by genetic factors of plants. Cd uptake by plant roots consist of two phases: apoplastic binding and symplastic uptake (Zhao et al. 2002). In the first phase, the positively-charged Cd2+ arises an electrostatic interaction with the negatively-charged carboxylate groups on cell walls, resulting in Cd accumulation in the root apoplast (Meychik and Yermakov, 2001). It is rapid and spontaneous, suggesting no energy requirement in this phase (a passive system). In the second phase, Cd is symplasticly taken up in a slow active process, which requires a great deal of energy and is highly dependent on the metabolic activity (Sloof et al., 1995). Cd transport across the root cell plasma membrane is the initial step for its symplastic uptake (Yin et al., 2015). It is a concentration-dependent process, reflected by a saturation kinetics in the relationship of Cd uptake velocity and concentration in the medium, indicating that Cd is taken up via a carrier (transporter)-mediated system (Verbruggen et al., 2009). InArabidopsis and rice, for instance, many transporters from the families of Natural Resistance-Associated Macrophage Proteins (NRAMPs, such as OsNRAMP1, OsNRAMP5, and AtNRAMP6) and Zinc/Iron-regulated transporter-like Protein (ZIP, such as AtIRT1) transporters are responsible for Cd transport in root cells (Schaaf et al., 2004; Lux et al., 2011; Sasaki et al., 2012). In addition, Cd may also enter root cells through the transport pathway for Ca (White and Broadley, 2003; Li et al., 2012; Chen et al., 2018), because of their similarities in charge and ionic radius. Adding La3+ and Gd3+ (the potent Ca channel inhibitors) or increasing Ca concentration in the culture solution suppressed the metabolically-dependent Cd uptake substantially in the Zn hyperaccumulatorThlaspi caerulescens (Zhao et al. 2002), the halophyte Suaeda salsa (Li et al., 2012) and rice (Chen et al., 2018). Recently, one member of the major facilitator superfamily (MFS), OsCd1, was also detected to be associated with root Cd uptake in rice (Yan et al., 2019). However, although a great number of membrane proteins have been demonstrated to be involved in Cd uptake, little is known about transport of Cd across plant root plasma membranes at the molecular level.

2.2 | Root-to-shoot Cd translocation

After its uptake at the root epidermis or exodermis, Cd is radially transported across the cortex, endodermis, and pericycle cells, and then loaded into root xylem for the subsequent translocation from root to shoot via xylem (Lu et al., 2009; Ueno et al., 2008; Uraguchi et al., 2009b). Loading Cd into root xylem is a crucial step for Cd translocation to the aerial plant parts (Uraguchi et al., 2009b; Ismael et al., 2019). The radial movement of Cd toward the root xylem occursvia symplastic and/or apoplastic transport in the form of free Cd2+ or Cd-complexes with various chelates (Shahid et al., 2017). Symplastic transport is considered as an energy-consuming (positive) pathway involving in both influx and efflux transporters (Clemens and Ma, 2016). Apoplastic transport is a passive pathway, usually driven by transpiration (Qiu et al., 2012). It was suggested that symplastic transport play the dominant role in the radial transport of Cd (Lu et al., 2009; Ueno et al., 2008). However, a recent study onSedum alfredii found that apoplastic pathway contributed up to 37% of Cd transported to the xylem (Tao et al., 2017). After crossing the barriers from root epidermis to root cortex, either free Cd2+ or Cd-chelates may enter symplasm and are then loaded into root xylem (Gallego et al., 2012). The xylem loading of Cd in roots is an energy-consuming process because it occurs against membrane potential (Clemens and Ma, 2016), and is mediated by heavy metal P1B-ATPases (HMAs) and possibly also by YSL proteins (Lux et al., 2011; Ismael et al., 2019).

2.3 | Cd sequestration

Cd sequestration has a significant contribution to preventing Cd translocation, thereby reducing Cd accumulation in cereal grains. The main sites for Cd sequestration in plants are cell walls and vacuoles. Call wall is the “first line of defense” against toxic metals from the external environment. When captured by root cells, metal ions are largely bound by some components of cell walls, such as cellulose, hemicellulose, lignin, and pectin (Shi et al., 2019). As a result, the highest Cd concentration in root tissues occurs in apoplasm, particularly in the cell wall of rhizodermis and cortical cells (Lux et al., 2011). Such binding of Cd to cell walls can efficiently prevent Cd from being transported across the plasma membrane into protoplasts, thereby reducing Cd translocation between cells and tissues.
Within the root cells, Cd is mainly located in vacuoles, which are generally considered to be the main storage site for metals (Salt and Rauser, 1995). Wu et al. (2005) found that 51% of Cd in barley roots was present in the soluble fraction of vacuole, and 36% was present in cell wall. After entering aerial plant parts, Cd is also mainly sequestered in the vacuoles of parenchyma cells in the leaf mesophyll, stem pith, and cortex (Qiu et al. 2011; Tian et al., 2017). Obviously Cd sequestration into vacuoles plays a vital role in reducing Cd concentrations in cytoplasm and alleviating Cd injury (Shi et al., 2019). Several families of transporters have been identified to be responsible for vacuolar sequestration of Cd, such as HMAs, Ca2+ exchangers (CAXs), NRAMPs, MTPs and ATP-binding cassette subfamily C proteins (ABCCs) (Korenkov et al., 2007; Park et al., 2012; Sharma et al., 2016). It is generally assumed that Cd2+ in cytosol firstly form the low molecular weight (LMW) complexes by binding with metal ligands, such as glutathione (GSH), phytochelatins (PCs), metallothioneins (MTs), nicotianamine, organic acids and amino acids, and subsequently the complexes are transported into vacuoles, where more Cd2+ and thiol-containing chelators like GSH and PCs are incorporated to produce the high molecular weight (HMW) complexes (Zhang et al., 2018). In addition, the formation of Cd-malate complex in vacuoles is also found to reduce the subsequent Cd efflux from vacuole to cytoplasm (Ueno et al., 2005).

2.4 | Cd accumulation in shoots and grains

Cd accumulation in shoots and grains (the edible parts in cereals) is the final destination of Cd transportation, which consists of three processes, including xylem unloading, phloem translocation and inter-vascular transfer (Clemens and Ma, 2016). Like xylem loading of Cd in root, Cd xylem unloading in shoot also occurs via both apoplastic and symplastic transport (Clemens et al., 2002). Phloem translocation represents the main pathway of Cd accumulation in plant shoots and grains. It was reported that Cd in phloem sap is mainly complexed with nicotianamine, glutathione (GSH), and phytochelatins (PCs) (Mendoza‐Cózatl et al., 2008). However, how these Cd-chelates are loaded into the phloem is poorly understood and no responsible transporter has been identified up to date (Khan et al., 2014).
Inter-vascular transfer is closely associated with Cd accumulation in grains (Clemens and Ma, 2016). In cereals, nodes are the complex but well-organized vascular systems, consisting of two major vascular bundles: enlarged vascular bundles (EVBs) and diffuse vascular bundles (DVBs) (Yamaji and Ma, 2014; Clemens and Ma, 2016). EVBs come from the lower nodes and are connected to leaves, whereas DVBs surrounding the EVBs start at the node and are connected to the upper nodes or panicles (Clemens and Ma, 2016). Fujimaki et al. (2010) performed a noninvasive detection of Cd in rice plant, and found that Cd was more concentrated in the nodes than in the internodes. In rice, increasing evidences suggest that phloem loading by inter-vascular transfer from the EVBs to the DVBs in the node I is a major pathway for Cd movement toward grains (Yamaji and Ma, 2014). OsHMA2 and low-affinity cation transporter 1 (OsLCT1) are involved in the inter-vascular transfer, with OsHMA2 loading Cd into the phloem of EVBs and DVBs and OsLCT1 exporting Cd from phloem parenchyma cells into the sieve tubes (Uraguchi et al., 2011; Yamaji et al., 2013). However, the molecular mechanisms underlying Cd distribution in eudicots shoots is still unclear.

3 | CADMIUM TRANSPORTERS

Since Ca is a non-essential element for plants and interferes with the uptake of other ions, it is likely to enter plant cells through the transporters for essential elements such as Fe2+, Zn2+, and Mn2+, due to the similarity of Cd2+ and these ions in chemical and physical properties. In recent decades, a large number of genes conferring Cd transportation across membranes in plants has been identified and functionally verified (Clemens and Ma, 2016; Shahid et al., 2017; Ismael et al., 2019; Shi et al., 2019). These transporters belong to Natural Resistance-Associated Macrophage Proteins (NRAMPs), Heavy Metal ATPases (HMAs), Zinc/Iron-regulated transporter-like Proteins (ZRT1/IRT1-Like Protein, ZIPs), Cation Diffusion Facilitators (CDFs), oligopeptide transporter family (OPTs), ATP-binding cassette subfamily C proteins (ABCCs) and Cation/H+ Exchangers (CAXs). The functions of these transporters, including uptake, translocation, sequestration, and distribution of Cd in plants, and their tissue-specific localization and substrate specificity are summarized in Table 1 and Table S1.

3.1 | NRAMPs

NRAMPs represent a family of metal transporters that are located at the membrane of root cells and evolutionarily conserved in a wide range of organisms, including bacteria, fungi, plants, and animals (Uiiah et al., 2018). In plants, NRAMP genes are participated in uptake of the divalent cations, such as Fe2+, Mn2+, Cu2+, Zn2+, and Cd2+ as well. In Arabidopsis , six NRAMP family members have been identified, with AtNRAMP3 andAtNRAMP4 being localized at tonoplast and responsible for Cd2+ efflux from vacuole to the cytosol (Lanquar et al., 2005). In rice, OsNRAMP1 and OsNRAMP5 are plasma membrane (PM)-localized transporters involved in taking up Cd from the external solution to root cells (Takahashi et al., 2011; Sasaki et al., 2012). The knockdown or CRISPR/Cas9-mediated editing of OsNRAMP5 caused a dramatic reduction of Cd and Mn concentrations in both rice roots and shoots (Sasaki et al., 2012; Tang et al., 2017). However, OsNRAMP3, OsNRAMP4 and OsNRAMP6 have no ability of Cd transportation (Yamaji et al., 2013; Peris-Peris et al., 2017; Xia et al., 2010). In barley, HvNRAMP5, which shares 84% identity with OsNRAMP5, can also mediate Cd uptake (Wu et al., 2016). In the hyperaccmulator Noccaea caerulescens , NcNRAMP1 is one of the main transporters involved in the influx of Cd2+across the endodermal PM, and thus plays a key role in Cd2+ influx into the stele and contributes to Cd root-to-shoot transport (Milner et al., 2014).

3.2 | HMAs

HMAs, also known as P1B –ATPase, are involved in transporting cations across the membrane by consuming energy from ATP hydrolysis (Williams and Mills 2005). Eight HMA members have been identified in Arabidopsis , with AtHMA1-AtHMA4 showing the ability of transporting divalent cations like Cd2+, Zn2+ and Pb2+ (Williams and Mills, 2005). Of them, AtHMA2 and AtHMA4 are predominately expressed in the tissues surrounding the vascular vessels of roots, and they mediate Cd2+ efflux from xylem parenchyma cells to xylem vessels, which is necessary for the root-to-shoot Cd translocation (Mills et al. 2003). In the hyperaccumulator Thlaspi caerulescens, Sedum plumbizincicola and Arabidopsis halleri ,HMA4 have much more gene copy numbers and transcript levels than that in Arabidopsis thaliana (Craciun et al., 2012; Hanikenne et al., 2008; Liu et al., 2017), and it is a candidate in determining the evolution of Cd hyperaccumulator phenotype (Hanikenne et al., 2008). In rice, OsHMA2 is localized at PM mainly in root cells and has been proven to play the crucial role in Cd xylem loading and root-to-shoot translocation, and the loss of its function significantly decreases Cd accumulation in leaves and grains (Satoh-Nagasawa et al., 2012; Takahashi et al., 2012). OsHMA3 is a tonoplast-localized transporter and involved in restricting Cd translocation by mediating Cd sequestration into the vacuoles (Miyadate er al., 2011). The lose-of-function of OsHMA3 could cause high Cd accumulation in rice shoots and grains (Yan et al., 2016), whereas its overexpression shows a great opportunity to produce Cd-free rice through reducing Cd concentration in brawn rice by 94-98% (Lu et al., 2019). In addition, several recent studies suggested that the natural variation in the promoter or coding region of HMA3 contributes to the genotypic difference in Cd accumulation in rice andBrassica rapa (Liu et al., 2019; Zhang et al., 2019).

3.3 | ZIPs

ZIPs family members are generally involved in Cd uptake and translocation in plants (Colangelo and Guerinot, 2006). IRT1 is the first identified member of the ZIP family in Arabidopsis , and participates in taking up Fe2+, Zn2+, Cu2+, Ni2+, and Cd2+ from soil (Vert et al., 2002). In rice, 17 ZIPs transporters have been identified. OsIRT1 , which is highly homologous to AtIRT1 , is predominantly expressed in roots and up-regulated by Fe deficiency and Cd exposure (Bughio et al., 2002; Chen et al., 2018). Recently, Zheng et al. 2018 showed a distinct difference between Arabidopsis and rice in the expression profiles of ZIPs genes in responses to Cd stress. In addition, other ZIP transporters, like OsZIP1 and OsZIP3, have been also proved to be involved in Cd uptake in rice (Zheng et al., 2018). It is noteworthy that the involvement of ZIP genes in Cd uptake was also found in hyper-accumulator plants. In Noccaea caerulescens ,NcZNT1 , a homolog of AtZIP4 , mediates low-affinity Cd uptake when expressed in Saccharomyces cerevisiae zhy3 cells (Pence et al. 2000). Recently, it was reported that NcZNT1 is a PM-localized Zn2+/Cd2+ transporter and its promoter is mainly active in cells of the cortex, endodermis, and pericycle of roots in Noccaea caerulescens (Lin et al., 2016).

3.4 | CDFs

CDF proteins, also known as Metal Tolerance Proteins (MTPs), are a family of heavy metal transporters involved in the transport of Zn2+, Cd2+, and Co2+ (Williams et al., 2000). They have been identified in the diverse organisms, including bacteria, fungi, animals, and plants. The CDFs found in plant cells, generally named as MTPs (Fu et al., 2017), are known to mediate heavy metals efflux from the cytoplasm either to the extracellular space or to vacuoles and organelles (Peiter et al., 2007). MTPs consists of seven phylogenetic groups, with Zn-CDFs in groups 1 (MTP1–MTP4), 5 (MTP5) and 12 (MTP12); the Fe/Zn-CDFs in groups 6 (MTP6) and 7 (MTP7); and Mn-CDFsg in groups 8 (MTP8) and 9 (MTP9–MTP11) (Gustin et al., 2011). In rice, OsMTP1 has been demonstrated to be a PM-localized transporter involved in translocation of Cd and other heavy metals in both roots and shoots (Yuan et al., 2012). Other MTPs, such as TgMTP1 from Thlaspi goesingense (Kim et al., 2004), CsMTP1 and CsMTP4 from Cucumis sativus (Migocka et al., 2015), and CitMTP1 from Citrus sinensis(Fu et al., 2017), have been proved to be involved in Cd sequestration into vacuoles or efflux from root cells.

3.5 | OPTs

OPT family, which contains Yellow Stripe-Like (YSL) transporters, is involved in transporting metal-nicotianamine (NA) complexes through the plant cell membrane. Thus, when Cd2+ is chelated, it can be taken up through the OPT or YSL proteins (Zheng et al., 2018). To enhance the availability of metal ions in rhizosphere, plant roots secrete LMW organic acids, such as mugineic acids (Mas) and phytosiderophore (PS), to form the metal-ligand complexes, which are then transported by YSL transporters (Negishi et al., 2002). This strategy is very efficient for some species of Poaceae to take up Fe from Fe-deficient soils (Morrissey and Guerinot, 2009). In addition, YSLs also play an important role in Cd transport. In Zea mays , ZmYS1 has been suggested to transport the complexes of Cd-PS and Cd-NA at a low rate (Schaaf et al., 2004). Two orthologues of ZmYS1isolated from rice and Cd hyperaccumulator Solanum nigrum ,OsYSL2 and SnYSL3 , have beenfound to transport Cd-NA complex when heterologously expressed in yeast (Koike et al., 2004; Ishimaru et al., 2010; Feng et a., 2017).

3.6 | ABCCs

ABC is one of the largest protein families in organisms (Higgins, 1992) and it has various substrates, including carbohydrates, lipids, xenobiotics, antibiotics, drugs, and heavy metals (Martinoia et al., 2002). Unlike NRAMP3/4, HMA3 and CAX2/4 transporting free Cd2+ ions, ABCCs are found to transport the Cd-PCs complex (Zhang et al., 2018). InArabidopsis , AtABCC1 and AtABCC2 are responsible for the transport of Cd-PCs into the vacuoles (Park et al., 2012). Likewise, AtABCC3 has been also suggested to mediate transport of Cd-PC complex (Bovet et al. 2003; Brunetti et al., 2015). Besides ABCCs, some other members of ABC transporters also confer Cd tolerance. AtABCG36/AtPDR8, a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters in Arabidopsis , is proposed to have a role in Cd tolerance by pumping Cd2+ or Cd complexes out of the plasma membrane of root epidermal cells (Kim et al., 2007). AtATM3, which belongs to the mitochondria subfamily of Arabidopsis ABC proteins, contributes to Cd tolerance by mediating the transport of glutamine synthetase-conjugated Cd across the mitochondrial membrane (Kim et al., 2006). In rice, OsABCG36/OsPDR9 has been recently demonstrated to be involved in Cd tolerance by exporting Cd2+ or Cd conjugates from the root cells (Fu et al., 2019).

3.7 | CAXs

The CAXs are tonoplast-localized transporters that export cations out of the cytosol to maintain ion homeostasis across biological membranes (Pittman et al. 2002). Most of CAX members are calcium (Ca2+) specific. However, two CXAs identified inArabidopsis , AtCAX1 and AtCAX2, have been found to be capable of pumping Ca2+ and other cations like Cd2+, Zn2+ and Mn2+ into vacuoles (Korenkov et al., 2007; Korenkov et al., 2009). Wu et al. (2011) reported that the ectopic expression of AtCAX1 inPetunia significantly increased Cd tolerance and accumulation (Wu et al., 2011). In the Cd hyperaccumulator Arabidopsis halleri , Cd tolerance is highly associated with the expression of AhCAX1(Baliardini et al., 2015), suggesting an involvement of AhCAX1 in conferring Cd tolerance in this plant. A CAX2-like protein in the hyperaccumulator Sedum alfredii , SaCAX2, confers Cd tolerance and accumulation when heterologously expressed in yeast and tobacco (Zhang et al. 2016).

3.8 | Other transporters

In addition to the above transporter families, some other transporters are also involved in Cd transport. OsLCT1, a rice homolog of wheat low affinity cation transporter 1, is localized at PM of the cells surrounding the EVBs and DVBs of node 1 and the phloem parenchyma cells of DVBs (Uraguchi et al., 2011). It mediates the efflux of various cations including Ca2+, Cd2+, K+, Mg2+, and Mn2+, with a high affinity for Cd2+(Uraguchi et al., 2011). Knockdown of OsLCT1 leads to a reduction up to 50% of grain Cd in rice plants grown in Cd contaminated soil, indicating that OsLCT1 is involved in xylem-to-phloem distribution of Cd (Clemens and Ma, 2016). Recently, a gene belonging to the major facilitator superfamily, OsCd1 , was proved to be involved in root uptake and grain accumulation of Cd in rice (Yan et al., 2019). Furthermore, a natural variation in OsCd1 caused by a missense mutation was found to be associated with the divergence of grain Cd accumulation between Indica and Japonica rice . Luo et al. (2018) identified a QTL in rice, namely CAL1, which encodes a defensin-like protein. CAL1 is preferentially expressed in root exodermis and xylem parenchyma cells, and acts by chelating Cd in the cytosol and facilitating Cd secretion into apoplast, thus lowing cytosolic Cd concentration (Luo et al., 2018).
Because of sharing a similarity with Ca2+ in charge and ionic radius, Cd2+ can enter the plant symplast by passive transport through channel proteins transporting Ca2+(Perfus-Barbeoch et al., 2002; Li et al., 2012; Chen et al., 2018). Indeed, several kinds of calcium-permeable channels, such as depolarization-activated calcium channels (DACCs), hyperpolarization-activated calcium channels (HACCs), and voltage-insensitive cation channels (VICCs), are capable of transporting Cd2+. Increasing evidences has been obtained using channel blockers and flux measurement to supporting the effects of Ca on Cd uptake and accumulation in plants (Perfus-Barbeoch et al., 2002; L.Z. Li et al., 2012; Chen et al., 2018). However, the functions of these channels in facilitating Cd transport is poorly understood. In a recent study on rice, the expression of the genes belonging to the two Ca channel families, annexins and glutamate receptors (GLRs), was shown to co-segregate with Cd2+ influx and uptake by root cells (Chen et al., 2018), suggesting the possibility to identify the candidate channels responsible for Cd transport.

4 | EVOLUTION OF CADMIUM ACCUMULATION IN PLANTS

4.1 | Phylogenetic variation in Cd accumulation in plants

The great variation in Cd accumulation has been found not only among species, but also among genotypes within a species (Cappa and Pilon‑Smits, 2014). Algae constitute an important component of our environment and ecosystem as a primary producer, contributing to nearly 40% of the global productivity of biomass (Issa et al., 2016). Although no evidence proves Cd is necessary for growth of algae, they have the ability to concentrate Cd from the environment, with a manner of algae species dependent (Klimmek et al., 2001). In fresh water green algae (Chlorophyta), such asScenedesmus spp. (Scenedesmaceae) andSpirogyra spp. (Zygnemataceae), the maximum concentration of Cd ranges between 3.0 to 11.4 mg/g dry biomass at 1.0-40.0 mg/L Cd in wastewater. (Brahmbhatt et al., 2012). However, the marine brown algae (Ochrophyta), such as Ecklonia radiate (Lessoniaceae),Macrocystis pyrifera (Laminariaceae), Laminaria digitata (Lessoniaceae) andSargassum spp. (Sargassaceae) have been shown to be the strong biosorbents of Cd2+, with the highest Cd concentration ranging from 76.4 to 1634 mg/g dry biomass (Davis et al., 2004; Park and Lee, 2002). Such a high capacity of Cd2+ adsorption for these marine brown algae could be attributed to high alginate content in cell wall matrix, as alginate is a biopolymer with high affinity for divalent metals (Davis et al., 2004; Park and Lee, 2002). On the other hand, the ability of algae to absorb Cd2+is strongly influenced by water pH. In the freshwater green algaPseudokirchneriella subcapitata (Selenastraceae), the intracellular flux of Cd2+ is at least 20 times higher at pH 7.0 than at pH 5.0 (Vigneault and Campbell, 2005). The similar result has been observed inScenedesmus obliquus (Coelastraceae), with the highest value of Cd absorption occurring at pH7.0 (Monteiro et al, 2009). Nevertheless, for the marine brown alga Ecklonia radiate (Lessoniaceae) considerable Cd uptake occurs at the pH ranging 2.0-6.0 and the maximum at pH 4.0 (Park and Lee, 2002).
Bryophytes are the first green plants to colonize the terrestrial environment (Nickrent et al. 2000), and they are usually divided into three large clades: the liverworts (Marchantiophyta), mosses (Bryophyta), and hornworts (Anthocerophyta) (Shaw et al. 2011). Both mosses and liverworts possess the ability to absorb heavy metal ions over their entire surface, due to the lack of cuticle layer, pronounced ion-exchange property and a large surface-to-weight ratio (reviewed by Stanković et al., 2018). Thus, both mosses and liverworts have been widely used as the biomoinitor or bioindicator of heavy metal pollution in both terrestrial and aquatic environments (Mahapatra et al., 2019; Stanković et al., 2018). Nevertheless, this ability is highly species-specific and also strongly dependent on the environmental conditions. Vukojević et al. (2005) reported that two moss species,Bryum capillare and Ceratodon purpureus , could accumulate up to 0.1% Cd DW (appx.1125-1250 mg/kg) in their shoots. However, in other two moss species Fabriona ciliaris and Leskea angustata , Cd concentrations only varies from 0.1 to 7.3 mg/kg, with an average value of 1.3 mg/kg (Macedo‑Miranda et al., 2016). Furthermore, Wells and Brown (1990) found that in the moss Rhytidiadelphus squarrosus low pH not only reduced the extracellular binding of Cd, but also inhibited its intracellular uptake.
Ferns and lycophytes are the largest groups in the vascular plants beside the seed plants, consisting of about 10,578 and 1,338 species, respectively (Pteridophyte Phylogeny Group I, 2016). They play the important role in early land plant evolution (Pryer et al., 2001), and are remarkably adapted to a wide range of environments, including both tropical and cold temperate climates, alpine and lowland regions, as well as aquatic and xeric conditions (Mehltreter, 2008). In general, both ferns and lycophytes can accumulate a large amount of Aluminum (Al) and Arsenic (As), but they have much less Cd accumulation (Meharg, 2002; Schmitt et al., 2017). However, Arora et al. (2006) measured Cd concentrations in threeAzolla (a small aquatic fern) species, and found thatAzolla would accumulate Cd as high as 2759 µg/g in shoot when exposed to 10 mg/L Cd, suggesting a potential of Azolla in Cd hyperaccumulation.
Gymnosperms are an ancient and widespread plant clade, which represents four of the five main lineages of seed plants, including cycads, ginkgos, gnetophytes, and conifers (De La Torre et al., 2017). Gymnosperm lineages separated from each other during the Late Carboniferous to the Late Triassic (311–212 Mya), earlier than the occurrence of the earliest extant angiosperms (around 300 Mya) (Magallón et al., 2013). Living gymnosperms comprise only a little more than 1000 species, with conifers (pines, cypresses and relatives) being the largest group (Wang and Ran, 2014). The available research on Cd accumulation in gymnosperms is mostly focused on conifers. Kim et al. (2003) reported that Pinus sylvestris would accumulate Cd up to 33.2 mg/kg in shoots under the treatment of 10 mg/kg Cd for 11 weeks. In a later study, however, another Pinusspecies, Pinus pinaster, was found to accumulate only 11.9 mg/kg Cd in shoots when exposed to 15 mg/kg Cd for 6 months (Sousa et al., 2014). Hashemi and Farajpour (2016) found that Picea abies, grown in the metal contaminated soils (Cd 6.8 mg/kg), could accumulate 1.5 mg/kg Cd in saplings, whereas Österås and Greger (2006) found the plant could accumulate Cd up to 72.4 mg/kg in barks and 26.1 mg/kg in wood after exposure to 0.5 μM Cd for 3 months. Guo et al. (2016) showed thatPlatycladus orientalis exhibited an exceptional ability of Cd tolerance, accumulating about 69.3 mg/kg Cd in stems and 406 mg/kg in roots after exposure to 100 mg/kg of soil for 220 days. Recently, Zeng et al. (2018) reported that the growth of Platycladus orientaliswas significantly inhibited under the soil containing 9.6 mg/kg Cd, and accumulated over 2 mg/kg Cd in stems and about 41.5 mg/kg in roots. Although the results of Cd accumulation were not consistent, both authors have suggested that Platycladus orientalis is a promising plant for phytostabilization of Cd-contaminated soil. In addition, a hybrid larch shows an extraordinary potential for Cd phytoextraction, as it can tolerate a 4-week exposure to 0.25 mM Cd (or 1-week exposure to 1.5 mM Ca) and accumulate as high as 208-220 mg/kg Cd in shoots (Bonet et al., 2016; Moudouma et al., 2013).
Angiosperms, appeared during the Early Cretaceous period (around 10-30 Myr), are the largest and most diverse group of vascular plants, consisting of about 400-500 families and perhaps as many as 400,000 species (Taylor et al., 2009). Angiosperms represent approximately 80% of all known living plants, including five major groups: Eudicots, Monocots, Magnoliids, Chloranthales, and Ceratophyllales ( APG IV, 2016). Angiosperms are able to occupy any environment on the earth, such as high mountaintops, deep oceans, freezing tundras, and warm, wet rainforests as well (Field and Arens, 2005). It is well documented that the angiosperm ecological incursion is highly driven by the environmental circumstances and biotic factors (Field and Arens, 2005). Broadley et al. (2001) summarized the early records of shoot Cd content in angiosperms, founding a significant variation in shoot Cd content among the tested 108 angiosperm species. All available results indicate that there is a large difference in absorbing Cd from the environment among the angiosperms. In fact, about 20 angiosperm species (~10 families) have been already identified as Cd hyperaccumulators (Cappa and Pilon‑Smits, 2014; Reeves et al., 2017; Qiu et al., 2012; Xu et al., 2020). These Cd accumulators are not randomly distributed over the families of angiosperms, and mainly belong to the families of Brassicaceae and Asteraceae. Two Brassicaceae species, Arabidopsis halleri andThlaspi caerulescens , which are the most intensively studied species of Cd hyperaccumulating plants, also show an extraordinary ecotypic variation in Cd accumulation (Reeves et al., 2018; Verbruggen et al., 2013). Likewise, the Cd hyperaccumulator Sedum alfrediiexhibits the fixed ecotypic difference in Cd sequestration and translocation (Cappa and Pilon‑Smits, 2014; Reeves et al., 2018), indicating the local adaptation of these species to the natural habitats (Reeves et al., 2018), or probably to the anthropogenic metal pollution (Sobczyk et al., 2017).
Overall, plants, especially angiosperms, display a wide adaptation to the presence of Cd in the environments. Such adaptation has occurred throughout their evolution (Broadley et al., 2001). The phylogenetic distribution of Cd accumulation across eight to ten orders indicates that Cd accumulation is a derived, polyphyletic trait that evolved convergently within eight to ten angiosperm clades (Broadley et al., 2001; Cappa and Pilon-Smits, 2014). The similar convergent patterns of evolution in angiosperms have also been observed for accumulation of other metals like selenium (Se) and nickel (Ni) etc. (Cappa and Pilon-Smits, 2014; Reynolds et al., 2017). Hence, here arises a question as to which selection pressures favor the evolution of Cd accumulation? It is well known that the emergence of Cd hyper-accumulation trait in some plant species like Arabidopsis halleri , Thlaspi caerulescens and Sedum alfredii was coincident with the appearance of anthropogenic metal-polluted sites in the mining regions (Reeves et al., 2017; Qiu et al., 2012; Xu et al., 2020). So it is generally believed that the colonization of plants in the anthropogenically heavy metal contaminated soils is likely to represent a recent event in the evolutionary history of plant species (Hanikenne and Nouet, 2011). However, a comparison study betweenArabidopsis halleri and Arabidopsis lyrata suggests that ecological differentiation may have occurred at the onset of speciation in this species pair (∼5 Mya, Koch et al., 2000) and Arabidopsis halleri has well fostered the spread of Cd-polluted areas long before the expansion of anthropogenic environments (Roux et al., 2011). Therefore, Cd accumulation might thus have evolved either on the calamine outcrops before metal pollution by mining activities, or on nonmetalliferous soils followed by a later colonization of metalliferous soils and an increase in metal tolerance (Hanikenne and Nouet, 2011; Reeves et al., 2018).

4.2 | Evolution of membrane transporters in plants

Evolution of the membrane transporters may play an important role in adapting to metalliferous environments in plants. As described above, there are many membrane transporters involved in Cd accumulation in plants, mainly including NRAMPs, HMAs, ABCCs, ZIPs, CDFs, CAXs and OPTs. Thus several intriguing evolutionary questions arise. Are these membrane transporters conserved in all plant lineages? Is there a stepwise evolution of membrane transporters in plants? If yes, do they occur from monophyletic or polyphyletic evolutionary events? The ancestors of modern land plants colonized the terrestrial habitat about 500 to 470 Mya. Since then, the dramatic changes have taken place for the living environments of land plants, with a large fluctuation of water availability, illumination, light intensity, temperature and the concentration ratio of carbon dioxide and oxygen, as compared to the aquatic environments for seagrasses grown in the ocean (Chen et al., 2017). As a consequence, an adaptive and stepwise evolution would happen persistently for the membrane transporters in plants.
We made a comparative genomics analysis of seven Cd transporter gene families using 41 plant species ranging from Rhodophyte to Eudicots to reveal the difference of these gene families accompanied by species evolution (Figure 1; Table 2). By applying the strict selection criteria (E-value < 10-10 and query coverage > 50%), the OPTs protein family is no longer detected in all algae species with the exception of Klebsormidium flaccidum , which has only one OPT homolog (Figure 1; Table 2). Hanikenne et al. (2005) attempted to find the YS1-like proteins in the genome sequences of green alga Chlamydomonas reinhardtii and red alga Cyanidioschizon merolae , but failed to identify any homolog. Likewise, a previous phylogenetic analysis of 325 OPTs family members from prokaryotes and eukaryotes revealed that OPT family members in eukaryotes were found only in fungi and land plants (Gomolplitinant and Saier Jr., 2011). Thus it may be suggested that the OPTs family have evolved after the emergence of land plants. By contrast, the families of NRAMPs, HMAs, ABCCs, ZIPs, CAXs and CDFs are identified across all the plant species, although CAX (in Cyanidioschyzon merolae and Mesotaenium endlicherianum ) and ZIP (in Porphyra yezoenesis and Volvox carteri ) homologs were not detected in several algae species (Table 2). As the ancient gene families that pre-date the origin of eukaryotes (Emery et al., 2012; Gustin et al., 2011; Hanikenne and Baurain, 2014; Hanikenne et al., 2005; Montanini, et al., 2007; Williams and Mills, 2005; Ullah et al., 2018), these Cd transporter families seem to be conserved throughout the evolutionary lineage of entire plantae, indicating an evolutionarily conserved function of them in metal homeostasis. Furthermore, it is notable that the vascular plants, including lycophytes, ferns, gymnosperms and angiosperms, contain much more family members of NRAMPs, HMAs, ABCCs, ZIPs, CAXs, CDFs and OPTs in their genomes than do algae and bryophytes (liverworts and mosses) (Table 2), suggesting that these transporter families underwent lineage specific expansion, which might be contributed by gene duplication resulting from segmental duplication [whole genome duplication (WGD) or duplications of large chromosomal regions] and/or tandem duplication (Cannon et al., 2004; Liu et al., 2012; Ullah et al., 2018). During the course of evolution, the expansion of membrane transporter families in the vascular plants may provide an adaptive advantage for colonizing new habitats like metalliferous soil before significant vascular development occurred in early land plants.
OsNRAMP5 and OsHMA2, which mediate Cd uptake and xylem loading respectively, are recognized as the most important transporters for Cd accumulation in rice (Clemens and Ma, 2016). In order to understand the origin of these two Cd transporters, a phylogenetic analysis was performed across the entire plant kingdom using oneKP database (One Thousand Plant Transcriptomes Initiative, 2019). In the phylogenetic tree with 396 species, the orthologues of OsHMA2 from algae, mosses, ferns, lycophytes, gymnosperms and angiosperms form a distinct cluster, but they are all basal to Rhodophyta species Rhodochaete parvula(Figure 2A), indicating that this transporter has experienced an early evolution in plants. While the angiosperms cluster can be grouped into two sub-clusters of monocots and eudicots, and all of them are basal toAmborella trichopoda , which is the only living species on the sister lineage to all other flowering plants (Amborella Genome Project, 2013). In addition, eudicots show a closer orthologous relationship withMyristica fragrans (Figure 2A). These results indicate that monocots and eudicots share a common origination of HMA2 transporter from Amborella trichopoda , which can be dated back to about 130 Mya. However, they have evolved separately thereafter, forming a monophyletic evolutionary lineage.
In phylogenetic analysis the orthologues of OsNRAMP5 proteins from 592 species are grouped into two clusters (Figure 2B). Cluster 1 includes green algae, Glaucomphyta, Rhodophyta and mosses; and Cluster 2 contains green algae, hornworts, liverworts, lycophytes, ferns, gymnosperms and angiosperms. Each cluster can be further divided into three sub-clusters; the sub-clusters in cluster 2 are deeply-branching, while those in cluster 1 are not. In the three sub-clusters of cluster 2, green algae form a distinct sub-subcluster; lycophytes, ferns and gymnosperms are included in the other two sub-subclusters; whereas hornworts, liverworts, and angiosperms can only be found in the last sub-subcluster. In addition, the higher plants are clearly divided into two groups, monocots and eudicots, occupying their specific ancestral gymnosperms and basal angiosperms (Figure 2B). It may be suggested that the orthologues of OsNRAMP5 in higher plants underwent a polyphyletic evolutionary lineage originated from different ancestors. Furthermore, the divergent presence of green algae, lycophytes, ferns and gymnosperms in the phylogenetic tree indicates a rampant occurrence of horizontal gene transfer during the evolution of the orthologues of OsNRAMP5 (Figure 2B), which has been previously evidenced in the studies on the evolution of NRAMPs in bacteria (Richer et al., 2003) and OPTs in plants (Gomolplitinant and Saier Jr., 2011). However, further studies are necessary to make insight into the molecular mechanisms and adaptive roles of horizontal gene transfer events in the evolution of Cd transporters in plants.
Our previous study revealed that the expression of OsGLR3.4 was co-segregated with Cd2+ influx and uptake into rice roots (Chen et al., 2018). Recently, the contribution of GLR channels to Cd2+ influx was verified using mammalian glutamate receptor antagonists AP-5, and our results showed that net Cd2+ influx in rice root epidermis was significantly inhibited by AP-5 (Figure 3A). Moreover, relative expression ofOsGLR3.4 was up-regulated when rice plants were exposed to 5 and 50 μM Cd stress (Figure 3B). All these results support the assumption that GLR channel is involved in mediating Cd uptake in rice roots (Lux et al., 2011; Clemens, 2019). Here we present the origin of OsGLR3.4 across the entire plant kingdom (Figure 3C). Accordingly, the phylogenetic analysis of OsGLR3.4 was conducted using 850 species-based oneKP database. In the phylogenetic tree, the orthologues of OsGLR3.4 from Streptophyta to Eudicots formed 5 distinct clusters. Interestingly, each phylogenetic cluster includes very specific clades of species (Figure 3C). Among the major clusters, GLR3.4 can be traced to Streptophyta species, and the significant presence are observed in hormworts, liverworts, mosses, lycophytes, ferns, gymnosperms and angiosperms (Figure 3C), in accordance with the phylogenetic analysis of OsHMAs (Figure 2A), indicating an ancient origin of OsGLR3.4 orthologues in plants (Bortoli et al., 2016). Moreover, angiosperms are grouped into 2 sub-clusters, with each sub-cluster containing the specific basal angiosperms, monocots and Eudicots, suggesting that GLR3.4 could have recently diverged into two evolutionary branches, possibly giving rise to the distinct functions between them, adapting to Cd-contaminated environments.

5 | CONCLUSIONS AND PERSPECTIVES

Cd is one of the most hazardous toxic heavy metals in the environments, posing great risks to human health. Cd accumulation in plants is controlled by both genetic and environmental factors via affecting the whole process, including uptake from soil, root-to-shoot translocation, sequestration and (re)distribution in shoots. In essence, all these processes are governed by membrane metal transporters, including NRAMPs, HMAs, ZIPs, CDFs, OPTs, ABCCs, CAXs and some other transporters or channels like OsLCT1, OsCd1 and CAL1. Across the entire plantae kingdom, Cd accumulation displays a wide phylogenetic variation among species and genotypes within a species. By applying oneKP database, the functional evolution of Cd accumulation in plants was carefully examined, indicating that Cd accumulation in plants is a derived and polyphyletic trait that has evolved convergently by several times. During the course of evolution, the membrane transporter families, such as NRAMPs, HMAs, ABCCs, ZIPs, CDFs and CAXs, are conserved throughout the evolutionary lineage of entire plantae species, indicating that their functions are evolutionarily conserved for metal homeostasis. However, the OPTs protein family is missed in algae species, suggesting that the OPTs family may evolve after the emergence of land plants. Moreover, the vascular plants contain much more family members of the above transporters in their genomes than do algae and bryophytes, suggesting that these transporter families underwent lineage specific expansion, which might be conferred by gene duplication due to segmental duplication and/or tandem duplication. Furthermore, the orthologues of OsHMA2 and OsGLR3.4 in higher plants are stepwisely evolved from monophyletic evolutionary lineage with one common ancestor; whereas the orthologues of OsNRAMP5 came from a polyphyletic evolutionary lineage with the different ancestors. In addition, the rampant intermixing have occurred for the orthologues of OsNRAMP5 in the phylogenetic clusters, indicating the horizontal gene transfer in its evolution. All these evolutionary patterns may provide an adaptive advantage for colonization of plants to new habitat like metalliferous soil. Due to lack of information on the known Cd hyperaccumulators in oneKP database, we still have no clear answer as to whether and what extent such evolutionary patterns of membrane transporters contributes to Cd (hyper)accumulation in plants. Ideally, the non-accumulator, accumulator and hyperaccumulator plants should be comprehensively compared, in terms of genome sequencing, ecological distribution patterns and the ability of Cd uptake and transport, so as to fully elucidate the evolutionary mechanisms associated with the accumulation and adaptive response of Cd in plants.

ACKNOWLEDGEMENTS

This work was supported by the National Key Technology R&D Program (2018YFD0800200), and Jiangsu Collaborative Innovation Center for Modern Crop Production (JCIC-MCP).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflict of interest.

AUTHOR CONTRIBUTIONS

F. R. Z. and G. P. Z. planned and designed the outline of this review.
G. C. and X. H. C. prepared the figures and tables.
F. R. Z., Q. L., X. J. W. and G. P. Z. wrote the manuscript.

SUPPLEMENTARY MATERIALS

Table S1 Information of NRAMPs, HAMs, ABCCs, ZIPs, CDFs, CAXs and OPTs families for evolutionary bioinformatics analysis.
Supplemental file 1 List of references for Table 1.

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FIGURE LEGENDS

Figure 1 Similarity heat map of key membrane Cd transporters in different species. Genesis software was used to estimate the similarity among protein sequences based on Tables 1 and S1. Candidate protein sequences were selected by BLASTP searches which satisfied E value <10-10 and query coverage >50%. Colored squares indicate protein sequence similarity from zero (blue) to 100% (red). White squares indicate that no homologous genes were found.
Figure 2 Phylogenetic analysis of OsHMA2 (A) and OsNRAMP5 (B). The mRNA sequences of OsHMA2 and OsNRAMP5 were queried from the One Thousand Plant Transcriptome (1KP) database (https://sites.google.com/a/ ualberta.ca/onekp/). Amino acid sequence of Oryza Sativa OsHMA2 and OsNRAMP5 were employed as the query sequences to access the transcriptome data with the criterion of E-value < 10-10 and coverage > 50% using BLASTP. The sequences were aligned with MAFFT and the phylogenies constructed with the online toolkit RAxML (Stamatakis, A., 2014) of CIPRES (Miller, M.A. et al., 2010). Genes sampled from Chromia algae were used as the outgroup (in the shade of light grayish magenta) and the root of the tree, and the Interactive Tree of Life resource (http://www.itol.embl.de) was used to annotate gene trees. Bootstraps (1-100) were displayed as the width of branches (1-10 px).
Figure 3 Kinetics of Cd2+ fluxes in response to glutamate receptor antagonists AP-5 (A), gene expression of OsGLR3.4 in response to external Cd (B) and phylogenetic analysis of OsGLR3.4 (C). Transient Cd2+ fluxes were measured from mature zone in rice root in response to 50 μM Cd pretreated with or without 100 μM AP-5, Data ±SE(n=8). Gene expression of OsGLR3.4 was quantified by qRT-PCR under 5 or 50 μM Cd at the exposure time indicated in the figure, , Data ±SE(n=3). Phylogenetic analysis of OsGLR3.4 was conducted similarly as described in Figure 2.
Table 1 The identified transporters mediating Cd uptake, translocation, sequestration and distribution in plants sequestration and distribution in plants