TY - JOUR
AU - Clemens,, Stephan
AB - Abstract Natural processes and human activities have caused widespread background contamination with non-essential toxic elements. The uptake and accumulation of cadmium (Cd), arsenic (As), and lead (Pb) by crop plants results in chronic dietary exposure and is associated with various health risks. Current human intake levels are close to what is provisionally regarded as safe. This has recently triggered legislative actions to introduce or lower limits for toxic elements in food. Arguably, the most effective way to reduce the risk of slow poisoning is the breeding of crops with much lower accumulation of contaminants. The past years have seen tremendous progress in elucidating molecular mechanisms of toxic element transport. This was achieved in the model systems Arabidopsis thaliana and, most importantly, rice, the major source of exposure to As and Cd for a large fraction of the global population. Many components of entry and sequestration pathways have been identified. This knowledge can now be applied to engineer crops with reduced toxic element accumulation especially in edible organs. Most obvious in the case of Cd, it appears likely that subtle genetic intervention has the potential to reduce human exposure to non-essential toxic elements almost immediately. This review outlines the risks and discusses our current state of knowledge with emphasis on transgenic and gene editing approaches. Crop engineering, food safety, health risk, metal homeostasis, metal transport, rice Introduction For plants as for other organisms, a number of elements can become bioavailable that are neither essential nor beneficial. Several of these elements without known biological function can be toxic even at very low concentrations. Their presence in the environment of plants is partially a consequence of geological processes but mostly of anthropogenic origin. For example, ever since the pioneering work of Clair Cameron Patterson and his co-workers, it is well documented that lead (Pb) release by human activities has been causing a nearly ubiquitous low-level contamination of the environment. While analyzing the concentration of mineral isotopes to determine the age of the earth, they found evidence for a widespread contamination of the biosphere with Pb (Settle and Patterson, 1980). Patterson concluded that the background concentrations of Pb found in human blood samples or animal tissues were by no means natural as claimed by the advocates of tetraethyl lead addition to gasoline. Instead, his analyses showed that Pb levels in human bodies were on average >100-fold higher than in prehistoric times, suggesting a considerable public health threat (Needleman, 1998). The main reason for the ubiquitous presence of Pb is atmospheric pollution through Pb-containing aerosols and particles emitted by mines, metal smelters, or motorized vehicles. Analysis of lake sediments in Northern Sweden demonstrated atmospheric pollution of remote areas since >2500 years ago. The chronology of emissions that can be derived from Pb isotope analysis of the annual sediment layers mirrors Pb production, with a first peak during the Roman empire, a second peak after the mining renaissance in Central Europe during the Middle Ages, and a sharp increase since ~1900 (Renberg et al., 1994; Brännvall et al., 1999). Sedimentary archives revealed an >100-fold enrichment of Pb in soil and water bodies in Europe and North America, with a peak in the 1950s to 1980s and a minor drop since then, which is explained by the phasing out of leaded gasoline and Pb-containing consumer products since the 1980s (Marx et al., 2016). Other elements of concern include cadmium (Cd), arsenic (As), and mercury (Hg). Together with Pb they constitute the ‘big four’ (Nriagu, 1988; Järup, 2003) and are the metals/metalloids present among the top 10 chemicals of major health concern on the respective WHO list (http://www.who.int/ipcs/assessment/public_health/chemicals_phc/en/). Like Pb they are potentially very toxic and, over the past two centuries, they have been massively released into the environment by human activities. The use of various metals is indispensable for a wide variety of products that constitute the basis of safe and enjoyable lives. Human civilization is unthinkable without the exploitation of metal ores and, appropriately, historical periods are named after the metals or alloys predominantly used (‘Bronze age’, ‘Iron age’). Principal sources of metals released into the environment are therefore mining, metal smelting, and the manufacturing of metal-containing products such as batteries and, more recently, electronic devices (Ogunseitan et al., 2009). A dramatic rise in metal pollution is associated with industrial development, the Industrial Revolution since about 1850 in Europe and North America, and corresponding processes in other regions of the world (Nriagu and Pacyna, 1988). The second source of unintended as well as intended toxic metal release into the environment is agricultural use. Cd has for decades been added to agricultural fields as an impurity in phosphate fertilizers, while As and Pb compounds such as sodium arsenite, dimethylarsinic acid (DMAV, also known as cacodylic acid; see below), or Pb-arsenate were widely applied as pesticides. An additional source especially of As pollution is the use of As-contaminated ground water for irrigation (Zhao et al., 2010b). In fact, awareness of non-occupational exposure to As causing a major public health threat grew when a crisis in Bangladesh unfolded in the late 1990s. An estimated 20–30 million people are at risk, because As had leached from natural As-rich minerals into aquifers and village wells (Bagla and Kaiser, 1996), leading to widespread As contamination of drinking water (Nordstrom, 2002). Potential risks associated with low-level exposure to potentially highly toxic elements have been investigated for a considerable time. This resulted in the above-mentioned banning of leaded gasoline, many Pb-containing consumer products, or organoarsencials during the 1970s and 1980s. Nonetheless, production remains high. According to the US Geological Survey, annual worldwide production in 2017 was 23 000 t for Cd, 37 000 t for As, and 4 700 000 t for Pb (https://minerals.usgs.gov/minerals/pubs/commodity/). Furthermore, a lowering of emission rates does not alter the fact that the toxic metals are ubiquitously present in the environment. In contrast to organic contaminants, they are non-degradable and persist in the soil. Depending on the element in question and on soil conditions, a variably small fraction of the total amount becomes available for uptake by plants (i.e. is dissolved in the soil solution). Uptake, loading into vascular tissues, and intracellular sequestration occur through transporters for essential or beneficial elements. Such inadvertent uptake of non-essential elements applies similarly to heterotrophic organisms that consume plant organs, for example humans. Digestion of plant-derived biomass releases these elements and at least a fraction becomes bioavailable for uptake into gut epithelial cells, enters the blood stream, and finally various organs. In fact, many of the transporters responsible for the toxic element uptake across plasma membranes, such as Nramps (Natural resistance-associated macrophage proteins), belong to the same protein families in plants and humans (Gunshin et al., 1997; Sasaki et al., 2012). For the general population, namely everybody not occupationally in contact with toxic metals, food consumption represents a major source of toxic element intake (e.g. United Nations Environment Programme, 2010). Plant-derived food contributes substantially, albeit at varying levels, to overall Cd, As, and Pb exposure, and these elements will therefore be the focus of this review. The most relevant entry pathway for Hg, in contrast, remains the consumption of seafood, because methylmercury, by far the most toxic form of mercury in the environment, bioaccumulates in aquatic food chains (Mergler et al., 2007). Nonetheless, there is documented accumulation of methylmercury also in rice grown in contaminated areas (Feng et al., 2008), and the underlying mechanisms clearly deserve research attention. After considering current exposure levels, associated health risks and current regulatory action, this review focuses on recent insights into mechanisms of toxic metal accumulation in plants, and addresses the question as to how these insights could be applied to lower human exposure. The findings of plant molecular physiology can be exploited either directly or indirectly, for example via direct genetic intervention or through informed changes in agronomic practices. However, both the extent of available evidence and the prospects of effectively tackling slow poisoning through dietary exposure vary widely for the elements in question. For Cd, the tools are available to achieve a drastic reduction in grain content at least in rice. In contrast, neither the actual importance of dietary exposure nor the mechanisms controlling accumulation in plant organs are understood at any satisfactory level for Pb. Tremendous progress has been achieved in recent years regarding the pathways and mechanisms especially underlying the accumulation of Cd and As. Thus, these two elements will be covered more extensively, and with particular emphasis on molecular insights published after recent reviews appeared (e.g. Clemens and Ma, 2016; Chen et al., 2017a; Lindsay and Maathuis, 2017). The problem: slow metal poisoning Principal mechanisms of toxicity for the considered non-essential elements are interaction with functional groups in proteins and interference with the homeostasis of essential elements. Furthermore, oxidative stress is practically always a symptom of exposure. Ions of Pb, Cd, As (in the form of arsenite, AsIII), and Hg all have a strong tendency to interact with thiols in proteins and other biomolecules. A large number of molecules can potentially be rendered inactive through interaction with Pb, Cd, As, or Hg. Thus, it is unclear whether primary targets of toxicity exist. Among the particularly sensitive proteins are cytosolic iron–sulfur (Fe–S) cluster proteins (Xu and Imlay, 2012). For Cd- and As-exposed cells, it is known that DNA repair enzymes are among the damaged proteins (Hughes, 2002; Maret and Moulis, 2013). Resemblance to micro- or macronutrients as a cause of toxicity is exemplified by AsV (arsenate), which is similar to phosphate and therefore can interfere with phosphate metabolism (Hughes, 2002). Likewise, CdII can replace ZnII in proteins that require this micronutrient as a cofactor (Maret and Moulis, 2013). PbII may block Ca-binding sites (Needleman, 2004). [Please note that from hereon only the oxidation state of As will be specified; for Cd, zinc (Zn), and Pb the element symbols will be used even when ions are meant as they do not undergo changes in oxidation state under physiological conditions.] Health threats Acute toxicity of metals and metalloids for humans has been known for a very long time at least in the case of Pb and As. Pb poisoning was already described by the Greek physician Nicander in the 2nd century BCE (Needleman, 2004). As is famous not only for making a high-profile homicidal poison, but also for medical uses that date back to antiquity and continue to this day. Hippocrates apparently used an As paste to treat various ailments. Fowler’s solution, 1% potassium arsenite, was since the late 18th century applied against, among others, diseases caused by parasites, such as malaria and syphilis. In 1910, Paul Ehrlich developed Salvarsan as an As-based drug to treat syphilis (Hughes et al., 2011). For a long time and to date, As compounds have been applied as chemotherapeutic drugs against certain forms of leukemia (Chen and Chen, 2017). Epidemics of acute poisoning have been documented since the early 20th century. An example is the Itai-itai disease, a bone disease causing severe pain, which broke out in the 1950s in Japan and was traced to the consumption of rice grown in Cd-contaminated paddy fields. Similarly, occupational exposure of workers in contact with Cd or Pb was associated with serious health effects (Nordberg, 2009). When production of tetraethyl lead started, hundreds of factory workers developed neurological symptoms and several of them died (Needleman, 1998). Relevant in the context of dietary intake, however, are the possible consequences of chronic low-level exposure not causing any noticeable symptoms. This arose as a new question in public health thinking, pioneered, among others, by Patterson. His analyses, which en passant demonstrated lead contamination by food processing, namely packaging in lead-soldered cans (Settle and Patterson, 1980), raised the possibility that a widespread slow poisoning might be underway due to the massive release of Pb into the environment. Over the past decades, a large body of evidence suggesting threats of low-level exposure for human health has accumulated. A variety of health risks affecting different organs are associated with metal or metalloid toxicity. The main organ affected by Pb is the brain. The early observations of Pb toxicity had already reported neurological deficits. Today, chronic Pb exposure is associated with a loss of IQ points and an increase in the incidence of behavioral disorders in children (Lanphear, 2015). A study published in 2003 had indicated negative consequences on intellectual abilities at blood Pb concentrations <10 µg/dl, which had until then been considered provisionally safe (Canfield et al., 2003). Several later studies arrived at similar conclusions (Lanphear et al., 2005). Cd and As are both class I carcinogens, meaning that there is sufficient evidence of carcinogenicity in humans according to the International Agency for Research on Cancer. Cd has been linked mostly to lung cancer and As to lung, skin, and bladder cancer (Straif et al., 2009). One mechanistic basis could be the inhibition of DNA repair. In the case of Cd, these suspected cancer risks are largely inferred from observations related to occupational exposure and smoking. Evidence for adverse health effects of exposure to lower Cd levels exists for the kidney and bones (Nordberg et al., 2018). Cd can bioaccumulate in kidneys for decades and cause damage in a dose-dependent manner. The most sensitive marker known to date for renal effects of Cd is an increase in the urinary secretion of β2-microglobulin (B2M) (see below). Chronic Cd exposure was later also associated with higher osteoporosis risk (Engström et al., 2011). A recent meta-analysis found an association of As, Pb, and Cd exposure with higher risk of cardiovascular disease and coronary heart disease (Chowdhury et al., 2018). Similarly, results for >14 000 adults enrolled in the Third National Health and Nutrition Examination Survey in the USA showed increased overall mortality and cardiovascular disease mortality in the percentile of the population with the highest blood Pb concentrations (Lanphear et al., 2018). Exposure to inorganic As has, in addition, been associated with a multitude of other adverse health effects, including skin lesions, neurological impairment, and multiple immune and endocrine effects (Naujokas et al., 2013). These and many other studies constitute growing evidence for adverse health effects of Pb, As, and Cd at lower exposure levels than previously assumed. They prompted several governmental agencies to revise risk assessments and to call for lowering the provisionally safe exposure limits, as detailed below. Dietary exposure, risk assessments, and regulatory action How exposure levels and epidemiological data on the association between exposure and adverse health effects are combined to assess risk can be illustrated using Cd as an example. Principal sources of metal exposure are water, air, soil, dust, and diet. For Cd, it is generally accepted that the consumption of food accounts for ~90% of total exposure in the non-smoking population (smokers are in addition exposed to the Cd accumulated in tobacco leaves) (Clemens et al., 2013). A comprehensive analysis by the European Food Safety Authority (EFSA) based on a food consumption database and >150 000 analytical results for Cd content of food items concluded that cereals, vegetables, tubers, and roots (i.e. plant-derived food) contribute most to dietary Cd exposure (EFSA, 2012). Average exposure for the European population as a whole is estimated at 2.04 μg kg–1 body weight (BW) per week and, among different age groups, is highest for toddlers, with an average of 4.85 μg kg–1 BW per week. Similar numbers were reported for other parts of the world (Clemens et al., 2013). In certain areas affected by metal pollution, dietary Cd exposure can be higher, as exemplified by a recent case study in Hunan province, China. Even when restricting the analysis to rice and vegetable consumption only, the average dietary intake for the general population was ~16 μg kg–1 BW per week (71.1 μg kg–1 BW per month) and for children ~26 μg kg–1 BW per week (116 μg kg–1 BW per month) (X. Chen et al., 2018). Such estimates have to be assessed in relation to data describing, ideally in a dose-dependent manner, toxic effects of metal exposure. The EFSA Panel on Contaminants in the Food Chain performed meta-analyses of available epidemiological studies (EFSA, 2009) focusing on renal effects, because here the data basis is by far the most extensive and dose–response relationships are established. Absorbed Cd accumulates mainly in the kidney, the critical target organ that shows the first signs of Cd toxicity. Early manifestation of renal damage is a reduced re-absorption of low molecular weight proteins such as B2M, resulting in enhanced urinary microprotein excretion. Urinary Cd, normalized to creatinine, serves as the most widely used biomarker reflecting Cd body burden, because it is highly proportional to Cd concentrations in the kidney especially at chronic intakes. The EFSA meta-analysis included 54 studies and calculated an average urinary Cd for elevated microprotein excretion of 4 μg g–1 creatinine. Because group means are used, interindividual variation had to be taken into account, which is quite substantial for renal effects. The conclusion was that below a urinary Cd of 1 μg g–1 creatinine, 95% of the population would not exceed the threshold for microprotein excretion. Similarly, a more recent risk assessment concluded that population exposures should not exceed what results in urinary Cd concentrations of more than 2 µg g–1 creatinine (Nordberg et al., 2018). Thus, the critical question becomes at which exposure level this urinary Cd concentration is reached. Indeed, a sizeable proportion of the non-smoking adult population around the world shows urinary Cd ≥0.5 µg g–1 creatinine, meaning that there is no margin of safety between the point of departure for adverse effects of Cd on health and the exposure levels of the general population. This led the EFSA CONTAM Panel to recommend a lowering of the provisionally tolerable intake (PTI) limit from the current value of 25 μg kg–1 BW per month—5.8 μg kg–1 BW per week—established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) (FAO/WHO, 2010), to 2.5 μg kg–1 BW per week. Furthermore, since for carcinogenic non-essential elements there are no thresholds below which negative effects can be ruled out, environmental exposure to Cd should be reduced as much as possible (Nordberg et al., 2018). Similar re-evaluations have been performed recently concerning human As exposure. In 2009 the EFSA CONTAM panel concluded, based on epidemiological studies linking As exposure to cancer risk, that the effective PTI of 15 μg kg–1 BW per week was no longer appropriate [EFSA Panel on Contaminants in the Food Chain (CONTAM), 2009]. The WHO/FAO JECFA arrived at the same conclusion in 2010 after modeling dose–response relationships based on epidemiological data for As exposure and cancer incidence (World Health Organization, 2011). It was found that the derived benchmark dose (lower confidence limit), namely the concentration at which a small negative effect of exposure is detectable, is 3.0 μg kg–1 BW d–1, only slightly above the previous PTI of 15 μg kg–1 BW per week. The contribution of diet to overall exposure is well documented for As. The initial attention for the As-related public health crisis in Bangladesh was focused on contaminated drinking water (Nordstrom, 2002). Soon, however, it became clear that consumption of rice represents another important route of As intake (Meharg and Rahman, 2003). For reasons discussed below, rice has a strong tendency to take up and accumulate much more As than other cereals such as wheat or barley (Williams et al., 2007). Moreover, irrigation of rice paddy fields with As-contaminated water caused a build up of As in the soil and concomitant accumulation in rice grains. Early analyses showed that even in regions of Bangladesh with drinking water high in As, rice contributes significantly to overall exposure (Meharg and Rahman, 2003). When drinking water As is in the normal range, rice becomes the main source of As intake, a situation that is by no means restricted to countries where rice is the staple food. A study with >200 pregnant women from New Hampshire, a US region with elevated As in drinking water, found a significant contribution of rice consumption to overall As exposure when urinary As secretion was used as biomarker (Gilbert-Diamond et al., 2011). Recent analyses confirmed these findings for the entire US population (Mantha et al., 2017) and for populations around the world (Davis et al., 2017). Mean dietary exposure in Europe ranges from 0.09 μg kg–1 to 0.38 μg kg–1 BW d–1 among the adult population, and from 0.20 μg kg–1 to 1.37 μg kg–1 BW d–1 for children (EFSA, 2014). Similar levels are reported for North America, slightly higher levels for Asian countries where rice is the main staple food, and ~3-fold higher levels for Bangladesh than for other countries in Asia (World Health Organization, 2011). In principle, these values apply to all age groups, but children are of particular concern, because they are more vulnerable and at the same time show the highest intake relative to body mass. Overall, these numbers translate into a small or no margin of safety between exposure levels and benchmark doses for possible negative health effects in many human populations. The revised risk assessment and exposure data led the EU in 2015 to pass legislation introducing maximum permissible levels of inorganic As. They are 0.1 mg kg–1 for rice-based baby food, 0.2 mg kg–1 for polished rice, 0.25 mg kg–1 for husked rice, and 0.3 mg kg–1 for products such as rice cakes or crackers [Commission Regulation (EC) No. 2015/1006 (future section 3.5 of the Annex to Regulation (EC) No 2006/1881)]. The 0.1 mg kg–1 threshold for baby food was also suggested by the US Food & Drug Administration (Docket No. FDA-2016-D-1099). With respect to Cd, the EU Regulation No. 2006/1881 was amended in 2014 by introducing maximum levels for cocoa-based products [Commission Regulation (EU) No. 488/2014]. Chocolate and other cocoa products are important contributors to overall Cd exposure especially of children. Therefore, effective 1 January, 2019, maximum Cd levels of 0.3 mg kg–1 were set for chocolate with ≥30% and <50% total dry cocoa solids, and 0.8. mg kg–1 for chocolate with ≥50% total dry cocoa solids. The latter threshold was adopted in 2018 by the Codex Alimentarius commission for WHO/FAO food standards (Joint FAO/WHO Food Standards Programme CODEX ALIMENTARIUS COMMISSION 41st Session, Rome, 2018). For Pb, the contribution of dietary exposure is much less clear than for Cd and As. Air-related routes such as the inhalation or ingestion of Pb-containing particles have been commonly seen as most relevant (e.g. Tchounwou et al., 2012; US EPA National Center for Environmental Assessment, 2013). The importance of air pollution for Pb exposure is illustrated by the decrease in average Pb blood levels from ~16 µg dl–1 to <9 µg dl–1 that accompanied the phasing out of leaded gasoline in the USA during the 1970s (Needleman, 2004). Pb intake through the consumption of plant organs has mostly been attributed to Pb deposited atmospherically on plant surfaces (McLaughlin et al., 1999). The reasons for this assumption are very low Pb bioavailability in soil and limited mobility of Pb within plants. Soil–liquid partitioning coefficients (Kd values) are much higher for Pb than for Cd or As (i.e. Pb is more tightly bound to soil particles) (Sauve et al., 2000). Furthermore, once taken up into plant roots, the rate of Pb translocation to aboveground organs is substantially lower than for Cd and As (McLaughlin et al., 2011). Nonetheless, a recent survey of rice across markets and field sites in several countries found a mean value of 0.031 μg kg–1 in brown rice when excluding samples from mining-impacted sites. The authors concluded that for people on a rice subsistence diet, the rice consumption can contribute significantly toward provisionally tolerable total Pb intake as established by the FDA (Norton et al., 2014). In addition, it is important to note that current PTI levels for Pb have been questioned as well. The EFSA CONTAM Panel found that the current PTI of 25 μg kg–1 BW per week is no longer appropriate when considering available evidence [EFSA Panel on Contaminants in the Food Chain (CONTAM), 2010)]. Taken together, recent analyses of exposure levels and associated risks have led various organizations to call for a lowering of dietary exposure. In the case of As and Cd, this has already resulted in legislative action. Molecular insights into the physiology of metal uptake and distribution form the basis for approaches aimed at achieving this goal, as there is no viable way to remove widespread background contamination by toxic, non-essential metals/metalloids. Plant science-based mitigation strategies: reducing dietary toxic element exposure The public health threats associated with Cd and As accumulation in crops have inspired research into the underlying mechanisms, and tremendous progress has been achieved in the past 12 years (reviewed, for example, in Clemens and Ma, 2016; Chen et al., 2017a; Lindsay and Maathuis, 2017). This applies especially to rice both as the most relevant crop species with respect to human exposure and as an established model system. Equally important for the discovery of mechanisms continues to be A. thaliana. Several important genes, proteins. and mechanisms, such as AsV reductases (Chao et al., 2014; Sánchez-Bermejo et al., 2014) or transporters for metal–phytochelatin (PC) complexes (Song et al., 2010), were discovered in this system and then elucidated further in rice. Pronounced variation in toxic element accumulation is well documented and known to exceed by far the variation in micronutrient content. Genetic analysis in a number of large-scale studies (e.g. Pinson et al., 2015; Duan et al., 2017) has demonstrated potential to breed low Cd or low As crops. For example, a recent study on a rice diversity panel of 529 accessions, cultivated over 3 years in two different locations in China, found high broad-sense heritability for As and Cd content of brown rice—while heritability was low for Pb (Yang et al., 2018). In populations derived from biparental crosses, several qantitative trait loci (QTLs) were detected that affect As or Cd accumulation in edible tissues of different crops, such as barley grains (Wu et al., 2015), potato tubers (Mengist et al., 2018), or rice grains (Norton et al., 2012; Abe et al., 2013). In wheat, the Cdu1 locus explains a large fraction of the difference between durum and bread wheat in grain Cd accumulation (Wiebe et al., 2010). To date, however, the genes underlying loci contributing to variation have been isolated only for Cd, and only in three cases restricted to rice. High grain Cd in certain rice cultivars is caused by the presence of defective OsHMA3 alleles (Ueno et al., 2010; Miyadate et al., 2011) (see below for more detail). A QTL contributing to variation in leaf Cd was cloned recently and found to encode a defensin gene (Luo et al., 2018). The difference in Cd accumulation between indica and japonica varieties was traced to a polymorphism in a gene encoding a major facilitator superfamily transporter hypothesized to be involved in Cd uptake (Yan et al., 2019). Factors explaining genotypic variation in As accumulation remain unknown (Chen et al., 2017a). The main sources of molecular insight have been mutant screens and candidate gene approaches. In the following, the uptake of Cd, As, and—with much less detail because of substantial knowledge gaps—Pb, as well as subsequent steps towards storage in edible plant organs will be covered. ‘Upstream’ plant processes influencing the rhizosphere and with that the availability of elements are important, but probably more amenable to agronomic than engineering strategies. An example is the formation of Fe plaques, which strongly affects As solubility (Zhao et al., 2010b). Upon flooding, the redox potential of soils drops, which can liberate As from Fe-(oxyhydr)oxides. This process is slowed down by the presence of manganese (Mn) oxides. As uptake into rice plants can thus be lowered by the addition of Mn oxides to soil (X. Xu et al., 2017). Also, the root physiology can strongly influence microbial activities in the soil, which in turn affect As availability for plants. For example, bacteria can methylate or oxidize AsIII, can reduce AsV as a means of detoxification, or can use it as an electron acceptor in anaerobic AsV respiration (Andres and Bertin, 2016). However, while they are clearly important, these processes are beyond the scope of this review. Once a toxic element is taken up into root cells, three principal fates can be distinguished. The element can be effluxed again, it can be sequestered somewhere in root tissue, or it can reach the long-distance transport routes (Fig. 1). The size of the fraction that reaches the vasculature largely determines accumulation in edible organs aboveground. The uptake into shoot tissues, loading of the phloem, unloading of the phloem, and, for seeds, the uptake into filial tissues are other important steps determining toxic element accumulation (Fig. 1). Fig. 1. Open in new tabDownload slide The movement of toxic elements from the soil solution to grains in rice. A schematic representation of processes involved in the uptake, distribution, and seed loading of non-essential toxic elements. Bottom panel: principally, the rates of uptake, efflux, vacuolar sequestration, and xylem loading determine root to shoot translocation for non-essential toxic elements; orange bars indicate Casparian strips. Above, three separate root cells highlighting specific steps and proteins involved in the principal steps of As (blue symbols), Cd (orange symbols), and Pb transport (violet symbols) are shown. Transporter names are in blue and bold font. Top panel: seeds are phloem fed. The phloem is loaded via xylem to phloem transfer and via remobilization from leaves. Particularly important for xylem to phloem transfer in rice is node I. For details of the structure of node I, which is not depicted here to reduce complexity, please refer to Yamaji and Ma (2014). Proteins implicated in phloem loading are shown (in blue and bold font). Please note that they are tentatively placed. The exact localization in particular cell types is in most cases not clear. The parenchyma cell is not specifically assigned to xylem or phloem. Phloem unloading and transport into filial tissues of the seed are molecularly not understood. Dotted lines indicate unknown transporters. For further details, see the text. Fig. 1. Open in new tabDownload slide The movement of toxic elements from the soil solution to grains in rice. A schematic representation of processes involved in the uptake, distribution, and seed loading of non-essential toxic elements. Bottom panel: principally, the rates of uptake, efflux, vacuolar sequestration, and xylem loading determine root to shoot translocation for non-essential toxic elements; orange bars indicate Casparian strips. Above, three separate root cells highlighting specific steps and proteins involved in the principal steps of As (blue symbols), Cd (orange symbols), and Pb transport (violet symbols) are shown. Transporter names are in blue and bold font. Top panel: seeds are phloem fed. The phloem is loaded via xylem to phloem transfer and via remobilization from leaves. Particularly important for xylem to phloem transfer in rice is node I. For details of the structure of node I, which is not depicted here to reduce complexity, please refer to Yamaji and Ma (2014). Proteins implicated in phloem loading are shown (in blue and bold font). Please note that they are tentatively placed. The exact localization in particular cell types is in most cases not clear. The parenchyma cell is not specifically assigned to xylem or phloem. Phloem unloading and transport into filial tissues of the seed are molecularly not understood. Dotted lines indicate unknown transporters. For further details, see the text. Regarded as most relevant for the elucidation of these steps are studies involving long-term experiments under realistic contamination conditions, namely growth in agricultural soils with only background or slightly elevated levels of the elements in question. When discussing engineering, the focus will be on reducing accumulation in edible organs, with the main emphasis on seeds. Uptake of non-essential elements Since no biological function is known for Cd, Pb, or As in plants, transporters for their uptake probably never evolved. Instead, they enter plant cells through transporters for chemically similar nutrients (Clemens, 2006). For Cd especially, various uptake routes have been postulated, including ZIPs (zinc-regulated transporter/iron-regulated transporter-like protein), calcium (Ca) channels, and non-selective cation channels (Lux et al., 2011). Supporting evidence has come from experiments addressing the effects of competing ions or channel blockers on Cd uptake. For example, flux measurements with Cd ion-selective microelectrodes along rice roots indicated an involvement of Ca channels (X. Chen et al., 2018). A second experimentally demonstrated route of Cd uptake is via Fe transporters. Several studies reported elevated Cd uptake under conditions of Fe deficiency (e.g. Nakanishi et al., 2006). Recently it was shown in a comparison of A. thaliana wild type and the irt1 mutant, defective in the main Fe uptake system, that both the absence of IRT1 and the presence of competing Fe ions reduce Cd uptake (He et al., 2017). Estimating the actual contributions of various pathways to Cd accumulation in crops, however, remains difficult because across studies vastly different external Cd concentrations have been applied in a multitude of cultivation systems. Nonetheless, the major route of Cd uptake into rice roots at least under realistic field conditions appears to be identified. Initially, transporters of the ZIP family, such as OsIRT1 (Lee and An, 2009), had been implicated in Cd uptake. However, the isolation of rice mutants with strongly reduced Cd accumulation in straw and grains enabled the identification of the quantitatively most relevant pathway. Three independent mutants all carried a loss-of-function mutation in OsNramp5 and showed grain Cd levels after growth in a Cd-contaminated paddy field that were >30-fold lower than in the respective wild-type plants (Ishikawa et al., 2012). In parallel, a study on an OsNramp5 insertion line demonstrated strongly reduced Cd accumulation in straw and grains of hydroponically and soil-grown plants. OsNramp5 was localized to plasma membranes at the distal side of both exodermis and endodermis cells. It transports Cd, as demonstrated in short-term uptake experiments (Sasaki et al., 2012). The physiological function of OsNramp5 is hypothesized to be the uptake of Mn ions. Loss of OsNramp5 function led to a strong reduction in Mn content of roots and shoots (Ishikawa et al., 2012; Sasaki et al., 2012). The OsNramp5 locus is underlying a major QTL for grain Mn (Liu et al., 2017). Contrasting views were published concerning the potential use of OsNramp5 loss-of-function alleles for breeding low Cd rice (Fig. 2B). Especially under conditions of limited Mn supply, a strong yield reduction was observed for the insertion line in Zhonghua 11, a japonica landrace (Sasaki et al., 2012), while field data for the three mutants in the Koshihikari background (an elite japonica cultivar) showed wild-type growth and grain yield (Ishikawa et al., 2012). Apparently, the reduced Mn content of shoots in the latter study was still sufficiently above the threshold required for optimal photosynthesis and other physiological activities. Field experiments with OsNramp5 knockout lines generated via CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] in an indica cultivar confirmed the lower Mn levels but again found no yield penalty (L. Tang et al., 2017), suggesting the applicability of OsNramp5 null alleles for breeding. Fig. 2. Open in new tabDownload slide Possible approaches to engineer crops with reduced toxic element accumulation. The objective is to lower the accumulation of non-essential toxic elements (red dots) without compromising the supply of essential or beneficial elements (green dots). (A) Overexpression of proteins mediating the sequestration of toxic elements, predominantly in root cells, but also in other cell types such as phloem companion cells. (B) The use of loss-of-function alleles, for example of genes encoding uptake transporters, in breeding programs. (C) The generation of transporters or other proteins with altered substrate specificity. (D) Heterologous expression of proteins mediating the sequestration or efflux of toxic elements. Respective examples are listed on the right. For details, see the text. Fig. 2. Open in new tabDownload slide Possible approaches to engineer crops with reduced toxic element accumulation. The objective is to lower the accumulation of non-essential toxic elements (red dots) without compromising the supply of essential or beneficial elements (green dots). (A) Overexpression of proteins mediating the sequestration of toxic elements, predominantly in root cells, but also in other cell types such as phloem companion cells. (B) The use of loss-of-function alleles, for example of genes encoding uptake transporters, in breeding programs. (C) The generation of transporters or other proteins with altered substrate specificity. (D) Heterologous expression of proteins mediating the sequestration or efflux of toxic elements. Respective examples are listed on the right. For details, see the text. Furthermore, available evidence indicates that Nramp5 orthologs may also account for a substantial fraction of Cd uptake in other monocots as well as in dicots, suggesting corresponding strategies for other crops. RNAi-mediated knockdown of the barley ortholog HvNramp5 resulted in a reduction of root and shoot Cd content (Wu et al., 2016). The contrasting Cd accumulation behavior of two tobacco cultivars was found to be associated with the presence of a non-functional Nramp5 allele in the genotype with lower Cd in roots and leaves. Again, the physiological function of the transporter appears to be Mn uptake as Mn accumulation was also reduced in the low Cd line (Z. Tang et al., 2017a). While Cd enters plant cells predominantly as the divalent Cd ion, the situation is more complex for As. Several different inorganic and organic As species can co-exist in soil solutions. Their relative abundance is a function of soil conditions and microbial activities. The main inorganic species are arsenite (H3AsIIIO3 and AsIII) and arsenate (H2AsVO4–/HAsVO42– and AsV) (Fig. 3). Under the reducing conditions caused by flooding, AsIII is the dominant form and, thus, the most relevant inorganic form of As in rice paddy fields. Conversely, most As is oxidized to AsV in well-aerated soil (Zhao et al., 2010b, 2013). Fig. 3. Open in new tabDownload slide Arsenic species known to occur in soil and to be taken up by plants. The oxyarsenic species (light blue background) have been extensively studied, while the thioarsenic species (yellow background) have only recently been detected in flooded rice fields and in plant tissues. Fig. 3. Open in new tabDownload slide Arsenic species known to occur in soil and to be taken up by plants. The oxyarsenic species (light blue background) have been extensively studied, while the thioarsenic species (yellow background) have only recently been detected in flooded rice fields and in plant tissues. AsIII enters plant root cells through nodulin26-like intrinsic channel proteins (NIPs), aquaglyceroporins dedicated to the uptake of the beneficial element Si (as silicic acid) or the micronutrient B (as boric acid) (Fig. 1). Examples are Lsi1 (OsNIP2;1) in rice (Ma et al., 2008) and AtNIP1;1 in A. thaliana (Kamiya et al., 2009). Like silicic acid (pKa1=9.51) and boric acid (pKa=9.25), AsIII has no charge under typical paddy field conditions because of its high pKa1 (9.2) and is therefore highly bioavailable (Pommerrenig et al., 2015). The other reason for strong As accumulation in rice is the exceptionally high activity of Si acquisition pathways relative to other cereals. Rice leaves can contain up to 10% of dry matter Si (Ma and Yamaji, 2015). When the major Si uptake channel Lsi1 is defective, short-term uptake of AsIII by rice roots is reduced by >50%, indicating that Lsi1 represents the main entry pathway (Ma et al., 2008). Still, other NIPs probably also contribute. Several NIPs were shown to mediate uptake of AsIII when expressed in oocytes. Among them was OsNIP3;2 (Chen et al., 2017b). Loss of OsNIP3;2 function caused a reduction of AsIII uptake especially by lateral roots. Given the major contribution of Lsi1 to the uptake of the predominant inorganic As species in rice paddy fields, AsIII, and of organic As species (see below), it is at least conceivable that low As rice can be developed via the use of non-functional Lsi1 alleles (Fig. 2B). However, as discussed further below, loss of Lsi1 function did not result in a reduction of grain As anywhere near the effects reported for Osnramp5 mutants and Cd. Grains harvested from plants grown in a field with background As concentrations contained about the same amount of total As in the wild type and the Lsi1 mutant (Ma et al., 2008). Furthermore, efficient Si uptake is highly beneficial for overall health and resilience of rice plants. Therefore, it appears unlikely that low As rice can be developed via the use of non-functional alleles of uptake transporter genes. Furthermore, mechanisms that regulate the pore selectivity of NIPs are still poorly understood (Pommerrenig et al., 2015), and attempts to engineer NIPs in order to reduce AsIII permeability (Fig. 2C) have not been successful (Mitani-Ueno et al., 2011). For crop plants growing in well-aerated soils, AsV is the most relevant chemical form of As (Fig. 3). Also, even in flooded rice fields, a fraction of the phytoavailable As is present as AsV. This applies especially to the immediate vicinity of roots because of their radial oxygen loss (Jia et al., 2014). Another factor contributing to the presence of AsV in flooded soil could be the activity of chemoautotrophic AsIII-oxidizing bacteria (Zhang et al., 2015). Attempts to achieve a reduction in As uptake thus could target phosphate transporters. Plant genomes encode families of Pht1 proteins that account for phosphate uptake across the plasma membrane. In rice, there are 13 Pht1 genes. Pht1 genes and proteins are subject to complex transcriptional and post-translational regulation. The majority are up-regulated under conditions of phosphate starvation. Several Pht1 proteins have been shown to be involved in phosphate acquisition (Gu et al., 2016). For three of them, OsPht1;1 (=OsPT1), OsPht1;4 (=OsPT4), and OsPht1;8 (=OsPT8), a significant contribution to root AsV uptake was demonstrated. Mutants for the respective transporters showed gains in AsV tolerance and reduced As accumulation at least in some tissues when exposed to AsV. However, an effect on grain As could be detected only for OsPht1;4 loss-of-function mutants. After cultivation of plants in soil spiked with As, inorganic As (i.e. the sum of AsIII and AsV) was significantly lower, while no effect on total As was found (Cao et al., 2017). In contrast, a T-DNA insertion mutant with reduced OsPht1;1 expression showed lower As levels in leaves, but no difference from the wild type in brown rice when total As was analyzed (Kamiya et al., 2013). For OsPht1;8 mutants, no data for soil experiments and grain have been reported yet. Overexpression resulted in higher AsV uptake rates without changing As accumulation in grains (Wu et al., 2011). Rice plants lacking the function of OsPHF1, a protein required for proper endoplasmic reticulum (ER) exit of Pht1 transporters, accumulated as much As in their grains as the respective wild type even though the function of several Pht1 proteins was presumably compromised and phosphate uptake was strongly reduced (Wu et al., 2011). Thus, it appears that overall the uptake of AsV via phosphate transporters has little influence on the accumulation of As in grains produced by plants grown in flooded soil (Wu et al., 2011). Consequently, reducing phosphate uptake capacity—if it were possible without risking yield penalties—is not likely to contribute much to reducing grain As accumulation at least in rice, the crop plant of highest concern. Nonetheless, for crops grown in aerated soils, it could be of substantial value to engineer the substrate specificity of Pht1 transporters (Fig. 2C) towards higher affinity for Pi and lower affinity for AsV (Gu et al., 2016). Usually much less abundant in soil solutions than inorganic As species are organic forms such as pentavalent monomethylarsonic acid [MMAV: CH3AsO(OH)2] and dimethylarsinic acid [DMAV: (CH3)2AsOOH] (Zhao et al., 2010b). Often they are alternatively referred to as monomethylarsenate [MMA: (CH3)H2AsVO3–/(CH3)HAsVO32–] and dimethylarsenate [DMA: (CH3)2H2AsVO2–/(CH2)HAsVO22–] (Fig. 3). Their occurrence is highly variable and are either attributable to anthropogenic influence, for example the application of organoarsenicals as pesticides, or are a result of microbial activity (Zhao et al., 2013). Detoxification of AsIII catalyzed by AsIIIS-adenosylmethionine methyltransferases is a widespread mechanism in bacteria (Zhu et al., 2014). Both DMAV and MMAV are taken up into plants, albeit less efficiently than the major inorganic species (Raab et al., 2007). This applies at least to plants cultivated at a pH of ~5.5 (Li et al., 2009). Under such conditions, the methylated As species with their lower pKa values relative to AsIII are partly dissociated. MMAV enters plant cells more readily than DMAV (Lomax et al., 2012). In rice it was shown that the transporter responsible for a major fraction of the AsIII uptake, Lsi1, also mediates MMAV and DMAV uptake into rice roots. In an Lsi1 mutant, uptake of the two species was reduced by ~80% and 50%, respectively, after 24 h exposure (Li et al., 2009). Recently, thioarsenates were detected as an additional class of inorganic As species in paddy fields (Planer-Friedrich et al., 2017) (Fig. 3). They are structural analogs of AsV yet form spontaneously from AsIII under sulfate-reducing conditions by OH–/SH– ligand exchange and oxidative addition of elemental sulfur (Planer-Friedrich et al., 2015). Most routine analyses are prone to missing them because they are unstable in the presence of oxygen or acid (Planer-Friedrich et al., 2007; Planer-Friedrich and Wallschläger, 2009). Uptake into plants was demonstrated for externally applied monothioarsenate, the most stable thioarsenate species. The proteins responsible, however, have not been identified yet. Competition experiments indicated a possible role for phosphate transporters (Kerl et al., 2018). Methylated forms exist not only for oxyarsenicals but also for their thiolated counterparts. Species such as monomethylmonothioarsenate (or monomethylmonothioarsenic acid, MMMTA) or dimethylmonothioarsenate (or dimethylmonothioarsenic acid, DMMTA) have been detected in various biological systems and in the environment (Fig. 3). Thus, they are now considered a major class of As metabolites (Fan et al., 2018). An initial investigation of their interaction with rice plants showed that DMMTA in particular can contribute to As accumulation (Kerl et al., 2019). In summary, when considering entry pathways, the prospects of applying molecular insights are much better for Cd than for As. Even though the most important uptake pathways for AsIII, AsV, and methylated As species are identified, it appears presently rather difficult to obtain low As crops through the engineering of uptake transporters. The uptake of Pb into plant cells is essentially not understood and thus cannot be engineered yet in any reasonable way. In general, very few genes have been identified to date that are involved in Pb accumulation (Fasani et al., 2018). A major reason for lack of knowledge regarding Pb–plant interactions is the technical challenge associated with exposing plants to Pb. As pointed out repeatedly (Huang and Cunningham, 1996; Kopittke et al., 2008b), the addition of Pb salts to widely used culture media such as Hoagland’s or Murashige and Skoog results in the precipitation of Pb-phosphate. It was therefore recommended to study Pb tolerance and accumulation at a pH <5.0 and in the presence of a phosphate concentration not exceeding 10 µM. However, most studies on plants in hydroponic or tissue culture used regular media compositions. Thus, in the vast majority of experiments described in the literature, there was no control over the actual Pb bioavailability and it is not clear how the reported results can be interpreted. A comparison of toxicity data illustrates the problem. When Pb bioavailability is controlled, inhibition of plant growth is observed at external Pb concentrations of ~1 µM. When applied in regular media, doses of several hundred µM or even >1 mM are needed to cause growth effects (Kopittke et al., 2010). For example, A. thaliana seedlings in half-strength MS medium showed only ~20% root growth reduction at 500 µM Pb(NO3)2 while in a medium with low pH and low phosphate concentration they showed >50% root growth reduction at 1 µM Pb(NO3)2 (Fischer et al., 2014). In the latter set-up, most of the added Pb was still in solution and therefore available for uptake. Nonetheless, there is some evidence for the uptake of Pb into roots. Early phytoremediation research, motivated by the relevance of Pb as an environmental contaminant, had already shown that Pb uptake rates are low, and root to shoot translocation rates are even lower (Huang and Cunningham, 1996). This had led to the concept of applying synthetic chelates such as EDTA to soil in order to enhance Pb accumulation. Pb uptake into the symplast appears to be largely restricted to the root meristem and this probably explains Pb toxicity, which manifests itself in root growth inhibition (Eun et al., 2000). In other parts of the root, Pb remains in the apoplast (Kopittke et al., 2008a). Available evidence suggests that Pb entry occurs at least in part through Ca channels. External Ca efficiently inhibited Pb uptake and protected rice roots from Pb toxicity (Kim et al., 2002). The Ca channel blocker lanthanum strongly reduced Pb uptake into wheat roots (Wang et al., 2007). A calmodulin-binding channel-like protein from tobacco (NtCBP4) was the first reported Pb-transporting protein from plants (Arazi et al., 1999). Overexpressing plants exposed to Pb at low pH (4.5) and in the presence of an appropriate Pi concentration (10 µM) showed Pb hypersensitivity. The NtCBP4 homolog from A. thaliana is a cyclic nucleotide-gated channel (CNGC1). A knockout mutant was slightly more Pb tolerant, consistent with reduced uptake of Pb (Sunkar et al., 2000). Since then very little progress has been made in identifying Pb uptake pathways at the molecular level. Efflux of non-essential toxic elements Once they are taken up into the roots, toxic elements can have three different fates: efflux out of the cell into the apoplast; sequestration in root cells; or long-distance transport (Fig. 1). Efflux for As is well documented. A large fraction of the As taken up into roots as AsV or AsIII is effluxed as AsIII. Prerequisite in situations of AsV exposure is the intracellular reduction of AsV as considered in more detail in the next section. Typically 60–80% of the AsV taken up is extruded as AsIII by rice roots (Xu et al., 2007). NIPs such as Lsi1 not only mediate uptake of AsIII, but they also possess bidirectional permeability and thus contribute to the detoxification of As via AsIII efflux as well (Zhao et al., 2010a) (Fig. 1). This might explain why As accumulation in shoots and grains of field-grown lsi1 mutant plants is barely or not at all reduced compared with wild-type plants (Ma et al., 2008). Engineering low As crops through the overexpression of aquaglyceroporins (Fig. 2A) is considered difficult because the transport is passive (Chen et al., 2017a). Still, a recent study found that the overexpression of two AsIII-permeable NIPs, OsNIP1;1 or OsNIP3;3, led to a reduction in shoot As by up to ~60% and in grains by up to ~40% (Sun et al., 2018). Importantly, accumulation of Si and all the tested macro- and micronutrients was not affected in the transgenic plants. In rice roots, nutrient transport follows the so-called coupled transcellular pathway (Barberon and Geldner, 2014) across the exodermis and endodermis via polarly localized uptake transporters (at the distal side of root cells, e.g. OsNramp5 and Lsi1) and efflux transporters (at the proximal side, e.g. Lsi2, see below). Overexpression of OsNIP1;1 or OsNIP3;3 as non-polarly localized transporters mediating efflux of AsIII is hypothesized to reduce the flux through the coupled transcellular pathway and thereby the loading into the xylem (Sun et al., 2018). An alternative approach to enhance As efflux is the heterologous expression of efflux transporters normally not encoded in the genomes of higher plants (Fig. 2D). The main As detoxification pathway in Saccharomyces cerevisiae employs ScACR3, a protein of the bile/arsenite/riboflavin transporter (BART) superfamily that mediates AsIII efflux (Wysocki and Tamás, 2010). Expression of ScACR3 led to an enhancement of As efflux in A. thaliana (Ali et al., 2012) and rice (Duan et al., 2012). However, a significant reduction in shoot and grain As was observed only for rice. Brown rice of transgenic plants showed up to 26% less As after cultivation in paddy fields irrigated with As-containing water (Duan et al., 2012). ACR3 homologs are not present in genomes of higher plants, but have been found in a few lower plant species including the As-hyperaccumulating fern Pteris vittata. PtACR3 was hypothesized to reside in the vacuolar membrane and to mediate sequestration of As (Indriolo et al., 2010). When overexpressed in A. thaliana, it was localized to the plasma membrane and conferred higher AsIII efflux (Chen et al., 2013). Root As levels were strongly reduced. Concomitantly, As translocation to the shoot was enhanced, because the efflux activity also stimulated xylem loading. Thus, effective lowering of As accumulation in grains and leaves will most probably require restricting the expression of efflux transporters to certain cell types such as epidermal and cortex cells (Chen et al., 2017a). Cd efflux across the plasma membrane of plant cells is predominantly mediated by P-type ATPases of the IB-2 type (Hanikenne and Baurain, 2014). They are found in bacteria, archaea, land plants, green algae, and a patchy group of other eukaryotes including, for example, diatoms. Representatives of this class of proteins in A. thaliana are HMA2 and HMA4 (for heavy metal ATPase). HMA3, another member of this group, resides in the tonoplast and sequesters Cd as discussed below. Efflux activity contributes to Cd detoxification as it lowers the cellular Cd load. However, with respect to accumulation in aboveground tissues, the same applies as discussed for PtACR3. HMA2 and HMA4 in A. thaliana are strongly expressed in the root vasculature, and HMA-dependent efflux across the plasma membrane loads Cd into the xylem, thereby enhancing translocation (Hussain et al., 2004; Wong and Cobbett, 2009). In the Cd-hyperaccumulating species Arabidopsis halleri, the triplicated AhHMA4 locus represents the main factor enabling strong shoot accumulation of Cd and is essential for Cd hypertolerance (Hanikenne et al., 2008). Thus, a possible use of efflux pumps in overexpression approaches would have to control the sites of expression carefully. Loss of function, on the other hand, has severe consequences for the Zn supply to the shoot (Hussain et al., 2004). An alternative Cd efflux pathway suggested to be potentially useful for the generation of low-Cd crops is dependent on the A. thaliana ABC-transporter ABCG36/PDR8/PEN3, expressed in epidermal cells of roots and leaves (Kim et al., 2007). Loss-of-function mutants for this plasma membrane-localized transporter showed compromised Cd tolerance and more accumulation of Cd in roots and shoots. Conversely, overexpressing lines were more Cd tolerant and accumulated less Cd. Cd flux assays with protoplasts derived from gain-of-function and loss-of-function lines showed higher or lower Cd efflux activity, respectively, than wild-type protoplasts. Corresponding observations at least with respect to tolerance were reported upon Pb exposure, while Pb content was not altered. Under conditions controlling Pb phytoavailability, however, the Pb tolerance effects of knocking out ABCG36/PDR8/PEN3 could not be confirmed (Fischer et al., 2014). Engineering of Pb efflux was achieved by expressing a bacterial P1B-ATPase metal pump, ZntA from Escherichia coli, in A. thaliana (Lee et al., 2003). Transgenic lines accumulated ~55% less Pb in their shoots, which was interpreted as evidence for effective ZntA-mediated export of Pb out of root epidermal and cortex cells. The trapping of toxic metals and metalloids Generally, the bulk of non-essential metals and metalloids taken up into the plant symplast stays in the root. Another portion never enters the symplast and remains in the apoplast. For some elements, such as Pb, the cell wall appears to represent the major site of accumulation (Bovenkamp et al., 2013). With few exceptions (such as methylated As species considered below), only a fraction of the elements that reach the root symplast is translocated to the shoot. Thus, the rate of accumulation in leaves, seeds, and fruits is largely determined by the ratio between the fraction available for xylem loading and the fraction sequestered in root cell vacuoles as the main storage site. Trapping in vacuoles is important for plants as it removes potentially toxic elements from far more sensitive sites in the cytosol or other organelles, and thereby confers tolerance (Zhang et al., 2018). The PC pathway represents the most important detoxification pathway for Cd, As, and Pb. Mutants with a defect in essential components of the PC pathway are hypersensitive to these elements (Cobbett and Goldsbrough, 2002; Song et al., 2010; Fischer et al., 2014). This was demonstrated in A. thaliana and in rice (Song et al., 2014b; Hayashi et al., 2017; Uraguchi et al., 2017; Yamazaki et al., 2018). When ions of these metals enter a plant cell, PC synthases are activated. These constitutively expressed enzymes use the ubiquitous redox buffering thiol glutathione (GSH) as substrate and synthesize PCs, peptides with the general structure (γ-glutamylcysteine)n-glycine. Correspondingly, a disturbance of GSH synthesis or homeostasis compromises metal/metalloid tolerance as, for example, recently shown when the AsV hypersensitivity of a rice mutant was traced to a defect in OsCLT1. The encoded protein is hypothesized to transport γ-glutamylcysteine and GSH from plastids—the site of synthesis—to the cytosol (Yang et al., 2016). PCs can form complexes with various metals. In plants exposed to AsIII or AsV, several different As–GS and As–PC complexes can be detected (Raab et al., 2005). Depending on plant species, exposure levels, and duration of the experiments, PC2 or PC3 complexes involving both classic PCs and derivatives such as hydroxymethyl-PC or des-glycine-PC have been detected as the dominant forms (Raab et al., 2005; Liu et al., 2010). Monomethylarsonic PC2 (MAIII–PC2) was found as well. As–PC complexes account for up to 80% of the total extractable As in roots. Correspondingly, in a study employing synchrotron-based fluorescence X-ray absorption near-edge spectroscopy of wheat and rice roots to determine lateral distribution of As, all As detected in the cortex and stele was AsIII, regardless of inorganic As species in the external medium, and complexed with thiols (Kopittke et al., 2014). AsV was restricted to the epidermal cell layer of AsV-exposed roots. Detection of Cd–PC and other metal–PC complexes is much more challenging. In contrast to As–PC complexes, Cd–PC complexes are not stable in an acidic environment such as the vacuole (Johanning and Strasdeit, 1998). The formation of high molecular weight Cd–PC complexes including sulfide has been reported predominantly in fission yeast, Schizosaccharomyces pombe, when treated with growth-inhibiting Cd doses (Cobbett and Goldsbrough, 2002). Thus, the presence of Cd–PC complexes in plant cells not exposed to extremely high Cd concentrations is probably more transient. They are therefore more difficult to extract from tissue and to analyze in an intact form. Consequently, data on their occurrence and exact chemical nature in plants not exposed to metal excess beyond the toxicity thresholds are missing. The final step of the PC pathway is the transport of metal–PC complexes into the vacuole (Fig. 1). The transporters responsible were first identified in A. thaliana. A double mutant with defects in the genetically redundant ABC-type transporters ABCC1 and ABCC2 was found to be highly As sensitive (Song et al., 2010) due to a deficiency in the ability to transport metal–PC complexes across the tonoplast. The As hypersensitivities of abcc1/2 and the cad1-3 mutant are equal, indicating that As can only be efficiently detoxified through vacuolar sequestration. This applies to Hg as well, while Cd exposure caused stronger growth inhibition of the cad1-3 mutant (Park et al., 2012). The latter observation suggested a partitioning of Cd–PC complexes between ABCC1/ABCC2-dependent vacuolar sequestration and an unknown route that achieves some degree of protection from toxicity even without transport into the vacuole. Given the effective vacuolar sequestration of metals via PC formation and transport of metal/metalloid–PC complexes, it is not surprising that the PC pathway strongly influences the accumulation of toxic metals in aboveground tissues including grains. Ample evidence supports the major role of PC-mediated trapping for restricting root to shoot translocation of As in plants exposed to various As species. When A. thaliana wild-type plants were exposed to AsV, a large fraction (~70%) of the As in roots was found to be complexed with PCs. This complexation reduces mobility as shoots of the cad1-3 mutant lacking a functional version of the major PCS gene, AtPCS1, contained ~3-fold higher As concentrations (Liu et al., 2010). Correspondingly, rice mutants with a defect in one of the two PCS genes in the rice genome (OsPCS1=Os05g0415200 and OsPCS2=Os06g0102300) accumulate more As in their grains when cultivated in a soil with an As content typical for As-polluted agricultural soil [please note: we initially designated the PCS genes in rice differently (Uraguchi et al., 2017); here the numbering used by other research groups (Hayashi et al., 2017; Yamazaki et al., 2018) is adopted]. One of the mutants (has2 for high arsenic, carrying a defect in OsPCS1) was identified in a screen for rice plants with elevated grain As. The mutant accumulated ~5-fold more As in grains than wild-type plants, while the As concentrations of node I were much lower. A T-DNA insertion line for OsPCS2 cultivated in soil with an environmentally relevant As contamination (0.73 mg kg–1 DW) accumulated ~2-fold more As in the grains than the respective wild type (Uraguchi et al., 2017). In a comparative study of rice cultivars with contrasting As accumulation behavior, a significant negative correlation was found between the level of PC production and the transfer factors for inorganic As from roots to grains and shoots to grains (Batista et al., 2014). The importance of the PC pathway for restricting As mobility was further demonstrated by the other mutant (has1) isolated in the screen for rice genotypes with elevated As accumulation (Hayashi et al., 2017). It carries a defect in OsABCC1, the ABC-type transporter responsible for the vacuolar sequestration of As–PC complexes. OsABCC1 is ubiquitously expressed in rice plants and had previously been shown to contribute significantly to restricting As movement towards the grains (Song et al., 2014). Knockout mutants grown to maturity in the presence of As contained >10-fold more As in grains than the respective wild-type plants, because sequestration of As–PC complexes in vacuoles of root cells and phloem companion cells is disabled (Fig. 1). As in the OsPCS1 mutant has2, much less As accumulated in node I. The shift in the distribution illustrates (i) the major role of node I in controlling the flow of nutrients as well as non-essential elements towards the grains in rice plants (Yamaji and Ma, 2014) (see below, grain loading); and (ii) the function of PC-mediated As trapping also in organs other than the root. As storage in node I was visualized by synchrotron μX-ray fluorescence (μ-XRF) and shown to be dependent on thiols as a strong reduction in node As accumulation was observed upon inhibition of GSH biosynthesis (Chen et al., 2015). AsV and MMAV do not directly interact with thiol peptides such as GSH and PCs. Instead, they are reduced to the trivalent AsIII and MMAIII, which are then complexed predominantly by PCs. For AsV-exposed plants, this is well documented; for MMAV-exposed plants, it is inferred from the detection of MMAIII inside plants (Lomax et al., 2012; Mishra et al., 2017) and the role of the PC pathway in protecting plants from MMAV toxicity. The cad1-3 and abcc1/2 mutants of A. thaliana are both hypersensitive not only to AsV but also to MMAV (Tang et al., 2016). Initial reports on the molecular identification of AsV reductases in plants were later falsified when knockout mutants for the suspected gene ACR2 were carefully analyzed (Liu et al., 2012). True AsV reductases were finally found when natural variation in AsV tolerance and leaf As content was genetically dissected in A. thaliana. Two studies independently identified HAC1/ATQ1. Loss-of-function alleles of HAC1/ATQ1 present in natural A. thaliana accessions cause AsV hypersensitivity (Sánchez-Bermejo et al., 2014) and a strong increase in leaf As accumulation when plants are exposed to AsV (Chao et al., 2014). AsV is no longer efficiently reduced to AsIII and thus escapes the PC detoxification pathway. More cellular damage is caused, explaining the hypersensitivity, and more As escapes the trapping in root cells, explaining the stronger translocation to the shoot. Twelve putative HAC1/ATQ1 orthologs were subsequently found in the rice genome. OsHAC1;1 and OsHAC1;2 both encode AsV reductases and show partly overlapping expression zones in roots. AsV-exposed mutant lines did not exhibit any differences from wild-type plants in AsV uptake, but effluxed less AsIII and accumulated more As in shoots and grains. Effects were exacerbated in a double mutant (Shi et al., 2016). Similar to the work with A. thaliana, an AsV-hypersensitive rice mutant turned out to carry a mutation in an AsV reductase gene, in this case HAC4 (Os02g06290) (J. Xu et al., 2017). OsHAC4 is strongly expressed in root epidermis and exodermis. It appears to represent the dominant AsV reductase in rice roots. Following AsV treatment, OsHAC4 mutant plants accumulated >5 times more As in shoots than the wild type. The importance of As sequestration in roots is further demonstrated by the much higher mobility of DMAV relative to inorganic species and the monomethylated organic As species MMAV. When exposures to AsV, AsIII, MMAV, and DMAV are compared, root to shoot translocation factors for DMAV are consistently higher than those for the other As species (Mishra et al., 2017). The same applies to the partitioning between vegetative tissues and grains. Differences of up to two orders of magnitude have been reported, with DMAV showing a much higher transfer rate than AsIII (Lomax et al., 2012). The reason is most probably that DMAV bypasses PC-mediated trapping in the roots and the vascular tissues. Only trivalent As is a soft acid prone to interact strongly with thiol groups. However, trivalent DMAIII has not been detected in extracts of plant tissues, indicating that plant cells do not reduce DMAV (Mishra et al., 2017). Several strategies to obtain crop plants low in toxic element accumulation have already been pursued or can be envisaged, involving the engineering of sequestration pathways. In line with the major role of PCs as ligands for trivalent As, the stimulation of PC-dependent As trapping in roots or along the vasculature represents a promising approach and can be achieved in various ways (Fig. 2). The constitutive overexpression of a PC synthase in a rice OsPCS1 mutant background was shown to reduce As accumulation in grains to levels below those in wild-type plants (Hayashi et al., 2017). Similarly, heterologous expression of a PC synthase from the aquatic plant Ceratophyllum demersum in rice resulted in ~50% lower grain As when plants were cultivated under flooded conditions in pots (Shri et al., 2014). Engineering vacuolar sequestration of As–PC complexes in rice was achieved by expressing OsABCC1 driven by the RCc3 promoter, which is specific for root cortical cells, internodes, and nodes (Deng et al., 2018). After cultivation in normal soil with basal As levels, total As accumulation in brown rice was reduced by ~25–40% for different transgenic lines. The effect could be slightly enhanced by the constitutive co-expression of a bacterial γ-glutamylcysteine synthetase, which presumably boosted PC synthesis by supplying a precursor of GSH. A further improvement was attained when YCF1 from S. cerevisiae, a vacuolar transporter for metal/metalloid–GS complexes, was added. Grain As was reduced by up to 70%. Important yield-related agronomic traits were not influenced. Interestingly, overexpression of OsABCC1 alone or in combination with the other genes under control of a ubiquitously active promoter (ubiquitin) did not result in a reduction of grain As. Overexpression of the step upstream of PC-mediated sequestration (i.e. reduction of AsV to AsIII) could be relevant when rice is grown in aerated soil or for targeting the fraction of As that is present even in flooded soil due to the release of oxygen from rice roots (Shi et al., 2016). Higher reductase activity may potentially enhance AsIII efflux or the trapping of AsIII as thiol complexes in AsV-exposed plants. Which process will dominate probably depends on the localization of the reductase within the root and the relative abundance of effluxing aquaglyceroporins versus components of the PC pathway in different cell types. For example, reduction in the epidermis is likely to favor efflux of AsIII (Salt, 2017). To date, only constitutive overexpression has been reported for a few of the >10 HAC genes in the rice genome. Overexpression of either OsHAC1;1 or OsHAC1;2 resulted in plants with elevated AsIII efflux and reduced As accumulation in roots and shoots. Following cultivation in As-amended soil with free drainage, thus under conditions that shift the balance of As species towards AsV, the grains of overexpressing lines contained up to 20% less As (Shi et al., 2016). Similar effects on the root and shoot accumulation were found for rice overexpressing OsHAC4 (J. Xu et al., 2017). While the accumulation of Cd in aboveground tissues is governed to a similar extent by the rate of root to shoot translocation (Uraguchi and Fujiwara, 2013), the contribution of the PC pathway to sequestration is less pronounced than in the case of As (Fig. 1). In fact, when rice plants were exposed to low, environmentally relevant concentrations of Cd in a soil experiment, an OsPCS2 T-DNA insertion line accumulated less Cd in leaves and grains than the wild type (Uraguchi et al., 2017). An OsPCS1 mutant (has2) grown hydroponically with external Cd accumulated about as much Cd in leaves and grains as the wild type (Hayashi et al., 2017). This shows that PC–Cd complexes are less likely to be transported by ABCC1 transporters. Instead, they can even facilitate the mobilization to the shoot. Tolerance data suggest that PC-mediated vacuolar sequestration of Cd is even less important in rice than in A. thaliana. While there was no loss of Cd tolerance observed for OsABCC1 mutants (Song et al., 2014b), the A. thaliana abcc1/2 double mutant showed at least a slight Cd hypersensitivity (Park et al., 2012). On the other hand, in species such as barley, vacuolar sequestration of Cd via PC complexation may be more important than in A. thaliana. Cd–PC and As–PC complexes were transported equally well into barley vacuoles (Song et al., 2014a). An approach to efficiently trap As and at the same time suppress the mobilization of Cd by PCs could be the engineering of kinetic properties. The enzyme PCS is activated by various metals and metalloids. While the structural basis for this activation is not yet understood, a truncated version of AtPCS1 was recently described that showed a stronger activation by AsIII relative to Cd than the wild-type protein (Uraguchi et al., 2018). Most important for restricting root to shoot translocation of Cd in rice appears to be another PIB-ATPase, namely OsHMA3 (Fig. 1). Its role became clear when the difference in grain Cd content between contrasting rice accessions was genetically dissected. In two independent studies, loss-of-function mutations in the OsHMA3 gene were found to be causal for higher Cd translocation rates and elevated grain Cd (Ueno et al., 2010; Miyadate et al., 2011). Later, the strong Cd accumulation capacity of additional rice cultivars was attributed to the presence of defective HMA3 alleles (Yan et al., 2016) or alleles with weaker promoter activity (Liu et al., 2019). OsHMA3 is expressed mainly in roots, and the encoded protein resides in the tonoplast, mediating the sequestration of Cd in root cell vacuoles. In A. thaliana, allelic variation in AtHMA3 explains some of the natural variation in leaf Cd accumulation (Chao et al., 2012). Non-functional alleles such as those found in Col-0 are associated with higher Cd accumulation. Given the dominant role of OsHMA3 in restricting root to shoot translocation of Cd in rice, an obvious strategy is the overexpression of HMA3 (Fig. 2A). A first attempt using the strong constitutive maize ubiquitin promoter already demonstrated a dramatic effect. Following cultivation in soil with 1.5 mg Cd kg–1, grain Cd was reduced from ~6 mg kg–1 in the Nipponbare wild type to <0.5 mg kg–1 in two overexpression lines (Ueno et al., 2010). Similarly, expression of OsHMA3 under control of the OsHMA2 promoter (for the role of OsHMA2 see below) strongly reduced grain Cd, this time from ~0.5 mg kg–1 to ~0.05 mg kg–1 (Shao et al., 2018). Importantly, OsHMA3 overexpression appears to be feasible without causing yield penalties. Concentrations of micronutrients were not affected in the transgenic lines even though OsHMA3 is known to transport Zn as well. This could be explained by the compensatory up-regulation of Zn uptake transporters in the OsHMA3-overexpressing lines (Sasaki et al., 2014). Further illustrating the potential of OsHMA3 overexpression for reducing rice grain Cd is a recent study reporting respective experiments with a popular indica variety. Grain Cd was consistently reduced by >90% in two field sites and in two different years with non-significant effects on yields (Lu et al., 2019). Radial transport and xylem loading When plants are exposed to one of the four As species on which practically all published studies have focused, three of these species can be detected in the xylem sap, namely AsIII, DMAV, and MMAV (Li et al., 2009). AsV is either not detected or only in traces even when applied to the growth medium, because it is reduced inside plant cells as discussed above. MMAIII formed in roots is also not found in the xylem. Molecular understanding of xylem loading is fragmentary. The anion permease Lsi2 accounts for most of the AsIII transport via the transcellular pathway towards the xylem of rice plants (Ma et al., 2008). Lsi2 is localized in the plasma membrane on the proximal side of exo- and endodermal cells and its physiological function is the transport of Si towards the xylem (Fig. 1). As concentrations in xylem sap of rice lsi2 mutants exposed to arsenite were strongly decreased (by nearly 50% in the absence of external Si). The effect on grain As was of about the same magnitude. However, similar to Lsi1, loss of Lsi2 function resulted in severe yield reductions because of the ensuing Si deficiency (Ma et al., 2007). Thus, the use of null alleles for breeding is not possible. Instead, an engineering of substrate specificity could be envisaged but has to date not been reported on. Aside from genetic interventions, the role of Lsi2 explains the beneficial effects of Si fertilization on lowering rice grain As. Externally applied Si reduced As concentrations in the xylem sap of wild-type but not of lsi2 mutant plants (Ma et al., 2008). Besides Lsi1 (=OsNIP2;1) and AtNIP1;1, several other NIPs have been shown to be permeable for AsIII. Among them is A. thaliana NIP3;1. It is expressed across the root, and loss-of-function mutants treated with AsIII accumulate less As in shoots (Xu et al., 2015). This is accompanied by less growth inhibition of shoots in the presence of toxic As concentrations. Mutants of A. thaliana NIP7;1 showed lower As content of xylem sap and seeds, implicating this transporter in the loading of AsIII into xylem and phloem (Lindsay and Maathuis, 2016). Additional NIPs could also contribute to the loading of AsIII into the xylem in rice (Lindsay and Maathuis, 2017). Candidates for the efflux of AsV would, in analogy to the uptake of AsV and to the role of Si transporters for AsIII, be respective phosphate transporters such as PHO1. However, root to shoot translocation is unaffected in A. thaliana pho1 mutants when AsV reduction is intact (Wang et al., 2017), indicating that AsV reduction is indeed nearly quantitative in wild-type plants. No AsV is thus available in xylem parenchyma cells for loading into the xylem. How methylated As species reach the xylem has not yet been elucidated (Fig. 1). Lsi2 can be ruled out based on the data available for the respective mutant lines. DMA-fed plants accumulated as much DMA in roots, xylem sap, and shoots as the wild type (Li et al., 2009). The peptide transporter OsPTR7 was recently described as a candidate for DMAV loading or unloading of xylem or phloem (Z. Tang et al., 2017b). When expressed in Xenopus laevis oocytes, OsPTR7 shows DMAV permeability. In OsPtr7 mutants, DMAV was less efficiently translocated from roots to shoots. Strikingly, no DMAV was detected in brown rice after cultivation in a paddy field, while the DMAV fraction of total As was ~35% in wild-type plants. Accumulation of inorganic As was unaffected. As mentioned above in the context of Cd efflux, loading of Cd into the xylem is largely attributable to the activity of PIB-ATPases. An A. thaliana hma2hma4 double mutant was found to retain only 2% of wild-type root to shoot translocation of Cd (Wong and Cobbett, 2009). In rice, as predicted based on homology to AtHMA4 (Nocito et al., 2011), OsHMA2 was identified as a major contributor to xylem loading. The protein is localized in the plasma membrane and most strongly expressed in roots (Satoh-Nagasawa et al., 2012; Takahashi et al., 2012). Insertion lines showed strongly reduced root to shoot translocation of Cd and Zn. After growth in paddy fields, grain Cd was up to ~50% lower (Takahashi et al., 2012). A contrasting hypothesis not concerning the effects of OsHMA2 on Cd distribution, but rather with respect to the transport direction was published after the initial reports (Yamaji et al., 2013). The observed reduction in grain Cd was even stronger than previously reported. However, in order to reconcile a lack of effects on Zn xylem sap concentrations and the immunodetection of the protein in phloem areas, the authors proposed an influx activity. Based on established knowledge about HMAs from bacteria to humans, this would be highly unusual as the consensus assumes efflux. Otherwise most reported physiological effects and the coupled binding of substrate metal and ATP hydrolysis would be difficult to explain (Argüello et al., 2007). HMA2 and 4 orthologs play a key role in xylem loading of the micronutrient Zn. Loss-of-function mutants are severely compromised in their Zn supply to the shoot, resulting, for example, in poor fertility. When targeting the Cd xylem-loading process, knockout of HMAs is therefore not an option. Instead, one would have to find and introduce variants able to better discriminate against Cd (Fig. 2C). A recent homology modeling of AtHMA4 based on the crystal structure of the E. coli ZntA protein identified amino acids presumably involved in transport (Lekeux et al., 2019). The respective mutated variants were expressed under control of the AhHMA4 promoter in the A. thaliana hma2hma4 double mutant. Testing of shoot Zn and Cd accumulation revealed one variant that still complemented the mutant but showed 80% reduced Cd. Also, two variants with the opposite effect were found. They no longer loaded Zn into the xylem while retaining wild-type Cd accumulation. From these data, it appears feasible to modulate substrate specificity and to generate HMAs with reduced or even neglible Cd transport activity. Seed loading Ultimately, consumption of seeds accounts for most of the human exposure to toxic elements. The importance of seeds, however, does not positively correlate with the amount of knowledge available on the immediate processes of seed loading. In fact, few proteins have to date been assigned a direct function in moving metals or metalloids into seeds or between seed tissues (Olsen et al., 2016). Most of the steps discussed above, such as uptake into roots, xylem loading, or the trapping in vacuoles, of course indirectly influence the rate of accumulation in seeds, and seed content is the most relevant endpoint when aiming at engineering crops low in toxic elements. This applies in a similar way to the loading of tubers where molecular knowledge is even more fragmentary. Seeds are fed via the phloem. Accordingly, ~90% of the AsIII reaches a rice grain through the phloem (Carey et al., 2011; Zhao et al., 2012). Thus, the first step to consider is the loading of the phloem. Furthermore, unlike in other sink tissues, there is no symplastic continuity. Elements have to leave the symplast of the maternal tissue into the seed apoplasmic space and then be taken up into the symplast of filial tissues (Patrick and Offler, 2001). Thus, at least two additional transport steps across membranes are needed (Lindsay and Maathuis, 2017) (Fig. 1). In A. thaliana, two inositol transporters, INT2 and INT4, were shown to control the loading of As into seeds (Duan et al., 2016). The proteins were predominantly found in plasma membranes of phloem companion cells and mediated phloem loading of inositols via proton-driven symport. Both can also transport AsIII. Plants carrying a knock-out allele of either INT2 or INT4 contain less As in phloem sap, shoots, siliques, and seeds, while no effect on xylem sap As concentrations was seen. Thus, these transporters most probably load AsIII into the phloem. It remains to be determined whether similar sugar transporters are involved in the transport of As in rice and other crops as well. Also, the uptake into cells of filial tissues is not understood. Aquaglyceroporins have been proposed as likely candidates (Lindsay and Maathuis, 2017). Proteins such as AtNIP7;1 could be involved in both the loading of phloem and the uptake into the seed (Lindsay and Maathuis, 2016). For Cd or Pb, no transporters have yet been implicated in phloem or seed loading in A. thaliana. By analogy, it appears likely that metal pumps HMA2 and HMA4, shown to be important for the export of Zn from the maternal tissues (Olsen et al., 2016), are mediating Cd export as well. However, no data addressing this question are available. When considering the minimum three steps of seed loading, the situation in rice is similar to that in A. thaliana. A few components of phloem loading are identified. However, proteins involved in phloem unloading and uptake into grain tissues remain unknown (Khan et al., 2014). Two principal pathways of phloem loading during the reproductive phase can be differentiated: (i) uptake from the soil, xylem transport, and intervascular exchange in nodes directing the elements; or (ii) remobilization of stored elements from flag leaves (Uraguchi and Fujiwara, 2013) (Fig. 1). A major control point of phloem loading in rice is the node. Especially in node I, xylem to phloem transfer occurs and the flow of nutrients or toxic elements is partitioned between leaves and grains (Yamaji and Ma, 2014). A large number of transporters are postulated to mediate the transfer between different vascular bundles in the node. Three of them have to date been implicated in Cd transfer to the grain. OsLCT1, a homolog of the first plant transporter shown to mediate Cd transport, TaLCT1 (Clemens et al., 1998), is strongly expressed around vascular bundles in node I, especially during the reproductive stage, and localized to the plasma membrane (Uraguchi et al., 2011). When OsLCT1 expression was knocked down by RNAi, the resulting plants were found to accumulate up to ~50% less Cd in grains than the wild type after cultivation in pots with normal soil. Since the Cd concentration was lower than that of the wild type in phloem sap, but not in xylem sap, OsLCT1 is hypothesized to be involved in the xylem to phloem transfer of Cd. Growth as well as grain elemental profiles—with the exception of Cd—were not altered relative to wild-type plants. Thus, OsLCT1 loss-of-function alleles could be useful tools to reduce grain Cd (Fig. 2B). Indeed, a recent study on rice lines carrying a OsLCT1 deletion generated by CRISPR/Cas9 showed 60% reduction in grain Cd after cultivation in a paddy field with low Cd contamination (0.9 mg kg–1 soil) (Songmei et al., 2019). Yield per plant was not significantly decreased. Still, more extended field trials are needed to show how useful a target gene OsLCT1 is for breeding low Cd rice. The metal pump OsHMA2 is expressed not only in root vascular tissue where it is involved in xylem loading (see above), but also in the phloem region of vascular bundles in nodes. After cultivation in a paddy field, the brown rice harvested from two OsHMA2 insertion mutants contained much less Cd than wild-type grains (Yamaji et al., 2013). Zn concentrations of organs above node I were also reduced, indicating a role for OsHMA2 in preferential loading of Zn into the panicle. Insufficient Zn supply to reproductive organs probably explains the severe yield reduction observed in insertion mutants. It was mostly due to much lower fertility, a phenomenon previously observed in A. thaliana hma2hma4 mutants (Hussain et al., 2004) and indicative of a particularly high Zn demand of reproduction. Simple OsHMA2 knockout is therefore not a viable strategy for developing low-Cd rice. A prerequisite would be the identification of variants with better discrimination against Cd as a substrate (see discussion of xylem loading above). The third node-expressed transporter implicated in grain Cd loading is the cation/calcium exchanger OsCCX2. Two knockout mutants generated with CRISPR/Cas9 produced grains with Cd reduced by nearly 50% (from ~0.24 mg kg–1 to ~0.14 mg kg–1) after cultivation in a paddy field with background Cd contamination. OsCCX2 is proposed to represent an efflux transporter residing in the plasma membrane (Hao et al., 2018). It may in fact also contribute to xylem loading of Cd in a similar fashion to OsHMA2 because xylem sap Cd was lower in OsCCX2 mutants. Wild-type elemental profiles of grains and a rather mild reduction in the 1000 grain weight of mutants make OsCCX2 a suitable engineering target. Here, as in many other cases, it would be highly informative to directly compare the effects of different mutants and their respective wild types under a variety of realistic field conditions in order to determine the relative contribution of distinct pathways to the loading of Cd into grains. Intervascular transfer in node I is also important for the phloem loading of AsIII. One of the required efflux activities in cells surrounding the xylem is most probably provided by Lsi2 (Fig. 1). The Lsi2 gene is strongly expressed in nodes where As (as well as Cd) accumulates. AsIII-fed excised panicles of mutant plants contained more As in nodes and less As in grains than wild-type panicles, suggesting an Lsi2-dependent flux of AsIII towards the grain (Chen et al., 2015). As considered above, the opposite effect is caused by PC-dependent trapping in vacuoles of phloem parenchyma cells in the node, which restricted mobility of AsIII in the phloem (Song et al., 2014b; Chen et al., 2015) (Fig. 1). How the actual phloem loading of AsIII occurs in rice remains to be elucidated. The role of inositol transporters and NIPs has to be clarified; additional factors need to be identified. For methylated As species, OsPTR7 is a candidate (Z. Tang et al., 2017b). Speciation of As and Cd within the phloem is only partially understood. Not only is the sampling of phloem sap difficult, but the analysis of metal–ligand complexes is also technically extremely challenging, especially in plants exposed to environmentally relevant concentrations (Alvarez-Fernández et al., 2014). In several studies, Cd was found to be associated with PCs, and small sulfur-containing proteins, for example when rice phloem sap was analyzed by size-exclusion chromatography and protease treatment (Kato et al., 2010). In contrast, AsIII, the main As species in phloem sap even of AsV-treated plants, is hypothesized to be mostly present in a non-complexed form. This was inferred from the analysis of the ‘phloem sap model’ Ricinus communis (Ye et al., 2010) and the limited stability of AsIII–PC complexes in alkaline pH (Johanning and Strasdeit, 1998). In castor bean plants treated with the organic As species MMA and DMA, these molecules were also detected in phloem sap (Ye et al., 2010). A second important question besides the seed loading processes and highly relevant with respect to food safety and human exposure is the partitioning of toxic elements between the different parts of the seed—in the case of a cereal grain, the husks, the bran, and the endosperm. There has been tremendous progress in the past years with regard to imaging and quantifying metals and metalloids in plant tissues (Zhao et al., 2014; Kopittke et al., 2018). Powerful X-rays produced in synchrotrons can be used to analyze the distribution of particular elements in a biological sample or to obtain information on the ligand environment. When inductively coupled plasma MS is coupled to laser ablation, multielement analyses of tissues can be performed with high spatial resolution. Even higher resolution is achieved with nano-secondary ion mass spectrometry (SIMS), which detects the ions emitted from a surface bombarded by an ion beam (Moore et al., 2012). In spite of tremendous progress, however, not all biologically relevant elements can be analyzed equally well. Sensitivities of most methods are element specific. For example, even though the concentrations of As species and Cd in grains of field-grown plants are of the same order of magnitude, the information on spatial distribution is restricted to As for which both X-ray-fluorescence and nano-SIMS are much more sensitive (Zhao et al., 2014). Several studies have addressed the distribution of As in rice grains (e.g. Lombi et al., 2009; Carey et al., 2010; Zheng et al., 2011). They agreed on finding the majority of inorganic As concentrated in the bran and, more specifically, in the ovular vascular traces on the dorsal side of the grain, which represent the principal loading pathway for nutrients. Thus, a transport barrier exists that restricts movement into filial tissues. For DMA, this apparently does not apply. When fed to rice plants, DMA is again highly mobile and distributed more evenly throughout the grain. The small fraction of inorganic As that reaches the filial tissues was found predominantly in the subaleurone cell layer and associated with thiols (Moore et al., 2010). Conclusions FAO and WHO, the EU, as well as numerous national authorities follow the ALARA principle (‘as low as reasonably achievable’) when regulating food safety. Concentrations of contaminants posing health threats should be kept to a minimum, provided the effort needed does not become unreasonably high. In light of documented health risks of dietary exposure to toxic non-essential elements and currently available knowledge on the pathways controlling accumulation of these elements in plants, the following conclusion suggests itself: honoring the ALARA principle urgently calls for the use of genome editing techniques and other genetic interventions to develop crops low in Cd and As. Genome editing with CRISPR/Cas in particular has enormous potential to dramatically accelerate the breeding of such crop varieties. Changes in one or a few nucleotides can be introduced precisely (Scheben et al., 2017) and will, at least in some cases, be sufficient to achieve respective goals, as described above. Furthermore, mutations introduced by CRISPR/Cas cannot be distinguished from naturally occurring mutations. and no foreign genes are present after segregating out the CRISPR/Cas cassette. Therefore, the resulting plants should not fall under the rigid regulations that are in place for transgenic plants. Some countries including Canada and the USA are establishing a corresponding product-based regulatory practice (Scheben and Edwards, 2018). In Europe, however, political action is needed to overcome the dire consequences of the ruling issued by the Court of Justice of the European Union (ECJ) in 2018 (Smyth and Lassoued, 2019). Background contamination with toxic elements is nearly ubiquitous and not always of anthropogenic origin. While emission should of course be limited as much as possible, removal of toxic elements from the environment is not feasible, making inadvertent uptake by plants practically unavoidable. Thus, the most effective and sustainable approach to lower dietary exposure via the consumption of plant-derived food is the breeding of crops with drastically reduced accumulation of non-essential, toxic elements. Genotype influence on within-species variation in Cd and As accumulation is strong and the molecular identification of underlying genes will provide useful alleles. Available mechanistic understanding already suggests that changes in a few genes would be sufficient to achieve major effects. Especially in the case of Cd, the path towards safer rice with dramatically reduced grain content is clear. OsNramp5 loss-of-function alleles, especially in the genetic background of widely grown elite cultivars such as Koshihikari, offer a practical approach. Overexpression of OsHMA3 represents a feasible and effective alternative. Cd exposure, unlike As exposure, is, however, not dominated by just one staple crop. Thus, it is urgent to translate the knowledge gained in A. thaliana and rice to other major crops such as wheat or potato. Identifying and targeting Cd uptake transporters, for example, would be an obvious strategy. Reducing As accumulation while maintaining all the important agronomic traits appears to be a bit more challenging. Nonetheless, tremendous progress has been made in the past years, and the best strategies are likely to emerge soon. Comparative large-scale field studies with a variety of rice mutants showing documented changes in grain As would be extremely helpful to decide on the most suitable engineering targets. The potential of genome editing techniques makes it extremely important to dissect the selectivity determinants of metal transporters. Tailored transporter proteins expressed in specific cell types and the right membranes can be expected to significantly improve food safety. Acknowledgements Research in the author’s laboratory on the accumulation of Cd and As in plants is supported by the Deutsche Forschungsgemeinschaft. I am grateful to Ursula Ferrera and Christian Hofmann for help with figure preparation. References Abe T , Nonoue Y , Ono N , Omoteno M , Kuramata M , Fukuoka S , Yamamoto T , Yano M , Ishikawa S . 2013 . Detection of QTLs to reduce cadmium content in rice grains using LAC23/Koshihikari chromosome segment substitution lines . Breeding Science 63 , 284 – 291 . Google Scholar Crossref Search ADS PubMed WorldCat Ali W , Isner JC , Isayenkov SV , Liu W , Zhao FJ , Maathuis FJ . 2012 . Heterologous expression of the yeast arsenite efflux system ACR3 improves Arabidopsis thaliana tolerance to arsenic stress . New Phytologist 194 , 716 – 723 . Google Scholar Crossref Search ADS PubMed WorldCat Alvarez-Fernández A , Díaz-Benito P , Abadía A , López-Millán AF , Abadía J . 2014 . Metal species involved in long distance metal transport in plants . Frontiers in Plant Science 5 , 105 . Google Scholar Crossref Search ADS PubMed WorldCat Andres J , Bertin PN . 2016 . The microbial genomics of arsenic . FEMS Microbiology Reviews 40 , 299 – 322 . Google Scholar Crossref Search ADS PubMed WorldCat Arazi T , Sunkar R , Kaplan B , Fromm H . 1999 . A tobacco plasma membrane calmodulin-binding transporter confers Ni2+ tolerance and Pb2+ hypersensitivity in transgenic plants . The Plant Journal 20 , 171 – 182 . Google Scholar Crossref Search ADS PubMed WorldCat Argüello JM , Eren E , Gonzalez-Guerrero M . 2007 . The structure and function of heavy metal transport P-1B-ATPases . Biometals 20 , 233 – 248 . Google Scholar Crossref Search ADS PubMed WorldCat Bagla P , Kaiser J . 1996 . India’s spreading health crisis draws global arsenic experts . Science 274 , 174 – 175 . Google Scholar Crossref Search ADS PubMed WorldCat Barberon M , Geldner N . 2014 . Radial transport of nutrients: the plant root as a polarized epithelium . Plant Physiology 166 , 528 – 537 . Google Scholar Crossref Search ADS PubMed WorldCat Batista BL , Nigar M , Mestrot A , Rocha BA , Barbosa Júnior F , Price AH , Raab A , Feldmann J . 2014 . Identification and quantification of phytochelatins in roots of rice to long-term exposure: evidence of individual role on arsenic accumulation and translocation . Journal of Experimental Botany 65 , 1467 – 1479 . Google Scholar Crossref Search ADS PubMed WorldCat Bovenkamp GL , Prange A , Schumacher W , Ham K , Smith AP , Hormes J . 2013 . Lead uptake in diverse plant families: a study applying X-ray absorption near edge spectroscopy . Environmental Science & Technology 47 , 4375 – 4382 . Google Scholar Crossref Search ADS PubMed WorldCat Brännvall ML , Bindler R , Renberg I , Emteryd O , Bartnicki J , Billstrom K . 1999 . The Medieval metal industry was the cradle of modern large scale atmospheric lead pollution in northern Europe . Environmental Science & Technology 33 , 4391 – 4395 . Google Scholar Crossref Search ADS WorldCat Canfield RL , Henderson CR Jr , Cory-Slechta DA , Cox C , Jusko TA , Lanphear BP . 2003 . Intellectual impairment in children with blood lead concentrations below 10 μg per deciliter . New England Journal of Medicine 348 , 1517 – 1526 . Google Scholar Crossref Search ADS PubMed WorldCat Cao Y , Sun D , Ai H , Mei H , Liu X , Sun S , Xu G , Liu Y , Chen Y , Ma LQ . 2017 . Knocking out OsPT4 gene decreases arsenate uptake by rice plants and inorganic arsenic accumulation in rice grains . Environmental Science & Technology 51 , 12131 – 12138 . Google Scholar Crossref Search ADS PubMed WorldCat Carey AM , Norton GJ , Deacon C , et al. 2011 . Phloem transport of arsenic species from flag leaf to grain during grain filling . New Phytologist 192 , 87 – 98 . Google Scholar Crossref Search ADS PubMed WorldCat Carey AM , Scheckel KG , Lombi E , Newville M , Choi Y , Norton GJ , Charnock JM , Feldmann J , Price AH , Meharg AA . 2010 . Grain unloading of arsenic species in rice . Plant Physiology 152 , 309 – 319 . Google Scholar Crossref Search ADS PubMed WorldCat Chao DY , Chen Y , Chen J , Shi S , Chen Z , Wang C , Danku JM , Zhao FJ , Salt DE . 2014 . Genome-wide association mapping identifies a new arsenate reductase enzyme critical for limiting arsenic accumulation in plants . PLoS Biology 12 , e1002009 . Google Scholar Crossref Search ADS PubMed WorldCat Chao DY , Silva A , Baxter I , Huang YS , Nordborg M , Danku J , Lahner B , Yakubova E , Salt DE . 2012 . Genome-wide association studies identify heavy metal ATPase3 as the primary determinant of natural variation in leaf cadmium in Arabidopsis thaliana . PLoS Genetics 8 , e1002923 . Google Scholar Crossref Search ADS PubMed WorldCat Chen H , Yang X , Wang P , Wang Z , Li M , Zhao FJ . 2018 . Dietary cadmium intake from rice and vegetables and potential health risk: a case study in Xiangtan, southern China . The Science of the Total Environment 639 , 271 – 277 . Google Scholar Crossref Search ADS PubMed WorldCat Chen X , Ouyang Y , Fan Y , Qiu B , Zhang G , Zeng F . 2018 . The pathway of transmembrane cadmium influx via calcium-permeable channels and its spatial characteristics along rice root . Journal of Experimental Botany 69 , 5279 – 5291 . Google Scholar Crossref Search ADS PubMed WorldCat Chen Y , Han YH , Cao Y , Zhu YG , Rathinasabapathi B , Ma LQ . 2017a. Arsenic transport in rice and biological solutions to reduce arsenic risk from rice . Frontiers in Plant Science 8 , 268 . Google Scholar PubMed WorldCat Chen Y , Moore KL , Miller AJ , McGrath SP , Ma JF , Zhao FJ . 2015 . The role of nodes in arsenic storage and distribution in rice . Journal of Experimental Botany 66 , 3717 – 3724 . Google Scholar Crossref Search ADS PubMed WorldCat Chen Y , Sun SK , Tang Z , Liu G , Moore KL , Maathuis FJM , Miller AJ , McGrath SP , Zhao FJ . 2017b. The Nodulin 26-like intrinsic membrane protein OsNIP3;2 is involved in arsenite uptake by lateral roots in rice . Journal of Experimental Botany 68 , 3007 – 3016 . Google Scholar Crossref Search ADS PubMed WorldCat Chen Y , Xu W , Shen H , Yan H , Xu W , He Z , Ma M . 2013 . Engineering arsenic tolerance and hyperaccumulation in plants for phytoremediation by a PvACR3 transgenic approach . Environmental Science & Technology 47 , 9355 – 9362 . Google Scholar Crossref Search ADS PubMed WorldCat Chen Z , Chen SJ . 2017 . Poisoning the Devil . Cell 168 , 556 – 560 . Google Scholar Crossref Search ADS PubMed WorldCat Chowdhury R , Ramond A , O’Keeffe LM , et al. 2018 . Environmental toxic metal contaminants and risk of cardiovascular disease: systematic review and meta-analysis . BMJ 362 , k3310 . Google Scholar Crossref Search ADS PubMed WorldCat Clemens S . 2006 . Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants . Biochimie 88 , 1707 – 1719 . Google Scholar Crossref Search ADS PubMed WorldCat Clemens S , Aarts MG , Thomine S , Verbruggen N . 2013 . Plant science: the key to preventing slow cadmium poisoning . Trends in Plant Science 18 , 92 – 99 . Google Scholar Crossref Search ADS PubMed WorldCat Clemens S , Antosiewicz DM , Ward JM , Schachtman DP , Schroeder JI . 1998 . The plant cDNA LCT1 mediates the uptake of calcium and cadmium in yeast . Proceedings of the National Academy of Sciences, USA 95 , 12043 – 12048 . Google Scholar Crossref Search ADS WorldCat Clemens S , Ma JF . 2016 . Toxic heavy metal and metalloid accumulation in crop plants and foods . Annual Review of Plant Biology 67 , 489 – 512 . Google Scholar Crossref Search ADS PubMed WorldCat Cobbett C , Goldsbrough P . 2002 . Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis . Annual Review of Plant Biology 53 , 159 – 182 . Google Scholar Crossref Search ADS PubMed WorldCat Davis MA , Signes-Pastor AJ , Argos M , Slaughter F , Pendergrast C , Punshon T , Gossai A , Ahsan H , Karagas MR . 2017 . Assessment of human dietary exposure to arsenic through rice . The Science of the Total Environment 586 , 1237 – 1244 . Google Scholar Crossref Search ADS PubMed WorldCat Deng F , Yamaji N , Ma JF , Lee SK , Jeon JS , Martinoia E , Lee Y , Song WY . 2018 . Engineering rice with lower grain arsenic . Plant Biotechnology Journal 16 , 1691 – 1699 . Google Scholar Crossref Search ADS PubMed WorldCat Duan G , Kamiya T , Ishikawa S , Arao T , Fujiwara T . 2012 . Expressing ScACR3 in rice enhanced arsenite efflux and reduced arsenic accumulation in rice grains . Plant & Cell Physiology 53 , 154 – 163 . Google Scholar Crossref Search ADS PubMed WorldCat Duan G , Shao G , Tang Z , Chen H , Wang B , Tang Z , Yang Y , Liu Y , Zhao F-J . 2017 . Genotypic and environmental variations in grain cadmium and arsenic concentrations among a panel of high yielding rice cultivars . Rice 10 , 9 . Google Scholar Crossref Search ADS PubMed WorldCat Duan GL , Hu Y , Schneider S , McDermott J , Chen J , Sauer N , Rosen BP , Daus B , Liu Z , Zhu YG . 2016 . Inositol transporters AtINT2 and AtINT4 regulate arsenic accumulation in Arabidopsis seeds . Nature Plants 2 , 15202 . Google Scholar Crossref Search ADS PubMed WorldCat EFSA (European Food Safety Authority) . 2009 . Cadmium in food—scientific opinion of the panel on contaminants in the food chain . EFSA Journal 7 , 980 . WorldCat EFSA (European Food Safety Authority) . 2012 . Cadmium dietary exposure in the European population . EFSA Journal 10 , 2551 . Crossref Search ADS WorldCat EFSA (European Food Safety Authority) . 2014 . Dietary exposure to inorganic arsenic in the European population . EFSA Journal 12 , 3597 . WorldCat EFSA Panel on Contaminants in the Food Chain (CONTAM) . 2009 . Scientific opinion on arsenic in food . EFSA Journal 7 , 1351 . Crossref Search ADS WorldCat EFSA Panel on Contaminants in the Food Chain (CONTAM). 2010 . Scientific opinion on lead in food: lead in food . EFSA Journal 8 , 1570 . Crossref Search ADS WorldCat Engström A , Michaëlsson K , Suwazono Y , Wolk A , Vahter M , Akesson A . 2011 . Long-term cadmium exposure and the association with bone mineral density and fractures in a population-based study among women . Journal of Bone and Mineral Research 26 , 486 – 495 . Google Scholar Crossref Search ADS PubMed WorldCat Eun S-O , Youn HS , Lee Y . 2000 . Lead disturbs microtubule organization in the root meristem of Zea mays . Physiologia Plantarum 110 , 357 – 365 . Google Scholar Crossref Search ADS WorldCat Fan C , Liu G , Long Y , Rosen B , Cai Y . 2018 . Thiolation in arsenic metabolism: a chemical perspective . Metallomics: Integrated Biometal Science 10 , 1368 – 1382 . Google Scholar Crossref Search ADS PubMed WorldCat FAO/WHO (Food and Agriculture Organization/World Health Organization) . 2010 . Evaluation of certain food additives and contaminants (Seventy-third report of the Joint FAO/WHO Expert Committee on Food Additives) . Rome : FAO . Google Preview WorldCat COPAC Fasani E , Manara A , Martini F , Furini A , DalCorso G . 2018 . The potential of genetic engineering of plants for the remediation of soils contaminated with heavy metals . Plant, Cell & Environment 41 , 1201 – 1232 . Google Scholar Crossref Search ADS PubMed WorldCat Feng X , Li P , Qiu G , et al. 2008 . Human exposure to methylmercury through rice intake in mercury mining areas, Guizhou province, China . Environmental Science & Technology 42 , 326 – 332 . Google Scholar Crossref Search ADS PubMed WorldCat Fischer S , Kühnlenz T , Thieme M , Schmidt H , Clemens S . 2014 . Analysis of plant Pb tolerance at realistic submicromolar concentrations demonstrates the role of phytochelatin synthesis for Pb detoxification . Environmental Science & Technology 48 , 7552 – 7559 . Google Scholar Crossref Search ADS PubMed WorldCat Gilbert-Diamond D , Cottingham KL , Gruber JF , Punshon T , Sayarath V , Gandolfi AJ , Baker ER , Jackson BP , Folt CL , Karagas MR . 2011 . Rice consumption contributes to arsenic exposure in US women . Proceedings of the National Academy of Sciences, USA 108 , 20656 – 20660 . Google Scholar Crossref Search ADS WorldCat Gu M , Chen A , Sun S , Xu G . 2016 . Complex regulation of plant phosphate transporters and the gap between molecular mechanisms and practical application: what is missing? Molecular Plant 9 , 396 – 416 . Google Scholar Crossref Search ADS PubMed WorldCat Gunshin H , Mackenzie B , Berger UV , Gunshin Y , Romero MF , Boron WF , Nussberger S , Gollan JL , Hediger MA . 1997 . Cloning and characterization of a mammalian proton-coupled metal-ion transporter . Nature 388 , 482 – 488 . Google Scholar Crossref Search ADS PubMed WorldCat Hanikenne M , Baurain D . 2014 . Origin and evolution of metal P-type ATPases in Plantae (Archaeplastida) . Frontiers in Plant Science 4 , 544 . Google Scholar Crossref Search ADS PubMed WorldCat Hanikenne M , Talke IN , Haydon MJ , Lanz C , Nolte A , Motte P , Kroymann J , Weigel D , Krämer U . 2008 . Evolution of metal hyperaccumulation required cis-regulatory changes and triplication of HMA4 . Nature 453 , 391 – 395 . Google Scholar Crossref Search ADS PubMed WorldCat Hao X , Zeng M , Wang J , Zeng Z , Dai J , Xie Z , Yang Y , Tian L , Chen L , Li D . 2018 . A node-expressed transporter OsCCX2 is involved in grain cadmium accumulation of rice . Frontiers in Plant Science 9 , 476 . Google Scholar Crossref Search ADS PubMed WorldCat Hayashi S , Kuramata M , Abe T , Takagi H , Ozawa K , Ishikawa S . 2017 . Phytochelatin synthase OsPCS1 plays a crucial role in reducing arsenic levels in rice grains . The Plant Journal 91 , 840 – 848 . Google Scholar Crossref Search ADS PubMed WorldCat He XL , Fan SK , Zhu J , Guan MY , Liu XX , Zhang YS , Jin CW . 2017 . Iron supply prevents Cd uptake in Arabidopsis by inhibiting IRT1 expression and favoring competition between Fe and Cd uptake . Plant and Soil 416 , 453 – 462 . Google Scholar Crossref Search ADS WorldCat Huang JW , Cunningham SD . 1996 . Lead phytoextraction: species variation in lead uptake and translocation . New Phytologist 134 , 75 – 84 . Google Scholar Crossref Search ADS WorldCat Hughes MF . 2002 . Arsenic toxicity and potential mechanisms of action . Toxicology Letters 133 , 1 – 16 . Google Scholar Crossref Search ADS PubMed WorldCat Hughes MF , Beck BD , Chen Y , Lewis AS , Thomas DJ . 2011 . Arsenic exposure and toxicology: a historical perspective . Toxicological Sciences 123 , 305 – 332 . Google Scholar Crossref Search ADS PubMed WorldCat Hussain D , Haydon MJ , Wang Y , Wong E , Sherson SM , Young J , Camakaris J , Harper JF , Cobbett CS . 2004 . P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis . The Plant Cell 16 , 1327 – 1339 . Google Scholar Crossref Search ADS PubMed WorldCat Indriolo E , Na G , Ellis D , Salt DE , Banks JA . 2010 . A vacuolar arsenite transporter necessary for arsenic tolerance in the arsenic hyperaccumulating fern Pteris vittata is missing in flowering plants . The Plant Cell 22 , 2045 – 2057 . Google Scholar Crossref Search ADS PubMed WorldCat Ishikawa S , Ishimaru Y , Igura M , Kuramata M , Abe T , Senoura T , Hase Y , Arao T , Nishizawa NK , Nakanishi H . 2012 . Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice . Proceedings of the National Academy of Sciences, USA 109 , 19166 – 71 . Google Scholar Crossref Search ADS WorldCat Järup L . 2003 . Hazards of heavy metal contamination . British Medical Bulletin 68 , 167 – 82 . Google Scholar Crossref Search ADS PubMed WorldCat Jia Y , Huang H , Chen Z , Zhu YG . 2014 . Arsenic uptake by rice is influenced by microbe-mediated arsenic redox changes in the rhizosphere . Environmental Science & Technology 48 , 1001 – 1007 . Google Scholar Crossref Search ADS PubMed WorldCat Johanning J , Strasdeit H . 1998 . A coordination-chemical basis for the biological function of the phytochelatins . Angewandte Chemie International Edition 37 , 2464 – 2466 . Google Scholar Crossref Search ADS PubMed WorldCat Kamiya T , Islam MR , Duan G , Uraguchi S , Fujiwara T . 2013 . Phosphate deficiency signaling pathway is a target of arsenate and phosphate transporter OsPT1 is involved in As accumulation in shoots of rice . Soil Science and Plant Nutrition 59 , 580 – 590 . Google Scholar Crossref Search ADS WorldCat Kamiya T , Tanaka M , Mitani N , Ma JF , Maeshima M , Fujiwara T . 2009 . NIP1;1, an aquaporin homolog, determines the arsenite sensitivity of Arabidopsis thaliana . Journal of Biological Chemistry 284 , 2114 – 2120 . Google Scholar Crossref Search ADS PubMed WorldCat Kato M , Ishikawa S , Inagaki K , Chiba K , Hayashi H , Yanagisawa S , Yoneyama T . 2010 . Possible chemical forms of cadmium and varietal differences in cadmium concentrations in the phloem sap of rice plants (Oryza sativa L.) . Soil Science and Plant Nutrition 56 , 839 – 847 . Google Scholar Crossref Search ADS WorldCat Kerl CF , Rafferty C , Clemens S , Planer-Friedrich B . 2018 . Monothioarsenate uptake, transformation, and translocation in rice plants . Environmental Science & Technology 52 , 9154 – 9161 . Google Scholar Crossref Search ADS PubMed WorldCat Kerl CF , Schindele RA , Brüggenwirth L , Colina Blanco AE , Rafferty C , Clemens S , Planer-Friedrich B . 2019 . Methylated thioarsenates and monothioarsenate differ in uptake, transformation, and contribution to total arsenic translocation in rice plants . Environmental Science & Technology 53 , 5787 – 5796 . Google Scholar Crossref Search ADS PubMed WorldCat Khan MA , Castro-Guerrero NA , Mendoza-Cozatl D . 2014 . Moving toward a precise nutrition: preferential loading of seeds with essential nutrients over non-essential toxic elements . Plant Nutrition 5 , 51 . WorldCat Kim DY , Bovet L , Maeshima M , Martinoia E , Lee Y . 2007 . The ABC transporter AtPDR8 is a cadmium extrusion pump conferring heavy metal resistance . The Plant Journal 50 , 207 – 218 . Google Scholar Crossref Search ADS PubMed WorldCat Kim Y-Y , Yang Y-Y , Lee Y . 2002 . Pb and Cd uptake in rice roots . Physiologia Plantarum 116 , 368 – 372 . Google Scholar Crossref Search ADS WorldCat Kopittke PM , Asher CJ , Blamey FP , Auchterlonie GJ , Guo YN , Menzies NW . 2008a. Localization and chemical speciation of Pb in roots of signal grass (Brachiaria decumbens) and Rhodes grass (Chloris gayana) . Environmental Science & Technology 42 , 4595 – 4599 . Google Scholar Crossref Search ADS PubMed WorldCat Kopittke PM , Asher CJ , Menzies NW . 2008b. Prediction of Pb speciation in concentrated and dilute nutrient solutions . Environmental Pollution 153 , 548 – 554 . Google Scholar Crossref Search ADS PubMed WorldCat Kopittke PM , Blamey FP , Asher CJ , Menzies NW . 2010 . Trace metal phytotoxicity in solution culture: a review . Journal of Experimental Botany 61 , 945 – 954 . Google Scholar Crossref Search ADS PubMed WorldCat Kopittke PM , de Jonge MD , Wang P , et al. 2014 . Laterally resolved speciation of arsenic in roots of wheat and rice using fluorescence-XANES imaging . New Phytologist 201 , 1251 – 1262 . Google Scholar Crossref Search ADS PubMed WorldCat Kopittke PM , Punshon T , Paterson DJ , Tappero RV , Wang P , Blamey FPC , van der Ent A , Lombi E . 2018 . Synchrotron-based X-ray fluorescence microscopy as a technique for imaging of elements in plants . Plant Physiology 178 , 507 – 523 . Google Scholar Crossref Search ADS PubMed WorldCat Lanphear BP . 2015 . The impact of toxins on the developing brain . Annual Review of Public Health 36 , 211 – 230 . Google Scholar Crossref Search ADS PubMed WorldCat Lanphear BP , Hornung R , Khoury J , et al. 2005 . Low-level environmental lead exposure and children’s intellectual function: an international pooled analysis . Environmental Health Perspectives 113 , 894 – 899 . Google Scholar Crossref Search ADS PubMed WorldCat Lanphear BP , Rauch S , Auinger P , Allen RW , Hornung RW . 2018 . Low-level lead exposure and mortality in US adults: a population-based cohort study . The Lancet. Public Health 3 , e177 – e184 . Google Scholar Crossref Search ADS PubMed WorldCat Lee J , Bae H , Jeong J , Lee JY , Yang YY , Hwang I , Martinoia E , Lee Y . 2003 . Functional expression of a bacterial heavy metal transporter in Arabidopsis enhances resistance to and decreases uptake of heavy metals . Plant Physiology 133 , 589 – 596 . Google Scholar Crossref Search ADS PubMed WorldCat Lee S , An G . 2009 . Over-expression of OsIRT1 leads to increased iron and zinc accumulations in rice . Plant, Cell & Environment 32 , 408 – 416 . Google Scholar Crossref Search ADS PubMed WorldCat Lekeux G , Crowet JM , Nouet C , et al. 2019 . Homology modeling and in vivo functional characterization of the zinc permeation pathway in a heavy metal P-type ATPase . Journal of Experimental Botany 70 , 329 – 341 . Google Scholar Crossref Search ADS PubMed WorldCat Li RY , Ago Y , Liu WJ , Mitani N , Feldmann J , McGrath SP , Ma JF , Zhao FJ . 2009 . The rice aquaporin Lsi1 mediates uptake of methylated arsenic species . Plant Physiology 150 , 2071 – 2080 . Google Scholar Crossref Search ADS PubMed WorldCat Lindsay ER , Maathuis FJ . 2016 . Arabidopsis thaliana NIP7;1 is involved in tissue arsenic distribution and tolerance in response to arsenate . FEBS Letters 590 , 779 – 786 . Google Scholar Crossref Search ADS PubMed WorldCat Lindsay ER , Maathuis FJM . 2017 . New molecular mechanisms to reduce arsenic in crops . Trends in Plant Science 22 , 1016 – 1026 . Google Scholar Crossref Search ADS PubMed WorldCat Liu C , Chen G , Li Y , et al. 2017 . Characterization of a major QTL for manganese accumulation in rice grain . Scientific Reports 7 , 17704 . Google Scholar Crossref Search ADS PubMed WorldCat Liu C-L , Gao Z-Y , Shang L-G , Yang C-H , Ruan B-P , Zeng D-L , Guo L-B , Zhao F-J , Huang C-F , Qian Q . 2019 . Natural variation in the promoter of OsHMA3 contributes to differential grain cadmium accumulation between Indica and Japonica rice . Journal of Integrative Plant Biology . WorldCat Liu W , Schat H , Bliek M , Chen Y , McGrath SP , George G , Salt DE , Zhao FJ . 2012 . Knocking out ACR2 does not affect arsenic redox status in Arabidopsis thaliana: implications for As detoxification and accumulation in plants . PloS One 7 , e42408 . Google Scholar Crossref Search ADS PubMed WorldCat Liu WJ , Wood BA , Raab A , McGrath SP , Zhao FJ , Feldmann J . 2010 . Complexation of arsenite with phytochelatins reduces arsenite efflux and translocation from roots to shoots in Arabidopsis . Plant Physiology 152 , 2211 – 2221 . Google Scholar Crossref Search ADS PubMed WorldCat Lomax C , Liu WJ , Wu L , Xue K , Xiong J , Zhou J , McGrath SP , Meharg AA , Miller AJ , Zhao FJ . 2012 . Methylated arsenic species in plants originate from soil microorganisms . New Phytologist 193 , 665 – 672 . Google Scholar Crossref Search ADS PubMed WorldCat Lombi E , Scheckel KG , Pallon J , Carey AM , Zhu YG , Meharg AA . 2009 . Speciation and distribution of arsenic and localization of nutrients in rice grains . New Phytologist 184 , 193 – 201 . Google Scholar Crossref Search ADS PubMed WorldCat Lu C , Zhang L , Tang Z , Huang X-Y , Ma JF , Zhao F-J . 2019 . Producing cadmium-free Indica rice by overexpressing OsHMA3 . Environment International 126 , 619 – 626 . Google Scholar Crossref Search ADS PubMed WorldCat Luo JS , Huang J , Zeng DL , et al. 2018 . A defensin-like protein drives cadmium efflux and allocation in rice . Nature Communications 9 , 645 . Google Scholar Crossref Search ADS PubMed WorldCat Lux A , Martinka M , Vaculík M , White PJ . 2011 . Root responses to cadmium in the rhizosphere: a review . Journal of Experimental Botany 62 , 21 – 37 . Google Scholar Crossref Search ADS PubMed WorldCat Ma JF , Yamaji N . 2015 . A cooperative system of silicon transport in plants . Trends in Plant Science 20 , 435 – 442 . Google Scholar Crossref Search ADS PubMed WorldCat Ma JF , Yamaji N , Mitani N , Tamai K , Konishi S , Fujiwara T , Katsuhara M , Yano M . 2007 . An efflux transporter of silicon in rice . Nature 448 , 209 – 212 . Google Scholar Crossref Search ADS PubMed WorldCat Ma JF , Yamaji N , Mitani N , Xu X-Y , Su Y-H , McGrath SP , Zhao F-J . 2008 . Transporters of arsenite in rice and their role in arsenic accumulation in rice grain . Proceedings of the National Academy of Sciences, USA 105 , 9931 – 9935 . Google Scholar Crossref Search ADS WorldCat Mantha M , Yeary E , Trent J , et al. 2017 . Estimating inorganic arsenic exposure from U.S. rice and total water intakes . Environmental Health Perspectives 125 , 057005 . Google Scholar Crossref Search ADS PubMed WorldCat Maret W , Moulis JM . 2013 . The bioinorganic chemistry of cadmium in the context of its toxicity . Metal Ions in Life Sciences 11 , 1 – 29 . Google Scholar Crossref Search ADS PubMed WorldCat Marx SK , Rashid S , Stromsoe N . 2016 . Global-scale patterns in anthropogenic Pb contamination reconstructed from natural archives . Environmental Pollution 213 , 283 – 298 . Google Scholar Crossref Search ADS PubMed WorldCat McLaughlin MJ , Parker DR , Clarke JM . 1999 . Metals and micronutrients—food safety issues . Field Crops Research 60 , 143 – 163 . Google Scholar Crossref Search ADS WorldCat McLaughlin MJ , Smolders E , Degryse F , Rietra R . 2011 . Uptake of metals from soil into vegetables. In: Swartjes FA , ed. Dealing with contaminated sites . Dordrecht : Springer, 325 – 367 . Google Preview WorldCat COPAC Meharg AA , Rahman MM . 2003 . Arsenic contamination of Bangladesh paddy field soils: implications for rice contribution to arsenic consumption . Environmental Science & Technology 37 , 229 – 234 . Google Scholar Crossref Search ADS PubMed WorldCat Mengist MF , Alves S , Griffin D , Creedon J , McLaughlin MJ , Jones PW , Milbourne D . 2018 . Genetic mapping of quantitative trait loci for tuber-cadmium and zinc concentration in potato reveals associations with maturity and both overlapping and independent components of genetic control . Theoretical and Applied Genetics 131 , 929 – 945 . Google Scholar Crossref Search ADS PubMed WorldCat Mergler D , Anderson HA , Chan LH , Mahaffey KR , Murray M , Sakamoto M , Stern AH ; Panel on Health Risks and Toxicological Effects of Methylmercury . 2007 . Methylmercury exposure and health effects in humans: a worldwide concern . Ambio 36 , 3 – 11 . Google Scholar Crossref Search ADS PubMed WorldCat Mishra S , Mattusch J , Wennrich R . 2017 . Accumulation and transformation of inorganic and organic arsenic in rice and role of thiol-complexation to restrict their translocation to shoot . Scientific Reports 7 , 40522 . Google Scholar Crossref Search ADS PubMed WorldCat Mitani-Ueno N , Yamaji N , Zhao FJ , Ma JF . 2011 . The aromatic/arginine selectivity filter of NIP aquaporins plays a critical role in substrate selectivity for silicon, boron, and arsenic . Journal of Experimental Botany 62 , 4391 – 4398 . Google Scholar Crossref Search ADS PubMed WorldCat Miyadate H , Adachi S , Hiraizumi A , et al. 2011 . OsHMA3, a P1B-type of ATPase affects root-to-shoot cadmium translocation in rice by mediating efflux into vacuoles . New Phytologist 189 , 190 – 199 . Google Scholar Crossref Search ADS PubMed WorldCat Moore KL , Lombi E , Zhao FJ , Grovenor CR . 2012 . Elemental imaging at the nanoscale: nanoSIMS and complementary techniques for element localisation in plants . Analytical and Bioanalytical Chemistry 402 , 3263 – 3273 . Google Scholar Crossref Search ADS PubMed WorldCat Moore KL , Schröder M , Lombi E , Zhao FJ , McGrath SP , Hawkesford MJ , Shewry PR , Grovenor CR . 2010 . NanoSIMS analysis of arsenic and selenium in cereal grain . New Phytologist 185 , 434 – 445 . Google Scholar Crossref Search ADS PubMed WorldCat Nakanishi H , Ogawa I , Ishimaru Y , Mori S , Nishizawa NK . 2006 . Iron deficiency enhances cadmium uptake and translocation mediated by the Fe2+ transporters OsIRT1 and OsIRT2 in rice . Soil Science and Plant Nutrition 52 , 464 – 469 . Google Scholar Crossref Search ADS WorldCat Naujokas MF , Anderson B , Ahsan H , Aposhian HV , Graziano JH , Thompson C , Suk WA . 2013 . The broad scope of health effects from chronic arsenic exposure: update on a worldwide public health problem . Environmental Health Perspectives 121 , 295 – 302 . Google Scholar Crossref Search ADS PubMed WorldCat Needleman H . 2004 . Lead poisoning . Annual Review of Medicine 55 , 209 – 222 . Google Scholar Crossref Search ADS PubMed WorldCat Needleman HL . 1998 . Clair Patterson and Robert Kehoe: two views of lead toxicity . Environmental Research 78 , 79 – 85 . Google Scholar Crossref Search ADS PubMed WorldCat Nocito FF , Lancilli C , Dendena B , Lucchini G , Sacchi GA . 2011 . Cadmium retention in rice roots is influenced by cadmium availability, chelation and translocation . Plant, Cell & Environment 34 , 994 – 1008 . Google Scholar Crossref Search ADS PubMed WorldCat Nordberg GF . 2009 . Historical perspectives on cadmium toxicology . Toxicology and Applied Pharmacology 238 , 192 – 200 . Google Scholar Crossref Search ADS PubMed WorldCat Nordberg GF , Bernard A , Diamond GL , Duffus JH , Illing P , Nordberg M , Bergdahl IA , Jin T , Skerfving S . 2018 . Risk assessment of effects of cadmium on human health (IUPAC Technical Report) . Pure and Applied Chemistry 90 , 755 – 808 . Google Scholar Crossref Search ADS WorldCat Nordstrom DK . 2002 . Public health. Worldwide occurrences of arsenic in ground water . Science 296 , 2143 – 2145 . Google Scholar Crossref Search ADS PubMed WorldCat Norton GJ , Pinson SR , Alexander J , et al. 2012 . Variation in grain arsenic assessed in a diverse panel of rice (Oryza sativa) grown in multiple sites . New Phytologist 193 , 650 – 664 . Google Scholar Crossref Search ADS PubMed WorldCat Norton GJ , Williams PN , Adomako EE , et al. 2014 . Lead in rice: analysis of baseline lead levels in market and field collected rice grains . The Science of the Total Environment 485-486 , 428 – 434 . Google Scholar Crossref Search ADS PubMed WorldCat Nriagu JO . 1988 . A silent epidemic of environmental metal poisoning? Environmental Pollution 50 , 139 – 161 . Google Scholar Crossref Search ADS PubMed WorldCat Nriagu JO , Pacyna JM . 1988 . Quantitative assessment of worldwide contamination of air, water and soils by trace metals . Nature 333 , 134 – 139 . Google Scholar Crossref Search ADS PubMed WorldCat Ogunseitan OA , Schoenung JM , Saphores J-DM , Shapiro AA . 2009 . The electronics revolution: from E-wonderland to E-wasteland . Science 326 , 670 – 671 . Google Scholar Crossref Search ADS PubMed WorldCat Olsen LI , Hansen TH , Larue C , et al. 2016 . Mother-plant-mediated pumping of zinc into the developing seed . Nature Plants 2 , 16036 . Google Scholar Crossref Search ADS PubMed WorldCat Park J , Song WY , Ko D , Eom Y , Hansen TH , Schiller M , Lee TG , Martinoia E , Lee Y . 2012 . The phytochelatin transporters AtABCC1 and AtABCC2 mediate tolerance to cadmium and mercury . The Plant Journal 69 , 278 – 288 . Google Scholar Crossref Search ADS PubMed WorldCat Patrick JW , Offler CE . 2001 . Compartmentation of transport and transfer events in developing seeds . Journal of Experimental Botany 52 , 551 – 564 . Google Scholar Crossref Search ADS PubMed WorldCat Pinson SRM , Tarpley L , Yan W , Yeater K , Lahner B , Yakubova E , Huang X-Y , Zhang M , Guerinot ML , Salt DE . 2015 . Worldwide genetic diversity for mineral element concentrations in rice grain . Crop Science 55 , 294 – 311 . Google Scholar Crossref Search ADS WorldCat Planer-Friedrich B , Härtig C , Lohmayer R , Suess E , McCann SH , Oremland R . 2015 . Anaerobic chemolithotrophic growth of the haloalkaliphilic bacterium strain MLMS-1 by disproportionation of monothioarsenate . Environmental Science & Technology 49 , 6554 – 6563 . Google Scholar Crossref Search ADS PubMed WorldCat Planer-Friedrich B , Kühnlenz T , Halder D , Lohmayer R , Wilson N , Rafferty C , Clemens S . 2017 . Thioarsenate toxicity and tolerance in the model system Arabidopsis thaliana . Environmental Science & Technology 51 , 7187 – 7196 . Google Scholar Crossref Search ADS PubMed WorldCat Planer-Friedrich B , London J , McCleskey RB , Nordstrom DK , Wallschläger D . 2007 . Thioarsenates in geothermal waters of Yellowstone National Park: determination, preservation, and geochemical importance . Environmental Science & Technology 41 , 5245 – 5251 . Google Scholar Crossref Search ADS PubMed WorldCat Planer-Friedrich B , Wallschläger D . 2009 . A critical investigation of hydride generation-based arsenic speciation in sulfidic waters . Environmental Science & Technology 43 , 5007 – 5013 . Google Scholar Crossref Search ADS PubMed WorldCat Pommerrenig B , Diehn TA , Bienert GP . 2015 . Metalloido-porins: essentiality of Nodulin 26-like intrinsic proteins in metalloid transport . Plant Science 238 , 212 – 227 . Google Scholar Crossref Search ADS PubMed WorldCat Raab A , Schat H , Meharg AA , Feldmann J . 2005 . Uptake, translocation and transformation of arsenate and arsenite in sunflower (Helianthus annuus): formation of arsenic–phytochelatin complexes during exposure to high arsenic concentrations . New Phytologist 168 , 551 – 558 . Google Scholar Crossref Search ADS PubMed WorldCat Raab A , Williams PN , Meharg A , Feldmann J . 2007 . Uptake and translocation of inorganic and methylated arsenic species by plants . Environmental Chemistry 4 , 197 – 203 . Google Scholar Crossref Search ADS WorldCat Renberg I , Persson M , Emteryd O . 1994 . Preindustrial atmospheric lead contamination detected in Swedish lake-sediments . Nature 368 , 323 – 326 . Google Scholar Crossref Search ADS WorldCat Salt DE . 2017 . Would the real arsenate reductase please stand up? New Phytologist 215 , 926 – 928 . Google Scholar Crossref Search ADS PubMed WorldCat Sánchez-Bermejo E , Castrillo G , del Llano B , et al. 2014 . Natural variation in arsenate tolerance identifies an arsenate reductase in Arabidopsis thaliana . Nature Communications 5 , 4617 . Google Scholar Crossref Search ADS PubMed WorldCat Sasaki A , Yamaji N , Ma JF . 2014 . Overexpression of OsHMA3 enhances Cd tolerance and expression of Zn transporter genes in rice . Journal of Experimental Botany 65 , 6013 – 6021 . Google Scholar Crossref Search ADS PubMed WorldCat Sasaki A , Yamaji N , Yokosho K , Ma JF . 2012 . Nramp5 is a major transporter responsible for manganese and cadmium uptake in rice . The Plant Cell 24 , 2155 – 2167 . Google Scholar Crossref Search ADS PubMed WorldCat Satoh-Nagasawa N , Mori M , Nakazawa N , Kawamoto T , Nagato Y , Sakurai K , Takahashi H , Watanabe A , Akagi H . 2012 . Mutations in rice (Oryza sativa) heavy metal ATPase 2 (OsHMA2) restrict the translocation of zinc and cadmium . Plant & Cell Physiology 53 , 213 – 224 . Google Scholar Crossref Search ADS PubMed WorldCat Sauve S , Hendershot W , Allen HE . 2000 . Solid-solution partitioning of metals in contaminated soils: dependence on pH, total metal burden, and organic matter . Environmental Science & Technology 34 , 1125 – 1131 . Google Scholar Crossref Search ADS WorldCat Scheben A , Edwards D . 2018 . Bottlenecks for genome-edited crops on the road from lab to farm . Genome Biology 19 , 178 . Google Scholar Crossref Search ADS PubMed WorldCat Scheben A , Wolter F , Batley J , Puchta H , Edwards D . 2017 . Towards CRISPR/Cas crops—bringing together genomics and genome editing . New Phytologist 216 , 682 – 698 . Google Scholar Crossref Search ADS PubMed WorldCat Settle DM , Patterson CC . 1980 . Lead in albacore: guide to lead pollution in Americans . Science 207 , 1167 – 1176 . Google Scholar Crossref Search ADS PubMed WorldCat Shao JF , Xia J , Yamaji N , Shen RF , Ma JF . 2018 . Effective reduction of cadmium accumulation in rice grain by expressing OsHMA3 under the control of the OsHMA2 promoter . Journal of Experimental Botany 69 , 2743 – 2752 . Google Scholar Crossref Search ADS PubMed WorldCat Shi S , Wang T , Chen Z , Tang Z , Wu Z , Salt DE , Chao DY , Zhao FJ . 2016 . OsHAC1;1 and OsHAC1;2 function as arsenate reductases and regulate arsenic accumulation . Plant Physiology 172 , 1708 – 1719 . Google Scholar Crossref Search ADS PubMed WorldCat Shri M , Dave R , Diwedi S , Shukla D , Kesari R , Tripathi RD , Trivedi PK , Chakrabarty D . 2014 . Heterologous expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in rice leads to lower arsenic accumulation in grain . Scientific Reports 4 , 5784 . Google Scholar Crossref Search ADS PubMed WorldCat Smyth SJ , Lassoued R . 2019 . Agriculture R&D implications of the CJEU’s gene-specific mutagenesis ruling . Trends in Biotechnology 37 , 337 – 340 . Google Scholar Crossref Search ADS PubMed WorldCat Song WY , Mendoza-Cózatl DG , Lee Y , Schroeder JI , Ahn SN , Lee HS , Wicker T , Martinoia E . 2014a. Phytochelatin–metal(loid) transport into vacuoles shows different substrate preferences in barley and Arabidopsis . Plant, Cell & Environment 37 , 1192 – 1201 . Google Scholar Crossref Search ADS PubMed WorldCat Song W-Y , Park J , Mendoza-Cózatl DG , et al. 2010 . Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters . Proceedings of the National Academy of Sciences, USA 107 , 21187 – 21192 . Google Scholar Crossref Search ADS WorldCat Song W-Y , Yamaki T , Yamaji N , Ko D , Jung K-H , Fujii-Kashino M , An G , Martinoia E , Lee Y , Ma JF . 2014b. A rice ABC transporter, OsABCC1, reduces arsenic accumulation in the grain . Proceedings of the National Academy of Sciences, USA 111 , 15699 – 704 . Google Scholar Crossref Search ADS WorldCat Songmei L , Jie J , Yang L , Jun M , Shouling X , Yuanyuan T , Youfa L , Qingyao S , Jianzhong H . 2019 . Characterization and evaluation of OsLCT1 and OsNramp5 mutants generated through CRISPR/Cas9-mediated mutagenesis for breeding low Cd rice . Rice Science 26 , 88 – 97 . Google Scholar Crossref Search ADS WorldCat Straif K , Benbrahim-Tallaa L , Baan R , et al. ; WHO International Agency for Research on Cancer Monograph Working Group . 2009 . A review of human carcinogens–Part C: metals, arsenic, dusts, and fibres . The Lancet. Oncology 10 , 453 – 454 . Google Scholar Crossref Search ADS PubMed WorldCat Sun SK , Chen Y , Che J , Konishi N , Tang Z , Miller AJ , Ma JF , Zhao FJ . 2018 . Decreasing arsenic accumulation in rice by overexpressing OsNIP1;1 and OsNIP3;3 through disrupting arsenite radial transport in roots . New Phytologist 219 , 641 – 653 . Google Scholar Crossref Search ADS PubMed WorldCat Sunkar R , Kaplan B , Bouché N , Arazi T , Dolev D , Talke IN , Maathuis FJ , Sanders D , Bouchez D , Fromm H . 2000 . Expression of a truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arabidopsis CNGC1 gene confer Pb2+ tolerance . The Plant Journal 24 , 533 – 542 . Google Scholar Crossref Search ADS PubMed WorldCat Takahashi R , Ishimaru Y , Shimo H , Ogo Y , Senoura T , Nishizawa NK , Nakanishi H . 2012 . The OsHMA2 transporter is involved in root-to-shoot translocation of Zn and Cd in rice . Plant, Cell & Environment 35 , 1948 – 1957 . Google Scholar Crossref Search ADS PubMed WorldCat Tang L , Mao B , Li Y , et al. 2017 . Knockout of OsNramp5 using the CRISPR/Cas9 system produces low Cd-accumulating indica rice without compromising yield . Scientific Reports 7 , 14438 . Google Scholar Crossref Search ADS PubMed WorldCat Tang Z , Cai H , Li J , Lv Y , Zhang W , Zhao FJ . 2017a. Allelic variation of NtNramp5 associated with cultivar variation in cadmium accumulation in tobacco . Plant & Cell Physiology 58 , 1583 – 1593 . Google Scholar Crossref Search ADS PubMed WorldCat Tang Z , Chen Y , Chen F , Ji Y , Zhao FJ . 2017b. OsPTR7 (OsNPF8.1), a putative peptide transporter in rice, is involved in dimethylarsenate accumulation in rice grain . Plant & Cell Physiology 58 , 904 – 913 . Google Scholar Crossref Search ADS PubMed WorldCat Tang Z , Kang Y , Wang P , Zhao F-J . 2016 . Phytotoxicity and detoxification mechanism differ among inorganic and methylated arsenic species in Arabidopsis thaliana . Plant and Soil 401 , 243 – 257 . Google Scholar Crossref Search ADS WorldCat Tchounwou PB , Yedjou CG , Patlolla AK , Sutton DJ . 2012 . Heavy metals toxicity and the environment . EXS 101 , 133 – 164 . Google Scholar PubMed WorldCat Ueno D , Yamaji N , Kono I , Huang CF , Ando T , Yano M , Ma JF . 2010 . Gene limiting cadmium accumulation in rice . Proceedings of the National Academy of Sciences, USA 107 , 16500 – 16505 . Google Scholar Crossref Search ADS WorldCat United Nations Environment Programme. 2010 . Final review of scientific information on cadmium - version of December 2010 . Geneva : United Nations . Google Preview WorldCat COPAC Uraguchi S , Fujiwara T . 2013 . Rice breaks ground for cadmium-free cereals . Current Opinion in Plant Biology 16 , 328 – 334 . Google Scholar Crossref Search ADS PubMed WorldCat Uraguchi S , Kamiya T , Sakamoto T , Kasai K , Sato Y , Nagamura Y , Yoshida A , Kyozuka J , Ishikawa S , Fujiwara T . 2011 . Low-affinity cation transporter (OsLCT1) regulates cadmium transport into rice grains . Proceedings of the National Academy of Sciences, USA 108 , 20959 – 20964 . Google Scholar Crossref Search ADS WorldCat Uraguchi S , Sone Y , Ohta Y , Ohkama-Ohtsu N , Hofmann C , Hess N , Nakamura R , Takanezawa Y , Clemens S , Kiyono M . 2018 . Identification of C-terminal regions in Arabidopsis thaliana phytochelatin synthase 1 specifically involved in activation by arsenite . Plant & Cell Physiology 59 , 500 – 509 . Google Scholar Crossref Search ADS PubMed WorldCat Uraguchi S , Tanaka N , Hofmann C , et al. 2017 . Phytochelatin synthase has contrasting effects on cadmium and arsenic accumulation in rice grains . Plant & Cell Physiology 58 , 1730 – 1742 . Google Scholar Crossref Search ADS PubMed WorldCat US EPA National Center for Environmental Assessment RTPN , Kirrane E . 2013 . Integrated Science Assessment (ISA) for Lead (Final Report, July 2013) . Washington, DC : U.S. Environmental Protection Agency . Google Preview WorldCat COPAC Wang C , Na G , Bermejo ES , Chen Y , Banks JA , Salt DE , Zhao F-J . 2018 . Dissecting the components controlling root-to-shoot arsenic translocation in Arabidopsis thaliana . New Phytologist 217 , 206 – 218 . Google Scholar Crossref Search ADS PubMed WorldCat Wang H , Shan X , Liu T , Xie Y , Wen B , Zhang S , Han F , van Genuchten MT . 2007 . Organic acids enhance the uptake of lead by wheat roots . Planta 225 , 1483 – 1494 . Google Scholar Crossref Search ADS PubMed WorldCat Wiebe K , Harris NS , Faris JD , Clarke JM , Knox RE , Taylor GJ , Pozniak CJ . 2010 . Targeted mapping of Cdu1, a major locus regulating grain cadmium concentration in durum wheat (Triticum turgidum L. var durum) RID A-6999-2009 . Theoretical and Applied Genetics 121 , 1047 – 1058 . Google Scholar Crossref Search ADS PubMed WorldCat Williams PN , Villada A , Deacon C , Raab A , Figuerola J , Green AJ , Feldmann J , Meharg AA . 2007 . Greatly enhanced arsenic shoot assimilation in rice leads to elevated grain levels compared to wheat and barley . Environmental Science & Technology 41 , 6854 – 6859 . Google Scholar Crossref Search ADS PubMed WorldCat Wong CKE , Cobbett CS . 2009 . HMA P-type ATPases are the major mechanism for root-to-shoot Cd translocation in Arabidopsis thaliana . New Phytologist 181 , 71 – 78 . Google Scholar Crossref Search ADS PubMed WorldCat World Health Organization, Food and Agricultiural Organization of the United Nations. 2011 . Evaluation of certain contaminants in food: seventy-second [72nd] report of the Joint FAO/WHO Expert Committee on Food Additives . Geneva : World Health Organization . Google Preview WorldCat COPAC Wu D , Sato K , Ma JF . 2015 . Genome-wide association mapping of cadmium accumulation in different organs of barley . New Phytologist 208 , 817 – 829 . Google Scholar Crossref Search ADS PubMed WorldCat Wu D , Yamaji N , Yamane M , Kashino-Fujii M , Sato K , Feng Ma J . 2016 . The HvNramp5 transporter mediates uptake of cadmium and manganese, but not iron . Plant Physiology 172 , 1899 – 1910 . Google Scholar Crossref Search ADS PubMed WorldCat Wu Z , Ren H , McGrath SP , Wu P , Zhao FJ . 2011 . Investigating the contribution of the phosphate transport pathway to arsenic accumulation in rice . Plant Physiology 157 , 498 – 508 . Google Scholar Crossref Search ADS PubMed WorldCat Wysocki R , Tamás MJ . 2010 . How Saccharomyces cerevisiae copes with toxic metals and metalloids . FEMS Microbiology Reviews 34 , 925 – 951 . Google Scholar Crossref Search ADS PubMed WorldCat Xu FF , Imlay JA . 2012 . Silver(I), mercury(II), cadmium(II), and zinc(II) target exposed enzymic iron–sulfur clusters when they toxify Escherichia coli . Applied and Environmental Microbiology 78 , 3614 – 3621 . Google Scholar Crossref Search ADS PubMed WorldCat Xu J , Shi S , Wang L , Tang Z , Lv T , Zhu X , Ding X , Wang Y , Zhao FJ , Wu Z . 2017 . OsHAC4 is critical for arsenate tolerance and regulates arsenic accumulation in rice . New Phytologist 215 , 1090 – 1101 . Google Scholar Crossref Search ADS PubMed WorldCat Xu W , Dai W , Yan H , Li S , Shen H , Chen Y , Xu H , Sun Y , He Z , Ma M . 2015 . Arabidopsis NIP3;1 plays an important role in arsenic uptake and root-to-shoot translocation under arsenite stress conditions . Molecular Plant 8 , 722 – 733 . Google Scholar Crossref Search ADS PubMed WorldCat Xu X , Chen C , Wang P , Kretzschmar R , Zhao F-J . 2017 . Control of arsenic mobilization in paddy soils by manganese and iron oxides . Environmental Pollution 231 , 37 – 47 . Google Scholar Crossref Search ADS PubMed WorldCat Xu XY , McGrath SP , Zhao FJ . 2007 . Rapid reduction of arsenate in the medium mediated by plant roots . New Phytologist 176 , 590 – 599 . Google Scholar Crossref Search ADS PubMed WorldCat Yamaji N , Ma JF . 2014 . The node, a hub for mineral nutrient distribution in graminaceous plants . Trends in Plant Science 19 , 556 – 563 . Google Scholar Crossref Search ADS PubMed WorldCat Yamaji N , Xia J , Mitani-Ueno N , Yokosho K , Feng Ma J . 2013 . Preferential delivery of zinc to developing tissues in rice is mediated by P-type heavy metal ATPase OsHMA2 . Plant Physiology 162 , 927 – 939 . Google Scholar Crossref Search ADS PubMed WorldCat Yamazaki S , Ueda Y , Mukai A , Ochiai K , Matoh T . 2018 . Rice phytochelatin synthases OsPCS1 and OsPCS2 make different contributions to cadmium and arsenic tolerance . Plant Direct 2 , e00034 . Google Scholar Crossref Search ADS PubMed WorldCat Yan H , Xu W , Xie J , et al. 2019 . Variation of a major facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies . Nature Communications 10 , 2562 . Google Scholar Crossref Search ADS PubMed WorldCat Yan J , Wang P , Wang P , Yang M , Lian X , Tang Z , Huang CF , Salt DE , Zhao FJ . 2016 . A loss-of-function allele of OsHMA3 associated with high cadmium accumulation in shoots and grain of Japonica rice cultivars . Plant, Cell & Environment 39 , 1941 – 1954 . Google Scholar Crossref Search ADS PubMed WorldCat Yang J , Gao MX , Hu H , Ding XM , Lin HW , Wang L , Xu JM , Mao CZ , Zhao FJ , Wu ZC . 2016 . OsCLT1, a CRT-like transporter 1, is required for glutathione homeostasis and arsenic tolerance in rice . New Phytologist 211 , 658 – 670 . Google Scholar Crossref Search ADS PubMed WorldCat Yang M , Lu K , Zhao FJ , et al. 2018 . Genome-wide association studies reveal the genetic basis of ionomic variation in rice . The Plant Cell 30 , 2720 – 2740 . Google Scholar PubMed WorldCat Ye WL , Wood BA , Stroud JL , Andralojc PJ , Raab A , McGrath SP , Feldmann J , Zhao FJ . 2010 . Arsenic speciation in phloem and xylem exudates of castor bean . Plant Physiology 154 , 1505 – 1513 . Google Scholar Crossref Search ADS PubMed WorldCat Zhang J , Martinoia E , Lee Y . 2018 . Vacuolar transporters for cadmium and arsenic in plants and their applications in phytoremediation and crop development . Plant & Cell Physiology 59 , 1317 – 1325 . Google Scholar PubMed WorldCat Zhang J , Zhou W , Liu B , He J , Shen Q , Zhao FJ . 2015 . Anaerobic arsenite oxidation by an autotrophic arsenite-oxidizing bacterium from an arsenic-contaminated paddy soil . Environmental Science & Technology 49 , 5956 – 5964 . Google Scholar Crossref Search ADS PubMed WorldCat Zhao FJ , Ago Y , Mitani N , Li RY , Su YH , Yamaji N , McGrath SP , Ma JF . 2010a. The role of the rice aquaporin Lsi1 in arsenite efflux from roots . New Phytologist 186 , 392 – 399 . Google Scholar Crossref Search ADS PubMed WorldCat Zhao FJ , McGrath SP , Meharg AA . 2010b. Arsenic as a food chain contaminant: mechanisms of plant uptake and metabolism and mitigation strategies . Annual Review of Plant Biology 61 , 535 – 559 . Google Scholar Crossref Search ADS PubMed WorldCat Zhao FJ , Moore KL , Lombi E , Zhu YG . 2014 . Imaging element distribution and speciation in plant cells . Trends in Plant Science 19 , 183 – 192 . Google Scholar Crossref Search ADS PubMed WorldCat Zhao F-J , Stroud JL , Khan MA , McGrath SP . 2012 . Arsenic translocation in rice investigated using radioactive As-73 tracer . Plant and Soil 350 , 413 – 420 . Google Scholar Crossref Search ADS WorldCat Zhao FJ , Zhu YG , Meharg AA . 2013 . Methylated arsenic species in rice: geographical variation, origin, and uptake mechanisms . Environmental Science & Technology 47 , 3957 – 3966 . Google Scholar Crossref Search ADS PubMed WorldCat Zheng MZ , Cai C , Hu Y , Sun GX , Williams PN , Cui HJ , Li G , Zhao FJ , Zhu YG . 2011 . Spatial distribution of arsenic and temporal variation of its concentration in rice . New Phytologist 189 , 200 – 209 . Google Scholar Crossref Search ADS PubMed WorldCat Zhu YG , Yoshinaga M , Zhao FJ , Rosen BP . 2014 . Earth abides arsenic biotransformations . Annual Review of Earth and Planetary Sciences 42 , 443 – 467 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Safer food through plant science: reducing toxic element accumulation in crops
JO - Journal of Experimental Botany
DO - 10.1093/jxb/erz366
DA - 2019-10-24
UR - https://www.deepdyve.com/lp/oxford-university-press/safer-food-through-plant-science-reducing-toxic-element-accumulation-uis19XLcpD
SP - 5537
VL - 70
IS - 20
DP - DeepDyve
ER -