**About the Editors**

**Luigi De Bellis** (Professor) completed his degree in agricultural science with distinction, at the University of Pisa on October 30th, 1981. He then went on to study for his PhD in crop and fruit trees science (curriculum propagation), also at the University of Pisa, 1983–1986. He was a researcher at the Dept. of Crop Biology, University of Pisa, from January 1st, 1988 to October 31st, 1998. He was employed as a postdoctoral fellow of the Japan Society for Promotion of Science (JSPS) in the lab directed by Prof. M. Nishimura, at the National Institute for Basic Biology (NIBB), Okazaki, Japan, for 18 months, from March 1990 to October 1991. He was then employed as a postdoctoral fellow in Dr. S.M. Smith's lab, Institute of Cell and Molecular Biology (ICMB), at the University of Edinburgh, UK, for 12 months, from October 1993 to October 1994; this fellowship was sponsored by the EU's Human Capital Mobility Program. He enrolled on a short-term training fellowship sponsored by the EU (Technical Priority Program) in Dr. S.M. Smith's lab, ICMB, at the University of Edinburgh, UK, for 2 months, from July to September 1995. He was then granted a professorship contract (Fellow of the Japanese Minister of Culture and Research) at Prof. M. Nishimura's lab, NIBB, Okazaki, Japan, for 12 months, from March 1997 to March 1998. De Bellis was an associate professor of plant physiology at the University of Lecce, Faculty of Science, starting on November 1st, 1998. On November 1st, 2002, he became Chair Professor of Plant Physiology at the University of Lecce, Faculty of Science. From March 2012 to February 2020, he was Head of the Dept. of Biological and Environmental Science and Technologies (DiSTeBA) at the University of Salento. Additionally, he has been a research unit leader for several regional, national and European/international research projects. He is also a referee of the main major plant physiology journals and other journals, and a member of the Editorial Board of Biology, Horticulturae, and Plants. He is the author of over 100 scientific publications. De Bellis' main scientific interests are the enzymes of the glyoxylate cycle, the intracellular localization of aconitase isoforms, carbohydrates and the control of gene expression, the study of wheat and barley flours for the preparation of fresh pasta, the valorisation of Salento olive oil and the development of oil wastewater purification systems, the valorisation of agro-food products and the evaluation of organoleptic qualities, the genetics of plant species such as olive trees, durum wheat and bread wheat, plant response to heavy metals, Xylella fastidiosa as an olive tree pest, and the selection of tolerant olive varieties.

**Alessio Aprile** is a researcher. He completed his PhD in 2008, after defending a thesis about the transcriptome changes after abiotic stresses in crops. Since 2015, he has been a plant physiology researcher at the Department of Biological and Environmental Sciences and Technologies at the University of Salento. He was the principal investigator of the project "Genetic and breeding evaluation of durum wheat cv Cappelli", funded by the Apulia region as part of the program "Future in Research". The research activity, documented by more than 30 publications, is focused on the study of the physiology of abiotic stresses in plants. Part of his works has investigated the effects of drought, heat stress and CO<sup>2</sup> in wheat. Recently, his research activity has been focused on the effects of heavy metals in durum wheat. He is also the co-author of papers describing olive secondary metabolites and the pathogen Xylella fastidiosa. He is an assistant professor in plant physiology at the University of Salento.

## *Editorial* **Editorial for Special Issue "Heavy Metals Accumulation, Toxicity, and Detoxification in Plants"** †

**Alessio Aprile and Luigi De Bellis \***

Department of Biological and Environmental Sciences and Technologies, University of Salento,

I-73100 Lecce, Italy; alessio.aprile@unisalento.it

**\*** Correspondence: luigi.debellis@unisalento.it

† This article is dedicated to Antonio Michele Stanca, eminent plant geneticist, friend, and mentor.

Received: 1 June 2020; Accepted: 5 June 2020; Published: 9 June 2020

"Heavy metals" is a collective term widely applied for the group of metals and metalloids with an atomic density above 4 g/cm<sup>3</sup> [1]. Non-essential toxic plant heavy metals include arsenic (As), cadmium (Cd), chromium (Cr), cobalt (Co), lead (Pb), mercury (Hg), nickel (Ni), and vanadium (V); whereas others are essential, such as copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). Heavy metals cause harmful effects in plants, animals, and humans as a result of long-term or acute exposure. Toxicity from heavy metals is increasing due to the extensive release from industrial, agricultural, chemical, domestic, and technological sources, which in turn contaminate the water, soil, and air. Natural phenomena, such as volcanic eruptions and sea movements, also contribute to the natural cyclization of metals on the earth, and human activities often alter the rate of release and transport by increasing emissions by a few orders of magnitude.

Heavy metals penetrate the human body through water, food, and air. Inside an organism, they bind to cellular structures, thereby damaging the performance of essential biological functions. Metals, for example, easily bind to the sulfhydryl groups of several enzymes that control the speed of metabolic reactions: the "new" metal-enzyme complex leads to the loss of the catalytic activity of the enzyme. The level of toxicity from heavy metals depends on several factors, including time of exposure, dose, and the health status of the people exposed.

The European Environment Agency (EEA) reported that of the 1000 industrial plants that released heavy metals into the air in 2016, eighteen accounted for more than half of the total pollution, suggesting a great responsibility on the part of a few large companies (Figure 1) [2].

An additional issue is the biomagnification (or bioaccumulation) caused by the very slow rate of elimination of heavy metals from an organism. Bioaccumulation, in ecology and biology, is the process whereby the accumulation of toxic substances in living beings increases in concentration following a rise in the trophic level: the higher the trophic level, the stronger the concentration of heavy metals. Biomagnification is also expressed as the concentration increase of a pollutant in a biological organism over time.

To limit the risks for humans and the environment, many countries have legislated limits for each heavy metal. Specific limits have been defined in drinking, waste, and surface waters (lakes, rivers, seas). There are also limits in foods and animal feed, because heavy metals can easily enter the food chain through plants (or algae) and are subsequently bioaccumulated into the higher trophic levels. The risk for human health is due to directly eating edible plant tissues, or indirectly through eating animals that have in turn fed on herbivores or directly on edible plant tissues. Understanding the mechanisms for regulating the storage and distribution of heavy metals in plants is the basis for improving the safety of the food chain.

**Figure 1.** Environmental pressures of heavy metal releases to air, 2016 [2]. An eco-toxicity approach (USEtox model, https://usetox.org/model) was applied to illustrate spatially the combined environmental pressures on Europe's environment caused by releases of the selected pollutants. This gives information about the location of source of heavy metals and the low or high levels in air as indicated in the upper left corner of the figure.

This special issue, entitled "Heavy Metals Accumulation, Toxicity, and Detoxification in Plants", explores three main issues concerning heavy metals: (a) the accumulation and partitioning of heavy metals in crops and wild plants; (b) the toxicity and molecular behaviors of cells, tissues, and their effects on physiology and plant growth; and (c) detoxification strategies, plant tolerance, and phytoremediation.

The issue contains a total of 19 articles (Table 1). There are four reviews covering the following topics: phytoremediation [3], manganese phytotoxicity in plants [4], cadmium effect on plant development [5], the genetic characteristics of Cd accumulation and the research status of genes and quantitative trait loci (QTLs) in rice [6], and fifteen original research articles, mainly regarding the impact of cadmium on plants [7–21].


**Table 1.** Contributors to the special issue "Heavy Metals Accumulation, Toxicity, and Detoxification in Plants". ABC: ATP-binding cassette.

Cadmium is therefore the predominant topic of this special issue, thus confirming the focus of the research community on the negative impacts determined by cadmium or cadmium associated with other heavy metals. Interestingly, we did not receive any manuscripts on other heavy metals such as arsenic, chromium and mercury despite their danger for human health.

The cadmium research articles come from China, Poland, Italy, Canada, Pakistan, and the United States. These studies investigate different molecular mechanisms or approaches, using model plants such as *Arabidopsis* and tobacco [17,18,20] or hyperaccumulator plant species [9,16,19,21] to unravel their molecular strategies in heavy metal accumulation. Other articles focus on how to prevent cadmium from entering the food chain by investigating edible plants such as *Zea mays* [7], durum and bread wheat [12,13], or animal feeding plants such as *Lolium multiflorum*.

The studies reveal some common strategies in terms of the molecular mechanisms involved. Some plants activate the production of small proteins such as glutathione S-transferase (GST) and

small heat shock protein (sHSP) [9,11,21] or antioxidants [16]. In order to alleviate heavy metal toxicity, other plants respond by activating a complex metabolism-like auxin pathway [7,8,17]. Plants also produce specific metallothionines and phytosiderophores [10,12] to chelate heavy metals or to activate heavy metals transporters such as heavy metal ATPase (e.g., HMA2 and HMA4) and ATP-binding cassette (ABC) transporters [12,13,18,19,21].

The studies in this special issue highlight considerable genetic variability, suggesting different possibilities for accumulation, translocation, and reducing or controlling heavy metals toxicity in plants.

Heavy metal pollution is still one of the world's great challenges. In the future, the main research objective should be to identify and characterize the genes controlling the uptake and translocation of heavy metals in a plant's above-ground organs in order to produce (i) phytoremediation plants that efficiently move heavy metals in the stem and leaves or (ii) plants dedicated to human nutrition that transport heavy metals only in trace amounts to seeds or fruits.

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

### **Abbreviations**



### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Heavy Metal Pollutions: State of the Art and Innovation in Phytoremediation**

**Giovanni DalCorso 1,\*, Elisa Fasani 1, Anna Manara 1, Giovanna Visioli <sup>2</sup> and Antonella Furini 1,\***


Received: 20 June 2019; Accepted: 10 July 2019; Published: 11 July 2019

**Abstract:** Mineral nutrition of plants greatly depends on both environmental conditions, particularly of soils, and the genetic background of the plant itself. Being sessile, plants adopted a range of strategies for sensing and responding to nutrient availability to optimize development and growth, as well as to protect their metabolisms from heavy metal toxicity. Such mechanisms, together with the soil environment, meaning the soil microorganisms and their interaction with plant roots, have been extensively studied with the goal of exploiting them to reclaim polluted lands; this approach, defined phytoremediation, will be the subject of this review. The main aspects and innovations in this field are considered, in particular with respect to the selection of efficient plant genotypes, the application of improved cultural strategies, and the symbiotic interaction with soil microorganisms, to manage heavy metal polluted soils.

**Keywords:** phytoremediation; heavy metals; hyperaccumulation; plant genotype improvement; soil management

### **1. Introduction**

Like all living organisms, plants require chemical elements that are used as cofactors in biochemical reactions, as components of structural proteins and macromolecules, and as regulators of the electrochemical balance of cellular compartments [1]. Soil availability of nutrient elements fluctuate due to temperature, precipitation, soil type and pH, oxygen content, and the presence or absence of other inorganic and organic compounds. Being sessile organisms, plants developed adaptive and flexible strategies for sensing and responding to fluctuations in element availability to optimize growth, development, and reproduction under a dynamic range of environmental conditions. In addition, once taken up, elements must be allocated to different organs, cell types, and tissues through tight homeostasis mechanisms to ensure metal requirement, storage, and re-mobilization under different environmental conditions [2].

Heavy metals are naturally occurring elements, which are widely distributed in the Earth's crust; they derive from rocks of volcanic, sedimentary or metamorphic origin, but in recent years, the prevalence of heavy metals in areas of agricultural and industrial activities has increased because of human activity [3]. A limited number of heavy metal ions are water soluble upon physiological conditions and thus bioavailable to plants and other living organisms, being either essential or potential risks for life [4]. Indeed, many heavy metals (mainly Fe, Zn, Cu, Ni, Co, and Mo), which are toxic when present in excess, are essential for plant and cellular biochemistry being involved in cell protection, gene regulation, and signal transduction and their absence (or deficiency) inhibits plant growth, reproduction, and tolerance to environmental stresses [5]. Other heavy metals such as Cd, Hg, Ag, Pb, and Cr are biologically non-essential and show toxicity even at low concentrations. The similarity of

certain non-essential metals to essential ones allows the latter to enter plants replacing their essential homolog and interfering with biological functions. To minimize the unfavorable effects of non-essential heavy metals, while maintaining the uptake of essential elements, plants have evolved a homeostatic network that controls metal uptake, trafficking, storage, and detoxification. Although a basal metal tolerance is usual, to guarantee the correct concentration of essential metal nutrients in different cell types at different stages of plant development, plants have acquired complex mechanisms to avoid or overcome the harmfulness of heavy metal excess. In metal-rich soils, plants have evolved mechanisms to tolerate, within a certain limit, metal toxicity. Plants encountering heavy metals employ two main approaches: the most common strategy is metal exclusion, in which metal accumulation is limited to the belowground organs. Uptake and root-to-shoot transport are regulated to maintain low shoot content over a wide range of external concentration. On the opposite, plants can accumulate metals, and an extreme evolution of this capacity is well represented in metal (hyper) accumulators, which are able to accumulate heavy metals in their shoots keeping low concentrations in roots. This trait is associated with the enhanced ability to detoxify high metal levels in the aboveground tissues [6]. Both strategies are regulated by finely tuned homeostatic mechanisms to guarantee sufficient metal uptake, transport, accumulation, and detoxification.

### **2. Plant Mechanisms for Heavy Metal Tolerance and Detoxification**

Relevant components of homeostatic networks underneath metal tolerance and detoxification include ion transporters, metallo-chaperons, and ligands that act in concert to ensure metal uptake, transport to different cell types and delivery inside cells. Membrane proteins are able to transport different metals across cellular membranes, playing a pivotal role in each influx-efflux step of the translocation from roots to shoots. The function of several transporters involved in import, trafficking, sequestration, and export of essential metals across the plasma membrane, tonoplast, or chloroplast envelope has been clarified [2,7,8]. Metal transporters have been classified into families according to sequence homology. For example, the ZIP family (ZRT-IRT-like proteins) is involved in several homeostatic processes including uptake and translocation from root to shoot [9,10]. The NRAMPs (naturally resistant associated macrophage proteins) comprises members such as: NRAMP1, which when in *A. thaliana* is localized in the plasma membrane, is involved in Fe transport, and also shows high-affinity Mn uptake from soil [11]; NRAMP3 and NRAMP4, which are localized in the tonoplast and are essential for exporting stored Fe from the vacuole during seed germination [12]. The HMA proteins (heavy metals P1B-type ATPases) contribute to pump cations out of the cytoplasm by ATP hydrolysis. HMA1 localizes in the chloroplast envelope and is possibly involved in plastid Zn detoxification under Zn excess [13]. Similarly, HMA3 is involved in the detoxification of Zn, Cd, Co, and Pb by regulating their sequestration into the vacuole, and HMA4, a plasma membrane transporter, plays a role in Zn efflux from the cytoplasm and xylem loading/unloading [14,15]. Another group of transporters that tightly regulate metal homeostasis ensuring the appropriate metal supply to tissues is represented by the CDF (cation diffusion facilitator) family whose members are involved in the translocation of metals towards internal compartments and extracellular space [7]. Among them, several MTPs (metal tolerance proteins) have been described in a variety of plant species. The best characterized is MTP1, which is a vacuolar Zn<sup>2</sup>+/H<sup>+</sup> antiporter involved in Zn tolerance, which in case of Zn excess accumulates Zn into the vacuole [16].

In addition to metal trafficking, plant responses to heavy metal stress include a variety of mechanisms, ranging from changes in gene expression and methylation to metabolic and biochemical adjustments, with the final goal of scavenging toxic metal ions, and ameliorating stress symptoms and damages. The production of hormones such as ethylene, jasmonic acid, and abscisic acid is also induced, as well as molecules involved in chelation of metal ions, such as organic acid, specific amino acids, phytochelatins, and metallothioneins [17,18]. Proline and histidine induce tolerance by chelating ions within cells and xylem sap [19]. The induction of phytochelatins occurs because of high levels of different heavy metals although Cd seems to be the most effective stimulator [20]. As opposed to phytochelatins, which are produced enzymatically, metallothioneins are gene-encoded polypeptides that play a role in the homeostasis and sequestration of intracellular metal ions [21,22]. Chelating compounds contribute to heavy metal tolerance by removing toxic ions from sensitive sites through sequestration and subsequent vacuolar compartmentalization by tonoplast-localized transporters. When the above-mentioned strategies are insufficient to contain the damage, cells trigger the production of reactive oxygen species (ROS), which might potentially result in massive oxidative stress with cell homeostasis disruption, inhibition of most cellular processes, DNA damage and protein oxidation [23]. As a result, cells activate the ROS-scavenging machinery with the production of antioxidant compounds such as glutathione, flavonoids, and carotenoids as well as antioxidant enzymes including superoxide dismutases, catalases, and peroxidases.

### **3. Phytoremediation**

As mentioned before, despite natural occurrence in soils, large quantities of heavy metals and metalloids have been dispersed into the environment by a variety of human activities including fertilizer use in agriculture, metal mining, and manufacturing by metallurgy, fossil fuel use, and military operations. Land contamination poses a serious risk to both human health and animal and plant biodiversity [24]. There are a variety of conventional approaches to reclaim contaminated sites that are usually based on physicochemical techniques, including soil washing, electric field application (electrokinetics), excavation and reburial of contaminated matrices, pumping and treating systems in case of polluted water. These approaches suffer from two main disadvantages, being expensive and frequently inefficient if pollutants are present at low concentrations. Moreover, harsh approaches cause significant changes to the physicochemical and biological characteristics of soils and landscapes. Ecological rehabilitation of contaminated sites may also be achieved by *phytoremediation*: an alternative in situ technology, which exploits plants and their rhizosphere to remove the contaminants or lower their bioavailability in soil and water with concurrent land revegetation [25].

### *3.1. Strategies for Phytoremediation*

Once placed in loco, plants deepen their root system into the contaminated soil matrix, establishing ecosystems with soil bacteria and fungi. Into this context, plants and the rhizosphere, i.e., soil and microorganisms associated to roots, employ mechanisms that altogether are responsible for the soil reclamation: phytodegradation, phytoextraction, phytovolatilization, phyto(rhizo)stabilization and phyto(rhizo)filtration (Figure 1). Such mechanisms are usually considered separately just for sake of clarity, even if they act in concert on the metal decontamination. In the following lines, each of these aspects will be highlighted individually, with the exclusion of phytodegradation, which is applicable to organic contaminants, rather than heavy metals, which are not degradable [26]. Plants acquire mineral elements from the soil primarily in the form of inorganic ions. The extended root system and its ability to absorb ionic compounds even at low concentrations make mineral absorption highly efficient. Obviously, heavy metals and metalloids can be absorbed by the plant root system, but since some of them, such as Cd and Pb, have no known biological function, it is likely that specific transporters do not exist. Indeed, toxic metals enter into the cells through cation transporters with a wide range of substrate specificity [18]. The ability of plants to take metals up and to accumulate them into the aboveground harvestable tissues is the rationale behind the second mechanism in phytoremediation, the so-called phytoextraction. Effective phytoextraction of metal-contaminated matrixes requires plants, which are characterized by a) efficient metal uptake and translocation to shoots; b) the ability to accumulate and tolerate high levels of metals; c) rapidly-growing and abundant shoots and deep root system. Some particular plants, commonly described as hyperaccumulator, show the ability to accumulate metals in aboveground tissues at very high concentration, without phytotoxic effects [27]. Unfortunately, most plant species displaying the hyperaccumulation trait are biennial or short-lived perennial herbs, shrubs, or small trees, characterized by a low biomass and a slow growth rate, which are major limitations for phytoextraction purposes [26]. Plants, which are suitable for effective

phytoremediation, are therefore selected considering their tolerance to metal stress and biomass of aerial organs. For instance, it has been shown that *Populus* spp. and *Salix* spp. are able to accumulate relatively high foliar concentrations of metals such as Cd and Zn [28] and are often associated with metal-contaminated lands in northern Europe [29]. Interestingly, the evaluation on a time span of ca. 30 years of natural colonization on a contaminated site by different plant species, i.e., *Populus* 'Robusta', *Quercus robur*, *Fraxinus excelsior*, and *Acer pseudoplatanus*, has shown that the tree species determined a redistribution of metals in the soil profile, which was dependent on two main processes: the accumulation of metals in the leaves (an enhanced metal deposit into leaves contributes to an increased metal amount in the upper soil layer, upon seasonal leaf fall) and species-specific soil acidification (higher soil acidification by the root metabolism resulted in higher metal leaching from the upper soil layer with subsequent lower metal concentrations in such soil layer) [28]. Other than such biological aspects, successful phytoextraction is also guaranteed by lowering the time constraint of the process itself, especially evaluating the rate of metal pollutant removal and any eventual pollutant inputs [30].

**Figure 1.** The main aspects of phytoremediation: the main steps involved in phytoremediation of heavy metals, which include (**a**) metal (yellow dots) adsorption on soil particles or cell walls (induced by rhizosphere metabolism) and compartmentalization of metals into root cell vacuoles (blue circles inside cells), preventing transport to the shoot; (**b**) metal accumulation in aerial organs (e.g., in vacuoles or trichomes) upon root-to-shoot xylem transport; (**c**) for particular metalloids (e.g., Se and As), leaf metabolism allows volatilization of the toxic compound.

When land contamination includes particular contaminants, such as Hg, As, and Se, plant metabolism is applicable to root absorption, translocation, and conversion of toxins into volatile compounds, which are released into the environment. Such a phenomenon, considered as a phytoremediation strategy, is called phytovolatilization. For instance, being chemically analog to sulfur, inorganic Se is converted to dimethyl selenide by plant enzymes involved in sulfur metabolism

pathways, assimilation, and volatilization. Dimethyl selenide is dispersed into the air as a gas, which is significantly less toxic than inorganic Se [31]. Arsenic is another carcinogen and its contamination in soils is mainly due to natural sources and anthropogenic activities. Arsenite, formed in soils by the microbial activity, is readily taken up by plants, and some crops, e.g., rice, showed particular attitude to mobilize arsenite through the silicon uptake pathway, resulting in serious As poisoning to consumers [32]. The third metal that can be converted into volatile compounds is Hg, which is present in soils, waters and in the atmosphere. Leaf Hg content, mainly in the form of methyl-Hg, seems to derive almost entirely from leaf absorption by the atmosphere, since Hg transport through vascular tissues is very limited even considered that in particular paddy soils, chemical forms of water-soluble Hg can be promptly adsorbed and transferred to shoots, as observed in rice [33]. Experiments upon controlled conditions with wild-type *Brassica juncea* plants hydroponically treated with HgCl2 confirmed that upon root uptake, phytovolatilization of Hg is indeed happening in roots, rather than shoots due to the low root-to-shoot transport of the metal, and seems to occur via the metabolic activity of the root-associated algal and microbial community [34].

In soil, plant metabolism may contribute to the chemical stabilization of metal ions within the vadose zone, limiting leaching, mobility, bioavailability, and ultimately hazard. This process is known as phytostabilization and is accomplished by both metal ions absorption and accumulation in and onto roots, and by their precipitation in the rhizosphere zone due to binding by organic compounds and changes of metal oxidative state. Positively charged metal ions effectively bind to pectins in plant cell walls and to the negatively charged plasma membranes [35]. Plant species that accumulate heavy metals in their belowground parts are recognized as the most effective for phytostabilization, also known as rhizostabilization. Enhancement of phytostabilization processes is commonly obtained by coupling biological activity with soil amendment, in particular when dealing with heavily polluted soils. The utilization of inorganic soil additives, which include phosphate fertilizers, manganese, and iron oxides, clay and other minerals, and organic compounds, such as coal, compost and manure, aids plants by metal sorption and/or chemical alteration, as well as by beneficial effects on plant growth [36,37]. By reducing contaminants mobility and eventually the associated risks without necessarily removing them from the site, phytostabilization does not produce contaminated waste, such as harvested materials, which would need further treatments.

The root metabolism of both terrestrial and aquatic plants can be also exploited to remediate polluted waters. This approach, named rhizofiltration, is used to absorb, concentrate, and precipitate metals from polluted water into plant biomass and its efficiency compares with currently employed water treatment technologies [38,39]. Early works demonstrated that a variety of aquatic plants, microorganisms, and seaweeds were able to biosorb metals and radionuclides dispersed in water (for a detailed description, refer to Section 3.2.2 in this review), but the lack of low-cost culturing, harvesting, and handling methods prevented full-scale testing [40]. It was in the early 1990s that the use of terrestrial plants grown hydroponically, able to achieve high above-water biomass and extensive root system to adsorb and absorb metals from contaminated liquids, got a foothold [41]. Mechanisms involved in rhizofiltration, also known as phytofiltration, mainly fall into three types characterized by different kinetics: a) sorption on the root surface, a quick component of metal removal, due to physical and chemical processes as chelation, ion exchange and specific adsorption (which do not include biological activity); b) processes that depend on plant metabolism, responsible for a slower metal removal from solutions and which rely on intracellular uptake, vacuolar deposition and eventually translocation to the shoots [42]; lastly, c) the slowest component of metal removal involves the release of root exudates which mediate metal precipitation from the solution in the form of insoluble compounds, as in case of phytostabilization.

An interesting corollary of phytofiltration is the use of microalgae to treat municipal, industrial, agro-industrial, and livestock wastewaters. Microalgal bioremediation has been effective in the removal of toxic minerals such as, Br, Cd, Hg, and Pb, from effluents of food-processing plants and different agricultural wastes [43]. Moreover, algal biomass has found application for the passive biosorption

of heavy metals in wastewater [43]. Recently, microalgae have drawn researchers' attention due to their abilities in CO2 mitigation with environmentally beneficial outcomes, considering that CO2 is the largest contributor to the greenhouse effect [44]. Other than the above-mentioned features of microalgae, the main product of algal culture, i.e., the biomass, can be valorized for the production of biofuels, including biodiesel, biomethane, and biohydrogen [45], which is noteworthy in the context of the circular economy.

### *3.2. Advancement in the Field of Phytoremediation*

### 3.2.1. Choosing the Best Plant Genotype

Enhancement of phytoremediation efficiency by increasing plant biomass, metal uptake or tolerance to metal toxicity is an important step in the development of new phytoremediation programs. The efficiency of phytoremediation can be improved through traditional approaches (such as plant breeding or hybridization and selection) or biotechnological techniques (i.e., the creation of engineered plants) that contribute to the development of plants with suitable phenotypes (Figure 2a).

**Figure 2.** Main aspects of human intervention to enhance phytoremediation. Steps in which human activity can operate are, (**a**) the selection, through varietal choice, classical breeding (e.g., somatic hybridization), or transgenic approach, of the most useful plant species to be applied; (**b**) the enhancement of rhizosphere interconnections between plant growth promoting rhizo- and endophytic bacteria (PGPR and PGPE, respectively, blue dots), and mycorrhizal fungi (drawn in red -arbuscular mychorrizae, AM - and orange), exploiting ex-novo inoculum or the native microflora; (**c**) the management of the polluted site, in terms of both soil conditions and growth techniques, which can change metal availability, plant growth, and remediation effectiveness.

Considering traditional approaches, improvement of plant phytoremediation efficiency was realized by the selection of wild non-edible ecotypes naturally growing in contaminated sites. Native plant species and populations, growing in metalliferous or contaminated sites, are able to cope with the high metal levels present in these soils; for this reason, they are much more resistant to these conditions than other plants and can be used for reclamation purposes [46]. Singh et al. [47] analyzed

native plants growing on a site near the Uranium mine tailing ponds in Jaduguda and Turamdih, in the Jharkand State (eastern India), contaminated with heavy metals (Al, V, Ni, Cu, Zn, Fe, Co, Se, Mn) and radionuclides. Among the plants able to accumulate toxic metals and remediate the contaminated site, the As hyperaccumulator *Pteris vittata* was identified as the most versatile as it could accumulate Al, V, Ni, Co, Se, and U. Barrutia et al. [48] identified and characterized native plants spontaneously growing on soils from an abandoned Pb-Zn mine containing toxic levels of Cd, Pb, and Zn in the Basque Country (northern Spain). Among these, 31 species were able to accumulate and tolerate metals, including *Festuca rubra*, *Noccaea caerulescens*, *Jasione montana*, *Rumex acetosa*, and *Plantago lanceolata*. Shoots of *N. caerulescens* accumulated the highest Zn concentrations. Moreover, in vitro and greenhouse selections are suitable for the obtainment of heavy metal-tolerant plants, useful for soil remediation. *Daphne jasminea* and *Daphne tangutica* shoots were cultivated in vitro in the presence of different concentrations of Pb(NO3)2. In these conditions, *D. tangutica* accumulated high Pb concentrations, and chlorophyll and carotenoid biosynthesis were higher in comparison to *D. jasminea* [49].

To enhance the phytoremediation efficiency, genetic determinants of heavy metal accumulation and tolerance associated with wild hyperaccumulator species can be introduced by introgression into the genome of plants with significantly higher biomass [50]. For instance, *Brassica juncea* protoplasts were fused with *N. caerulescens* protoplasts to transfer the metal-resistant ability of *N. caerulescens* into *B. juncea* by somatic hybridizations. Hybrid plants showed the high Zn and Ni accumulation potential and tolerance derived from *N. caerulescens*, and the high biomass production specific of *B. juncea* [51].

Despite positive results obtained using classical breeding and genetic approaches, molecular engineering may be helpful to enhance plant phytoremediation potential and efficiency for the reclamation of polluted sites. Recombinant DNA technologies currently used for both nuclear and cytoplasmic genome transformation and the availability of genome sequences for different plant species allow the transfer of desirable determinants from hyperaccumulator species to sexually incompatible and high-biomass crops, suitable for in field phytoremediation. For instance, genetic engineering can be exploited to enhance metal tolerance and accumulation by the introduction of genes responsible for metal uptake, transport, accumulation, and detoxification, and for the response to oxidative stress, or to increase biomass production of hyperaccumulator plants [26]. Considering the reclamation of metal contaminated sites, good prospects come from the genetic engineering of high biomass species and trees, such as poplar. Eastern cottonwood trees (*Populus deltoides* Bartr. ex Marsh.) were genetically engineered by the introduction of the bacterial *merA* (mercuric ion reductase that reduces Hg2<sup>+</sup> to the less toxic Hg0, which is volatized by plants) and *merB* (organomercury lyase that converts organic Hg to Hg2+) genes isolated and modified from *Escherichia coli* for the reclamation of mercury-contaminated sites. In vitro, *merA*/*merB* plants were more resistant to phenylmercuric acetate than wild-type controls and could detoxify organic Hg more efficiently [52]. Furthermore, genes that are currently widely used to improve plant phytoremediation potential are those that encode transporters of metal ions [26]. For example, Shim et al. [53] produced genetically engineered Bonghwa poplar (*Populus alba* x *P. tremula* var. *glandulosa*) lines expressing the yeast *ScYCF1* gene (*Saccharomyces cerevisiae -* yeast cadmium factor 1), which encodes a vacuolar transporter involved in toxic metal sequestration into the vacuole. When grown on a heavy metal contaminated soil from a mining site in South Korea, *ScYCF1*-expressing plants showed reduced Cd toxicity symptoms and accumulated much more Cd in comparison to wild plants. When plants were tested in the field on contaminated soil, dry weight and accumulation of Cd, Zn, and Pb in transgenic roots were significantly higher than in wild-types, demonstrating a potential utilization of these lines in long-term phytoextraction and phytostabilization of highly contaminated lands [53].

Among the genetic engineering tools, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is highly attractive to introduce a wide range of genes in different candidate organisms. CRISPR-Cas9 technology is a revolutionary and versatile gene-editing tool that can be used to enhance selected traits in plants targeting highly specific sequences of DNA [54]; for this reason, the technique could be used to transfer or modulate a desired set of genes in the plant genome to enhance the phytoremediation potential toward polluted soils and waters. The availability of genome sequences from model hypertolerant/hyperaccumulator species that may be considered for phytoremediation (such as the Cd, Ni, and Zn hyperaccumulator *N. caerulescens*, the Cd and Zn hyperaccumulator *Arabidopsis halleri* or the As hyperaccumulator *P. vittata*) and the improvement of bioinformatic tools have opened new opportunities for the use of CRISPR-Cas9 genome editing in the improvement of plant phytoremediation potential [55]. CRISPR-Cas9 technology could be used to introduce or modulate the expression of genes coding for metal transport proteins or involved in the synthesis of metal ligands [55]. Nowadays, despite the potential of this technique, its application in genome editing is still at an early stage in this field. However, there are few reports to date in which the CRISPR/Cas9 system has been successfully used for the reduction of metal content in plants. New rice lines knockout for the metal transporter gene *OsNRAMP5* were generated using the CRISPR/Cas9 system. These plants showed low Cd accumulation in shoots, roots, and in grains, upon hydroponic culture and in Cd-contaminated paddy field trials, maintaining biomass similar to wild-type [56]. This work provides a good example of the potential of the CRISPR/Cas9 system for the development of plants with a modified heavy metal content that could be used to achieve sustainable environmental cleanup via phytoremediation. In addition, phytoremediation could also benefit from the plant association with plant growth-promoting rhizobacteria [PGPR] (see following Section 3.2.3), where the CRISPR technology could be used to create more competent bacterial strains [55]. The application of the CRISPR-Cas9 technology to plants and PGPR, therefore, could be used to increase biomass yield and/or heavy metal tolerance, accumulation and detoxification, and thus to enhance their potential for the application in phytoremediation programs.

### 3.2.2. Changing the Growth Conditions

In addition to the plant genotype used, cultural strategies have an enormous impact on phytoremediation efficiency (Figure 2c). Several phytotypologies, i.e., planting strategies, can be suitable to treat different pollutants taking into account the characteristics of the polluted matrixes [57]. Among the systems for the decontamination of liquid polluted matrixes, such as sewages, landfill leachates or storm water, constructed wetlands have been extensively applied. This strategy relies on floating and/or rooted hydrophytes and the associated microbiota to detain and remove pollutants mainly by rhizofiltration, although other remediation processes such as phytostabilization and extraction also occur. Free-floating macrophytes (e.g., *Lemna* spp., *Eichhornia crassipes,* and others) have been demonstrated to be excellent metal accumulators [58]; however, the rapid growth and invasiveness of many of these species are a double-edged sword and raise controversies regarding their application for phytoremediation [59]. For this reason, rooted plant species have found a wider application. Rooted hydrophytes (e.g., *Phragmites* spp., *Thypha* spp., *Cyperus alternifolius*, and others) and flood-tolerant species (e.g., *Chrysopogon zizanioides*) have been employed with excellent results for the construction of surface- and subsurface-flow wetlands (reviewed by [60,61]), as well as floating bed systems [62,63]. In constructed wetlands, the composition of growth beds plays a pivotal role. Almost complete removal of metal pollutants has been achieved by using porous materials such as crushed sea shell grits [64], stratified pomice and loamy soil [65], and zeolite [66], whereas composted green waste and gravel limit the remediation performance [64,66]. However, it is important to notice that this fundamental role of growth bed materials is imputable to their different absorption capacities. Indeed, evidence show that, in constructed wetlands, metal pollutants are partially retained by the medium depending on its properties, opening the question of growth bed disposal after the treatment [60].

Another strategy proposed for liquid waste treatment and disposal is that of land treatment, i.e., the application of polluted wastewater as irrigation for plant cultures. Although this system has been tested on different scales with some success [67,68], serious issues remain regarding its influence on soil characteristics, leaching in groundwater and overall impact on the environment [69,70]. As for polluted soils, the application of plant covers has been widely used for inorganic contaminants and

relies on a variety of phytoremediation mechanisms, including phytoextraction and phytostabilization (refer to Section 3.1). Indeed, the plant covers effectively prevent pollutant dispersion and leaching in the groundwater, in addition to playing a more active role by removing it through uptake and detoxification [71]. Since metal-polluted soils often offer prohibitive growth conditions due to low nutrient content in addition to high metal levels, phytoremediation can be aided by good soil management practices to enhance global soil quality. In particular, the application of organic amendments, as for example manure or waste compost, has a positive effect on plant growth in phytoremediation [71,72]. Moreover, amendments can alter metal speciation, solubility, and bioavailability by altering water holding capacity, pH, and redox status of the soil [71], influencing the predominant phytoremediation strategy and the efficiency of the system. For example, cow manure, sewage sludge, and forest litter have been reported to enhance As extractability in As-polluted soils, favoring phytoextraction by ryegrass [73]. On the contrary, the application of sewage sludge and municipal waste compost reduced Cu, Zn, and Pb mobility in acidic metal-contaminated soil, leading to a more phytostabilization-oriented strategy [74].

Another widely considered practice to alter metal availability in polluted soils is the chelate-induced phytoextraction. The synthetic chelating agent ethylenediaminetetraacetic acid (EDTA) has proven highly efficient in metal mobilization [75], but its poor biodegradability causes concern due to its persistence in the environment, possible metal leaching and negative effects on soil properties and microbial communities [76]. As an alternative, biodegradable chelating agents as ethylenediaminedisuccinic acid (EDDS) and nitrilotriacetic acid (NTA), as well as organic acids, have been employed in several studies, enhancing phytoextraction efficiency [77–79].

Another supplementation that has been considered is that of exogenous phytohormones, with the aim of enhancing plant biomass, fitness and stress tolerance, thus improving phytoremediation efficiency. Cytokinins were demonstrated to produce generally positive effects in terms of plant growth and phytoextraction capacity [80], whereas conflicting results were achieved using auxins [81,82]. The application of gibberellins or some commercial growth regulator mixtures reduced metal accumulation in plant tissues, but the increased plant biomass brought still to a general enhancement of metal extraction per plant [83]. Moreover, the combined application of phytohormones with other treatments, such as nitrogen fertilization and chelating agents, produced a synergistic action resulting in a significant increase in metal phytoextraction [83].

In recent years, a novel strategy has been proposed to overcome some of the flaws of traditional phytoremediation, namely the long time and limited treatment depths. This technique, named electro kinetic-enhanced phytoremediation, relies on the combined application of plants and a physicochemical treatment, i.e., low-intensity electric fields, to the metal-polluted soil, favoring metal mobilization and bioavailability [84]. The application of different electrode materials and distribution, and different types of electric field have been considered with variable results [84]; however, a significant increase in Pb, As, and Cs phytoextraction has been achieved by DC electric field applied with inert low-cost graphite electrodes, due to the alteration of soil pH and metal solubility resulting from the electric field [85]. Interestingly, the application of a solar cell-powered electric field has been tested in a real-scale field trial for the phytoremediation of a metal polluted electronic waste recycling center by *Eucalyptus globulus*: the application of the electric field resulted in increased plant growth and metal accumulation. Moreover, although traditional power supply systems are more efficient in metal mobilization and containment, solar cells make this strategy significantly more sustainable on the economical level [86].

Finally, *nanoparticles* have also been considered for their possible use in assisting phytoremediation. Different types of nanomaterials have been applied for the decontamination of metal-polluted substrates thanks to their absorption capacity or redox catalytic activity [87]. In combination with plants, nanoparticles can be employed to improve the effectiveness of phytostabilization by absorbing metal ions [88], or phytoextraction by improving plant fitness and stress tolerance and increasing metal bioavailability [89,90].

### 3.2.3. Enhancing the Plant-Microorganism Interactions

In recent years, researchers have focused their attention on the interactions between plant and metal resistant soil microorganisms, in particular, those colonizing roots (i.e., the rhizobiome) [91]. The synergism between plant roots and microorganisms can implement the remediation process by enhancing phytostabilization, as in the case of arbuscular mycorrhizae (AM) fungi, and phytoextraction employing plant growth promoting rhizo- and endo-bacteria [91] (Figure 2b). AM fungi establish mutual symbiosis with higher plants, improving mineral nutrition. Thus, AM contributes to plant growth in heavy metal contaminated sites by increasing plant access to nutrients such as P, by improving soil texture through the stable aggregation of soil particles and by binding heavy metals into roots restricting their translocation to shoot tissues. In that respect, AM fungi have been reported to reduce metal uptake and distribution in sunflower plants [92,93]. Therefore, AM fungi promote phytostabilization of heavy metals, accelerating the revegetation of severely degraded lands, such as coalmines or waste sites [94].

On the other hand, plant growth promoting rhizobacteria and endophytes (PGPR and PGPE, respectively) are able to increase the phytoremediation competence of plants by promoting their growth and health even under hazardous levels of heavy metals, by means of traits, such as organic acid production, secretion of siderophores, indole-3-acetic acid (IAA) production and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. In most metalliferous soils, metals are strongly bound to soil particles, being not promptly available for plant uptake. Various PGPR and PGPE (e.g., *Arthrobacter*, *Microbacterium*, *Bacillus*, *Kocuria* and *Pseudomonas* spp.) can solubilize water-insoluble Zn, Ni, and Cu by local soil acidification through the secretion of protons and/or organic anions (e.g., acetate, lactate, oxalate, tartrate, succinate, citrate, gluconate, ketogluconate, and glycolate) [95]. Moreover, metal bioavailability in soils can be further increased by inoculating PGPR able to secrete biosurfactants, which can aid in metal ion release from soil particles [96]. Under iron-limiting conditions, PGPR secrete low molecular weight siderophores, which are iron chelators with an exceptionally strong affinity for ferric iron (Fe3+), enhancing its availability to both microorganisms and, indirectly, plants [97]. Siderophores are able to chelate several other metal species, such as Mg, Mn, Cr(III), Cd, Zn, Cu, Ni, As, and Pb with variable affinity. For instance, *B. juncea* plants inoculated with the mutant SD1 of the phosphate-solubilizing *Enterobacter* sp. NBRI K28, characterized by an enhanced siderophore production, showed increased biomass and phytoextraction of Ni, Zn, and Cr [98]. In addition to altering metal availability, a great majority of root-associated PGPR also produces the main bacterial auxin IAA, which promotes plant growth, stimulating root cell proliferation, lateral root initiation and overproduction of root hairs. Generally, bacterial IAA facilitates the adaptation of host plants in metal-contaminated sites by triggering physiological changes in plant cell metabolism under metal stress and helping plants to withstand high concentrations of heavy metals [99]. Several PGPR and PGPE are also able to synthesize the enzyme ACC deaminase, which degrades ACC (an immediate precursor of plant ethylene) into 2-oxobutanoate and ammonia, hence inhibiting ethylene production in plants, which is usually induced by heavy metal stress. It has been demonstrated that inoculation with ACC deaminase-producing PGPR resulted in extensive root proliferation in hyperaccumulator plants and efficient phytoremediation in metal-polluted soils [100]. From a technological point of view, microorganisms, which can be exploited for soil remediation or phytoextraction technologies, are usually members of complex metal-tolerant populations associated with tolerant and/or hyperaccumulator plant species growing in metalliferous soils. In some cases, PGPR and PGPE originally isolated from hyperaccumulator plants have been shown to promote growth and phytoextraction of diverse plant species grown in single and multiple metal-contaminated soils [101,102]. However, the impact of PGPR and AM fungi on different plants varies depending on the plant and microbial species and soil types. Several authors have tested PGPR and PGPE as bio-inoculum to remove different heavy metals from soils [103–105]. Zn-mobilizing bacteria, isolated from serpentine soils, promoted Zn, Cu, and Ni accumulation in *Ricinus communis* [106], while *Rahnella* sp. JN6, isolated from *Polygonum pubescens,* can promote growth and Cd, Pb, and Zn uptake in

*B. napus* [107]. A bacterial consortium, isolated from the rhizosphere of the pseudometallophyte *Betula celtiberica* growing in an As-polluted site, enhanced As accumulation in leaves and roots, whereas the rhizobacterium *Ensifer adhaerens* strain 91R mainly promoted plant growth upon laboratory conditions [108]. Moreover, field experimentation showed that additional factors, such as soil As content and pH, influenced As uptake in the plant, attesting the relevance of field conditions in the success of phytoextraction strategies [108]. As for a phytostabilization-oriented strategy, AM fungi associated to the metallophyte non-accumulator *Viola calaminaria* inhabiting Zn- and Pb-rich soils were shown to improve maize growth in a polluted soil reducing heavy metal concentrations in plant tissues [109,110].

Nevertheless, it must be considered that the details of the interaction between plant roots and root-associated microorganisms are still rather unknown. Moreover, the rhizosphere is an extremely complex and still poorly characterized community: roughly, 99% of soil microbial taxa are yet to be cultured and can only be investigated using culture-independent methods [111]. At this purpose, approaches with *Omics* technologies, based on DNA, RNA, and protein sequencing have advanced our understanding of plant and microbial responses to pollutants and of plant–microbe interactions. For instance, high-throughput sequencing of bacterial 16S rDNA allows defining the composition of the microbial community and how heavy metals drive the selection toward microorganisms, which are more suitable for phytoremediation purposes [92]. In addition, transcriptomic and proteomic studies on rhizosphere communities in contaminated soils are instrumental to predict valuable microbial functions directly [112,113]. The integration of these strategies allows creating a complete picture of how cohabiting and symbiotic biological communities interact to adapt to metal stress and could enhance phytoremediation [114]. Eventually, these data need to be combined with high-throughput isolation and screening for key microbial characteristics such as growth rate, to target microbes that are perhaps not naturally dominant but have valuable traits for their application in phytoremediation [114].

Despite all the research in the field of plant-microorganism interaction, applications of PGPR and mycorrhizal consortia in assisted phytoremediation in contaminated soils are still scarce and the performance of these microorganisms under natural conditions needs to be more deeply investigated. A particular consideration is the biosafety linked to the release of non-autochthonous bacterial strains. In addition, even though such strains might be superior in terms of metal resistance and mobilization effectiveness, the competition with the native microbial population can reduce the efficacy of the inoculated strains. Despite these concerns, co-inoculation with PGPR and mycorrhizal consortia might partially mimic the natural conditions of contaminated soils, in which multiple microorganism interactions occur, helping plants to cope with the toxic effects of heavy metals. Co-inoculation can also improve the phytostabilization or phytoextraction efficiency for various metals at the same time, which indicates the possibility of exporting the technology to multi-metal contaminated sites [67,105].

### **4. Conclusions**

Summarizing, plants and associated microorganisms surely are of great interest for their potential application in polluted soil reclamation. A variety of options are available when considering a phytoremediation approach, including the utilization of wild plant-microorganisms associations, or the implementation by applying particular planting and culturing techniques. Researchers can develop the best suitable plant lines or microorganisms to be exploited, as well as the best fertilizers or soil conditioners. Interestingly, attention moved also on the fate of contaminated biomass, particularly when dealing with approaches of phytoextraction. Indeed, recent research is aimed to valorize metal-rich biomass rather than simply dispose of it, coupling land reclamation with non-food products (e.g., timber) and energy production, a concept known as integrated phytoremediation. In this view, harvested plant biomass has a substantial calorific value in terms of renewable energy production. Therefore, long-term operations of planting, maintaining the phytoremediation site (otherwise unsuitable for remunerative and productive uses) and fruitfully converting harvested

biomass, are grouped into the new idea of phyto-management, in which the major goal is mitigating environmental risk and making contaminated lands economically valuable [26,30].

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

### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **OsMSR3, a Small Heat Shock Protein, Confers Enhanced Tolerance to Copper Stress in** *Arabidopsis thaliana*

**Yanchun Cui 1,\*, Manling Wang 1, Xuming Yin 1, Guoyun Xu 2, Shufeng Song 3, Mingjuan Li 1, Kai Liu <sup>1</sup> and Xinjie Xia <sup>1</sup>**


Received: 21 October 2019; Accepted: 30 November 2019; Published: 3 December 2019

**Abstract:** Copper is a mineral element essential for the normal growth and development of plants; however, excessive levels can severely affect plant growth and development. *Oryza sativa* L. multiple stress-responsive gene 3 *(OsMSR3)* is a small, low-molecular-weight heat shock protein (HSP) gene. A previous study has shown that *OsMSR3* expression improves the tolerance of *Arabidopsis*to cadmium stress. However, the role of *OsMSR3* in the Cu stress response of plants remains unclear, and, thus, this study aimed to elucidate this phenomenon in *Arabidopsis thaliana*, to further understand the role of small HSPs (sHSPs) in heavy metal resistance in plants. Under Cu stress, transgenic *A. thaliana* expressing *OsMSR3* showed higher tolerance to Cu, longer roots, higher survival rates, biomass, and relative water content, and accumulated more Cu, abscisic acid (ABA), hydrogen peroxide, chlorophyll, carotenoid, superoxide dismutase, and peroxidase than wild-type plants did. Moreover, *OsMSR3* expression in *A. thaliana* increased the expression of antioxidant-related and ABA-responsive genes. Collectively, our findings suggest that *OsMSR3* played an important role in regulating Cu tolerance in plants and improved their tolerance to Cu stress through enhanced activation of antioxidative defense mechanisms and positive regulation of ABA-responsive gene expression.

**Keywords:** *Arabidopsis*; small heat shock protein; *OsMSR3*; copper stress; reactive oxygen species

### **1. Introduction**

Copper is an essential mineral element for the normal growth and development of plants. In plants, Cu functions as an important cofactor for metalloproteins and participates in numerous biological processes, including photosynthesis, respiration, oxygen superoxide scavenging, cell wall metabolism and lignification, and ethylene perception [1–6]. Cu-deficient soils not only affect the quality and quantity of plant food crops but also reduce their nutritional value as the main source of essential minerals for humans [7]. In addition, exposure of plants to excess Cu interferes with normal growth, proliferation, and differentiation of most plant cells [8–14]. One of the earliest and most obvious symptoms of Cu stress is inhibition of primary root elongation [15–17], while its prominent manifestations are decreased proliferation of root meristem cells [18], impaired cell integrity [19], and cell death [20]. Excessive accumulation of Cu in plants leads to the production of reactive oxygen species (ROS), which are toxic owing to their high redox activity [21].

Plants have developed specific mechanisms to prevent Cu toxicity by tightly regulating Cu homeostasis, including Cu uptake, translocation, efflux, and sequestration [22]. Plants also activate antioxidant defense responses to mitigate oxidative damage caused by free Cu ions in the cytosol [23]. The defense system includes ROS-removing enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as low-molecular-weight antioxidants, such as ascorbic acid (ASC) and glutathione (GSH) [24]. These antioxidant compounds and enzymes can be used as physiological indicators for evaluating plant antioxidant defense ability [15–26].

The stress hormone abscisic acid (ABA) plays an important role in plant stress tolerance. Heavy metals such as Cd, Hg, and Cu can induce the expression of ABA synthesis genes, which in turn increase the endogenous level of ABA [27,28]. A previous study showed that cadmium treatment increases endogenous ABA levels in cattail and reed roots [29], potato tubers [30], and rice plants [31]. Exposure to high Cu concentrations also increased ABA levels in rice [31]. During the germination of wheat seeds, ABA levels increased in the presence of Hg, Cd, and Cu stress [32]. Plants exposed to heavy metal stress showed an increased concentration of ABA, which indicates that the hormone is involved in the protective mechanism against heavy metal toxicity [27,33,34].

In plants, small heat shock proteins (sHSPs) with monomer sizes ranging from 12 to 42 kDa are more diverse and abundant than those in other organisms. There are 23 sHSPs in rice, and these are proposed to be categorized into fourteen classes [35]. Classes CI-CXI (nine subfamilies) are localized in the nucleus or cytoplasm, whereas the other five are positioned in the endoplasmic reticulum, mitochondria, plastid, and peroxisome [25–36]. These sHSPs are stimulated in response to a wide range of abiotic stresses. For instance, *Oshsp26*, which encodes a chloroplast-localized sHSP, has been shown to enhance tolerance against oxidative and heat stress in tall fescue [37]. Overexpression of sHSP17.7 enhances drought tolerance in transgenic rice [38]. Overexpression of *OsHsp18.0-CI*, an sHSP-CI family gene, enhances resistance to bacterial leaf streak in rice [39]. However, there are only a few studies on the sHSPs involved in heavy metal resistance.

Our previous studies have shown that OsMSR3 belongs to the class I sHSP family [40]. The expression of the *OsMSR3* gene significantly enhances tolerance of *Arabidopsis thaliana* (L.) Heynh (*A. thaliana*) to cadmium stress [41]. However, the molecular mechanism of *OsMSR3*-induced Cu tolerance is poorly understood. In this study, we determined that the expression of *OsMSR3* was upregulated by Cu stress. Therefore, we speculated that *OsMSR3* plays an important role in plant tolerance to Cu. Expression of *OsMSR3* enhanced the Cu stress tolerance of *A. thaliana*. Our research enhances the understanding of the role of sHSPs in heavy metal resistance in plants.

### **2. Results**

### *2.1. Expression of OsMSR3 is Induced by Cu Stress*

The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) showed that the expression of *OsMSR3* increased rapidly after 6 h of Cu stress and peaked at 12 h in Pei'ai 64S rice seedlings (Figure 1). Subsequently, *OsMSR3* expression decreased by nearly 3.5-fold compared to the control levels at 48 h (Figure 1).

**Figure 1.** Expression analysis of the *Oryza sativa* L. multiple stress-responsive gene 3 (*OsMSR3*) gene. Expression analysis of *OsMSR3* in Pei'ai 64S rice seedlings at different time points (0, 1, 3, 6, 12, 24, and 48 h) under Cu stress using quantitative reverse transcription-polymerase reaction (qRT-PCR). The *ACTIN1* gene was used as an internal control. Error bars indicate standard deviations (SD) of three independent experiments.

### *2.2. Expression of OsMSR3 Enhances Cu Tolerance of Transgenic A. thaliana*

To assess whether upregulation of *OsMSR3* enhances tolerance to Cu, transgenic *Arabidopsis* plants expressing *OsMSR3* were generated and analyzed. Based on a previous study [41], two independent transgenic lines, L-5 and L-7, were chosen for further experiments. We examined survival rates and root lengths of both *OsMSR3*-expressing and wild-type plants treated with Cu (50 μM). As shown in Figure 2A, there was no significant difference in survival rate and root length between wild-type and transgenic seedlings in the absence of stress. However, in half-strength Murashige and Skoog ( <sup>1</sup> <sup>2</sup> MS) medium supplemented with Cu, transgenic plants showed higher Cu tolerance than wild-type plants did, with a higher survival rate and longer root length (Figures 2B and 3B). Furthermore, the fresh and dry weight of wild-type and transgenic plants measured under normal and Cu stress did not differ significantly in the absence of stress, but fresh and dry weight were higher in both transgenic lines than they were in wild-type plants under Cu stress (Figure 3C,D). After the application of Cu stress, the relative water content (RWC) of transgenic plants was >35%. As shown in Figure 3E, under control conditions, the RWC was almost the same for all tested lines. In the presence of 50 μM copper chloride (CuCl2), all plants showed a reduction in RWC. However, water loss was higher in the wild-type than it was in the transgenic lines.

**Figure 2.** Performance of transgenic plants and wild-types under normal and Cu stress conditions. (**A**) Left panel, seedlings reared under normal conditions (0 μM CuCl2) for 30 days; right panel, seedlings exposed to 50 μM CuCl2 for 30 days. WT, wild-type *Arabidopsis thaliana*; L-5 and L-7, transgenic lines 5 and 7. (**B**) Survival rates of plants after growth under Cu stress for 30 days. Each column represents an average of three replicates, and bars indicate ± standard deviation (SD); and \*\* *p* < 0.01 indicate significant differences compared to wild-type T plants under the same conditions determined using Student's *t-*tests.

**Figure 3.** Improved tolerance to Cu stress induced by *Oryza sativa* L. multiple stress-responsive gene 3 (*OsMSR3*) expression. (**A**) Images of representative plants grown on half-strength Murashige and Skoog ( 1 <sup>2</sup> MS) medium with or without 50 μM copper chloride (CuCl2) for 21 days. (**B**) Effect of Cu treatment on root length of plants presented in panel A. (**C**) Fresh weight (FW) and (**D**) dry weight (DW) of wild-type and transgenic line plants treated with or without 50 μM CuCl2 for 21 days. (**E**) Relative water content (RWC) of wild-type and transgenic plants treated with or without 50 μM CuCl2 for 21 days. Values are means ± standard deviation (SD) of three independent biological replicates; \* *p* < 0.05 and \*\* *p* < 0.01 indicate significant differences from the wild-type determined using Student's *t*-test.

### *2.3. Expression of OsMSR3 in Arabidopsis Causes Higher Accumulation of Cu*

To determine whether the enhanced Cu tolerance of transgenic plants was associated with their lower Cu accumulation, Cu content was determined in the different lines at the end of Cu treatment. Cu accumulation was higher in the roots and shoots of transgenic plants than that of wild-type plants. As shown in Figure 4A,B, the Cu content of transgenic lines L-5 and L-7 was approximately 1.2 and 1.1 times higher in the roots, and 1.66 and 1.59 times higher in the shoots, respectively, than it was in the wild-type plants.

**Figure 4.** Quantitative analysis of various physiological indexes in wild-type and transgenic plants. (**A**,**B**) Cu content in wild-type and transgenic plant shoots and roots treated with 50 μM copper chloride (CuCl2) for 21 days, respectively. (**C**) abscisic acid (ABA), (**D**) malondialdehyde (MDA), and (**E**) hydrogen peroxide (H2O2) content in wild-type and transgenic plants treated with or without 50 μM CuCl2 for 24 h. (**F**,**G**) Chlorophyll and carotenoid content in wild-type and transgenic plants treated with or without 50 μM CuCl2 for 21 days. Values are means ± standard deviation (SD) of three independent biological replicates; \* *p* < 0.05 and \*\* *p* < 0.01 indicate significant differences from wild-type plants under the same conditions determined using Student's *t*-test.

### *2.4. E*ff*ects of OsMSR3 Expression on ABA, Malondialdehyde (MDA), and Hydrogen Peroxide (H2O2) Content in A. thaliana*

To determine whether OsMSR3 affects ABA content in *A. thaliana* under Cu stress, endogenous ABA content in transgenic and wild-type plants was measured. Under normal conditions, there was almost no difference in ABA content between the wild-type and transgenic lines, whereas Cu treatment increased the levels in both plant types (Figure 4C). Specifically, the mean ABA content, which was 4.12 ng g−<sup>1</sup> fresh weight (FW) in wild-type plants, increased to 5.18 and 5.21 ng g−<sup>1</sup> FW in the L-5 and L-7 lines, respectively (Figure 4C).

To examine the oxidative damage induced by excess Cu, we monitored the accumulation of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in wild-type and transgenic plants. Under normal conditions, differences in MDA levels were not apparent between wild-type and transgenic plants, but levels were significantly increased by Cu stress (Figure 4D). Wild-type plants had a higher MDA content than transgenic plants did (Figure 4D). As shown in Figure 4D, MDA levels were approximately 1.61 and 1.59 times higher in wild-type plants than they were in transgenic lines L-5 and L-7 plants, respectively.

Cu stress can lead to H2O2 generation, which can be used to examine the oxidative damage induced by excess Cu [42]. In our study, H2O2 levels were not significantly different between transgenic and wild-type plants under controlled conditions. However, under Cu stress, H2O2 levels were lower in both transgenic lines than in wild-type plants, but no significant difference was observed between the L-5 and L-7 lines (Figure 4E).

### *2.5. E*ff*ects of OsMSR3 Expression in A. thaliana on Chlorophyll and Carotenoid Content*

To determine whether the chlorophyll content is altered in transgenic plants under salt stress, we detected chlorophyll and carotenoid content in the leaves of wild-type and transgenic seedlings under normal conditions and Cu stress. As shown in Figure 4F,G, there was no significant difference in chlorophyll and carotenoid content between the transgenic and wild-type lines under normal growth conditions. In contrast, following Cu treatment, the chlorophyll content in the leaves of the *OsMSR3* transgenic lines (L-5 and L-7) was 1.59 and 1.57 times higher than that in the wild-type plants, although the content decreased in both transgenic and wild-type plants. The carotenoid content in L-5 and L-7 plants was 1.26 and 1.23 times higher than that in the wild-type.

### *2.6. Antioxidant Enzyme Activities are Altered in Transgenic A. thaliana*

To determine whether increased Cu tolerance in transgenic plants is related to changes in oxidase activity in vivo, SOD, POD, and CAT activities were measured in wild-type and transgenic plants grown in medium without (CK) or with 50 μM CuCl2. The data showed that under normal conditions, SOD and POD activities in the transgenic lines were slightly higher than those in wild-type plants (Figure 5A,B), whereas CAT activity was slightly lower in the transgenic lines than in the wild-type plants (Figure 5C). Under Cu stress, SOD and POD activities increased in both wild-type and transgenic lines. However, the SOD activity of L-5 and L-7 was 1.07 times higher (Figure 5A), and the POD activity was 1.12 and 1.14 times higher (Figure 5B) than that of wild-type plants, respectively. Cu stress decreases CAT activity in both wild-type and transgenic lines with a greater decrease in the transgenic lines than in the wild-type plants (Figure 5C). The POD activity of the two transgenic lines was only 0.87 and 0.88 times higher than that of the wild-type plants (Figure 5C).

**Figure 5.** *Cont*.

**Figure 5.** Antioxidant enzyme activity determination. Quantitative analysis of (**A**) superoxide dismutase (SOD), (**B**) peroxidase (POD), (**C**) and catalase (CAT) activity in wild-type and transgenic plants treated with or without 50 μM copper chloride (CuCl2) for 24 h. Values are means ± standard deviation (SD) of three independent experiments; \* *p* < 0.05 and \*\* *p* < 0.01 indicate significant differences from wild-type plants under the same conditions determined using the Student's *t*-test.

### *2.7. Expression of OsMSR3 Increases Expression of Antioxidant-Related and ABA-Responsive Genes*

To determine the performance of transgenic plants under Cu stress and elucidate the molecular mechanism underlying the resistance of transgenic plants to Cu stress, the transcript levels of antioxidant-related (*AtCSD1*, *AtCSD2*, and *AtPOD*) and ABA-responsive (*AtRD29A*, *AtABA1*, and *AtABI5)* genes were assayed in wild-type and transgenic plants under normal and stress conditions. The expression levels of *AtCSD1*, *AtCSD2*, and *AtPOD* were higher in wild-type plants than they were in transgenic plants under control conditions. However, higher gene expression was observed in transgenic plants than in wild-type plants under Cu stress conditions (Figure 6A–C). Compared to the expression under normal conditions, Cu stress inhibited the expression of these genes in wild-type plants but activated their expression in transgenic lines. The ABA-responsive genes, *AtRD29A*, *AtABA1*, and *AtABI5,* showed no significantly different expression levels between transgenic and wild-type plants under normal conditions (Figure 6D–F). Under Cu stress conditions, the expression levels of the three genes were higher in the transgenic lines than in the wild-type plants (Figure 6D–F).

**Figure 6.** Quantitative reverse transcription-polymerase reaction (qRT-PCR) gene analysis. qRT-PCR analysis of relative expression of (**A**) *AtCSD1*, (**B**) *AtRD29A*, (**C**) *AtCSD2*, (**D**) *AtABA1*, (**E**) *AtGSH*, (**F**) and *AtABI5*, in two-week-old transgenic and wild-type (WT) plants treated with (Cu) or without (Control) 50 μM copper chloride (CuCl2) for 24 h. Values are means ± standard deviation (SD) of three independent biological replicates; \* *p* < 0.05 and \*\* *p* < 0.01 indicate significant differences from wild-type plants under the same conditions, determined using the Student's *t*-test.

### **3. Discussion**

Heavy metal pollution in soils is an emerging worldwide threat owing to its adverse effects on environmental safety [43]. Currently, cadmium and lead pollution in soils and their harmful effects on humans are attracting the attention of researchers globally. Cu pollution has become an important problem in the soil environment; however, studies of this phenomenon are still in infancy [44]. With the development of modern molecular biology, transgenic technology has emerged as an effective method to discover new Cu-tolerant genes in plants and cultivate plants that are highly efficient at repairing damage due to Cu contamination. In the previous study, we found that the expression of *OsMSR3* in *Arabidopsis* significantly enhanced tolerance to cadmium stress [41]. As Cu and cadmium belong to the group of heavy metal elements, we evaluated if the transgenic lines expressing *OsMSR3* in *Arabidopsis* could enhance the ability of copper tolerance.

The expression of *OsMSR3* was enhanced by Cu stress (Figure 1), which indicated that *OsMSR3* is involved in the response to Cu. Then, two transgenic lines L-5 and L-7 were used to perform the Cu tolerance experiment. The results showed that the expression of *OsMSR3* enhanced the tolerance of *A. thaliana* to Cu stress than that of wild-type plants, manifested as higher survival rate (Figure 2), higher biomass (Figure 3), and longer root length (Figure 3B). Moreover, transgenic plants accumulated less MDA than wild-type plants under Cu stress (Figure 4D). It is well known that Cu damages cell membranes by inducing lipid peroxidation [45]. The MDA level is used to detect membrane lipid peroxidation and permeability [46]. These results suggest that the expression of *OsMSR3* alleviated Cu-induced damage to the cell membrane of *A. thaliana*. Cell membrane stability is a major factor contributing to the maintenance of water status in plants during water deficit [47,48]. Therefore, the RWC of transgenic lines under Cu stress was higher than that of wild-type plants (Figure 3E).

Although the transgenic plants accumulated more Cu in the root and shoot (Figure 4A,B), their growth was significantly better than that of the wild-type. The accumulation of Cu in plant roots may inhibit the development of fine roots and reduce the absorption of iron and other trace elements [49]. To some extent, OsMSR3 protein may reduce the inhibition of Cu transport from the root to the shoot. We suggest that OsMSR3 may be helpful in maintaining the homeostasis of Cu metal ions at the cell and plant levels. In addition, we found that chlorophyll and carotenoid content in the leaves of *OsMSR3* transgenic lines was higher than that in those of the wild-type plants under Cu stress (Figure 4F,G). Chlorophyll is an important part of the light-harvesting complex (LHCII). As an antenna for capturing light energy and transferring it to the reaction center, the chlorophyll content reflects the intensity of plant photosynthesis [50]. Carotenoids play an important role in plant growth and development. For example, they can act as a haptokine by transferring captured light to chlorophyll and can also act as a scavenger of free radicals in plant cells [51,52]. We speculated that the expression of *OsMSR3* reduced the damage to chlorophyll and carotenoids under Cu stress.

SOD activity and SOD-related gene expression in *OsMSR3* transgenic lines were significantly higher than those in wild-type plants (Figure 5A, Figure 6A,C). The presence of excess Cu causes the generation of ROS, such as superoxide radical (O2 <sup>−</sup>), H2O2, singlet oxygen (1O2), and hydroxyl radicals (OH) [53]. To scavenge ROS and alleviate their deleterious effects, plants stimulate ROS-scavenging systems such as CAT, SOD, and POD [24], to combat the oxidative injury induced by heavy metal exposure [54]. SOD is the first line of defense against ROS and catalyzes O2 <sup>−</sup> to produce H2O2 and O2 [55]. An increase in SOD activity in stressed plants is an important indicator of superoxide ion production and enhancement of oxidative tolerance [55]. Therefore, the enhanced Cu tolerance of transgenic lines is related to the expression of SOD-related genes and SOD activity in *A. thaliana* under Cu stress induced by *OsMSR3* expression.

Cu stress can lead to the generation of ROS, such as H2O2 [56]. In this study, we found that H2O2 accumulated in wild-type and transgenic seedlings significantly during Cu stress, albeit to a lower extent in the two *OsMSR3* transgenic lines than in the wild-type (Figure 4E). The primary H2O2-scavenging enzymes in plant cells are CAT and POD; the former degrades H2O2 into water and oxygen. No studies, to date, have confirmed that a change in CAT activity is necessary to eliminate

H2O2 in rice plants under Cu stress [55]. However, the current study revealed that Cu significantly increased POD activity in the transgenic lines but had little effect on CAT activity (Figure 5B,C). Moreover, *POD* gene expression was upregulated under Cu stress (Figure 6E). This is consistent with the increase in POD activity. Studies have shown that CAT has a high capacity but low affinity, whereas POD has a high affinity for H2O2 [57]. Thus, POD is the most effective H2O2-scavenging enzyme to reduce H2O2 content in plant cells under Cu stress.

Heavy metal exposure induces the expression of ABA synthesis-related genes in plants, which eventually leads to an increase in endogenous ABA levels [28]. In this study, under Cu stress conditions, ABA content and ABA-related gene expression levels in transgenic plants were significantly higher than those in wild-type plants (Figures 4C and 6B,D,F). Therefore, we also propose that the expression of *OsMSR3* leads to the upregulation of ABA-related genes and an increase in endogenous ABA level under Cu stress, which may partly explain the increased tolerance of transgenic plants to Cu stress.

### **4. Materials and Methods**

### *4.1. Plant Material and Growth Conditions*

The seeds of rice (*Oryza sativa* ssp. *indica*) cultivar Pei'ai 64S were surface-sterilized with 75% ethanol for 2 min, treated with 50% sodium hypochlorite for 20 min, and then washed with distilled water at least thrice. The sterilized seeds were germinated on half-strength Murashige and Skoog ( 1 <sup>2</sup> MS) medium and grown in a greenhouse under conditions of a light intensity of 600 μmol/m2/s 70% relative humidity, and 28 ◦C temperature with a 12-h light/dark photoperiod. For the Cu stress experiment, two-week-old seedlings were exposed to a nutrient solution containing 50 μM CuCl2 for 48 h. The leaves were harvested as a pool for each sample at 0, 1, 3, 6, 12, 24, 36, and 48 h after Cu treatment.

### *4.2. Cu Tolerance Assay*

We used 50 μM and 100 μM CuCl2 to do the pre-experiment and then selected the concentration of 50 μM CuCl2 as the most suitable. The seeds of T3 transgenic and wild-type *A. thaliana* (ecotype Columbia-0) were surface-sterilized and sown in Petri dishes containing <sup>1</sup> <sup>2</sup> MS media with or without 50 μM CuCl2. The seeds were incubated in the dark at 4 ◦C for two days to break the dormancy and then transferred to a growth chamber. After incubation for 30 days, the survival rate of *A. thaliana* was determined. For measurement of root growth under Cu treatment, three-day-old *A. thaliana* seedlings were transferred onto <sup>1</sup> <sup>2</sup> MS medium with or without 50 μM CuCl2, in vertically placed dishes. After incubation for 21 days, the root length (from the base of the root to the tip) and FW of six plants were measured.

Next, whole plants were rehydrated with distilled water at 4 ◦C for 12 h, blotted dry, and then the turgid weight (TW) was recorded. Rehydrated whole plants were oven-dried at 80 ◦C for 24 h, and the dry weight (DW) was recorded. RWC was calculated as follows: RWC (%) = (FW − DW)/(TW − DW) × 100. For the qRT-PCR analysis of selected genes, three-day-old *A. thaliana* seedlings were transferred onto <sup>1</sup> <sup>2</sup> MS medium with or without 50 μM CuCl2. After 21 days of treatment, plant materials were harvested, and qRT-PCR was performed. The detailed procedure is provided in the next section.

### *4.3. RNA Extraction and qRT-PCR Analysis*

Total RNAs were extracted with TRIzol reagent (Invitrogen, Burlington, ON, Canada), as described previously [58]. qPCR analysis was conducted using AceQ qPCR SYBR Green Master Mix (Vazyme Biotech, Nanjing, China), and the reactions were performed using an ABI7900HT (Applied Biosystems, Foster City, CA, USA) and run on the following schedule: 95 ◦C for 10 min, followed by 40 cycles at 95 ◦C for 15 s and 58 ◦C for 30 s. The internal controls were *ACTIN1* and β*-TUBULIN* for rice and *A. thaliana*, respectively. The data for relative expression were analyzed using the comparative Ct method [59]. The primer pairs used in the qPCR analysis are listed in Table S1.

### *4.4. Measurement of Cu Content*

Cu content was determined according to the method described by Li et al. [24]. Briefly, three-day-old *A. thaliana* seedlings were transferred to <sup>1</sup> <sup>2</sup> MS medium with or without 50 μM CuCl2. After 21 days of treatment, the roots and shoots were harvested and dried at 80 ◦C for two days. Dried plant tissues (50–100 mg roots; 100–200 mg shoots) were digested with 11 N HNO3 at 200 ◦C for 10 h. The digested samples were then diluted with 0.1 N HNO3 and analyzed using an atomic absorption spectrometer (Solaar M6; Thermo Fisher, Boston, MA, USA). The experiments were performed in triplicate.

### *4.5. Measurement of MDA Content*

Two-week-old transgenic and wild-type plants were cultivated on <sup>1</sup> <sup>2</sup> MS medium with or without 50 μM CuCl2 for 24 h. Then, 0.3 g of the seedlings was harvested and ground into a powder for the determination of MDA content, which was measured according to a previously standardized method [24].

### *4.6. Measurement of ABA Content*

Two-week-old transgenic and wild-type plants were cultivated on <sup>1</sup> <sup>2</sup> MS media with or without 50 μM CuCl2 for 24 h. Approximately 0.2 g of the leaf tissue was harvested, ground into a powder, and then suspended in 1.8 mL 100 mM sodium phosphate buffer (PBS, pH = 7.4) for ABA leaf content detection, using a previously published method [60].

### *4.7. Measurement of Chlorophylls and Carotenoids*

The chlorophyll and carotenoid content were determined according to a previous method [60]. Briefly, three-day-old *A. thaliana* seedlings were transferred onto <sup>1</sup> <sup>2</sup> MS medium with or without 50 μM CuCl2 in vertically placed dishes. After incubation for 21 days, chlorophyll and carotenoids were extracted from the rosette leaves of the wild-type and transgenic plants with 100% alcohol. An ultraviolet-visible (UV-vis) spectrometer (UV-2600; Shimadzu Co., Kyoto, Japan) was used to measure the absorption of the extracts. The total chlorophyll and carotenoid content were calculated according to a previously published method [61].

### *4.8. Quantitative Analysis of H2O2*

The H2O2 concentration was determined using a commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, two-week-old plants (wild-type and transgenic lines) cultivated in <sup>1</sup> <sup>2</sup> MS medium were treated for 24 h with or without 50 μM CuCl2. Then, 0.5 g of the seedlings was harvested, weighed, immediately ground, and then suspended in 5 mL 0.9% sodium chloride solution. The supernatant was collected after centrifugation for 10 min at 4 ◦C and 3000× *g*, and the H2O2 content was measured according to the protocol provided by the manufacturer of the kit.

### *4.9. Assay of Antioxidant Enzyme Activities*

To measure antioxidant enzyme activities, two-week-old plants (wild-type and transgenic lines) cultivated in <sup>1</sup> <sup>2</sup> MS medium were treated for 24 h with or without 50 μM CuCl2. Seedling samples (0.5 g) were frozen in liquid nitrogen, rapidly ground into powder, and then homogenized in 100 mM sodium phosphate buffer (pH 7.4) on ice. After centrifugation at 3000× *g* for 15 min at 4 ◦C, the supernatant samples were immediately used for the detection of antioxidant enzymes. The activities of SOD, POD, and CAT were measured using specific assay kits (A001-1, A084-3, and A007-1, respectively) from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer's instructions.

### *4.10. Statistical Analysis*

The experimental data were analyzed using the statistical package for the social sciences (SPSS) 17.0 statistical software (*SPSS* Inc., Chicago, IL, USA). At least three independent experiments were performed, and the average results are presented. Error bars represent standard deviation (SD, n > 3). Furthermore, \* *p* < 0.05 or \*\* *p* < 0.01 indicate statistically significant means.

### **5. Conclusions**

In conclusion, we showed the involvement of *OsMSR3* in Cu tolerance in *A. thaliana*. *OsMSR3*-expressing lines exhibited enhanced Cu stress tolerance, possibly through enhanced activation of antioxidative defense mechanisms and positive regulation of ABA-responsive gene expression. In view of the good performance of the transgenic lines, *OsMSR3* can be used to modify plants for remediation of Cu pollution in the soil. Therefore, this study provides an important insight into plant biology and mechanisms to overcome increasing heavy metal pollution in soils.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/23/ 6096/s1. Table S1. Primer sequences used for quantitative reverse transcription polymerase reaction (qRT-PCR).

**Author Contributions:** Y.C. designed and performed the experiments; Y.C. and X.X. analyzed the data and wrote the paper; M.W., X.Y., G.X., S.S., M.L., and K.L. contributed reagents/materials/analysis tools; X.X. supervised the work and revised the manuscript. All the authors agreed on the contents of the paper and declared no conflicting interests.

**Funding:** This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 31301253 and 31671671) and Youth Innovation Team Project of ISA, CAS (2017QNCXTD\_GTD).

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

### **Abbreviations**


### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Cadmium and Plant Development: An Agony from Seed to Seed**

### **Michiel Huybrechts, Ann Cuypers, Jana Deckers, Verena Iven, Stéphanie Vandionant, Marijke Jozefczak and Sophie Hendrix \***

Environmental Biology, Centre for Environmental Sciences, Hasselt University, B-3590 Diepenbeek, Belgium **\*** Correspondence: sophie.hendrix@uhasselt.be

Received: 5 July 2019; Accepted: 9 August 2019; Published: 15 August 2019

**Abstract:** Anthropogenic pollution of agricultural soils with cadmium (Cd) should receive adequate attention as Cd accumulation in crops endangers human health. When Cd is present in the soil, plants are exposed to it throughout their entire life cycle. As it is a non-essential element, no specific Cd uptake mechanisms are present. Therefore, Cd enters the plant through transporters for essential elements and consequently disturbs plant growth and development. In this review, we will focus on the effects of Cd on the most important events of a plant's life cycle covering seed germination, the vegetative phase and the reproduction phase. Within the vegetative phase, the disturbance of the cell cycle by Cd is highlighted with special emphasis on endoreduplication, DNA damage and its relation to cell death. Furthermore, we will discuss the cell wall as an important structure in retaining Cd and the ability of plants to actively modify the cell wall to increase Cd tolerance. As Cd is known to affect concentrations of reactive oxygen species (ROS) and phytohormones, special emphasis is put on the involvement of these compounds in plant developmental processes. Lastly, possible future research areas are put forward and a general conclusion is drawn, revealing that Cd is agonizing for all stages of plant development.

**Keywords:** cadmium; oxidative stress; cell cycle; cell wall; germination; reproduction; plant growth and development

### **1. Introduction**

Cadmium (Cd) pollution, as a consequence of both geological and anthropogenic activities, affects many regions worldwide [1]. Although Cd is non-essential and no specific Cd uptake mechanisms have been identified in plants, it is taken up in root cells through transporters for essential bivalent cations such as calcium (Ca), iron (Fe), manganese (Mn) and zinc (Zn) [2]. Depending on the plant species and growth conditions, plants can endure low Cd concentrations, but in general Cd disturbs photosynthesis, respiration and the uptake of water and nutrients. As a consequence, Cd pollution negatively affects plant growth and development, thereby significantly reducing crop yield [3].

This Cd-induced phytotoxicity is related to its ability to bind to thiol, histidyl and carboxyl groups of structural proteins and enzymes, thereby interfering with their function. Furthermore, Cd can also disturb protein function by replacing essential ions in their active sites due to its strong chemical similarity with other divalent cations. Despite its non-redox-active nature, Cd indirectly induces the production of reactive oxygen species (ROS), resulting in an oxidative challenge, which is defined as an imbalance between cellular pro- and antioxidants in favor of the former [4,5]. This Cd-induced ROS production is achieved through multiple mechanisms. Firstly, Cd is able to replace Fe in various proteins, thereby increasing free cellular Fe levels. As a redox-active metal, Fe can directly induce ROS production through Fenton and Haber-Weiss reactions [6]. Furthermore, Cd indirectly induces ROS production by depleting cellular levels of the non-enzymatic antioxidant glutathione (GSH), as a consequence of increased phytochelatin (PC) synthesis. The latter contributes to Cd chelation but reduces the amount of GSH available for antioxidative defense [7,8]. In addition, indirect Cd-induced ROS production can result from its ability to inhibit enzymes involved in antioxidative defense mechanisms [8]. Cadmium can also contribute to ROS production through its effects on ROS-producing enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and increases ROS production in plant organelles by interfering with metabolic processes such as photosynthesis, respiration and photorespiration [4].

When present in elevated concentrations, ROS can evoke damage to a multitude of cellular macromolecules including lipids, proteins and DNA. However, ROS are not only damaging agents, but are also key players in signal transduction during physiological processes as well as responses to biotic and abiotic stresses [9–11]. In order to enable ROS-induced signaling and prevent damage to cellular macromolecules, ROS levels should be tightly controlled. To this end, plants have developed an extensive antioxidative defense system consisting of both enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and several peroxidases and non-enzymatic antioxidants such as the water-soluble GSH and ascorbate (AsA) and the lipid-soluble carotenes and α-tocopherol [9].

Besides interfering with ROS homeostasis, Cd exposure also affects phytohormone signaling [12,13]. It was shown, for example, that Cd exposure induces a fast and transient increase in ethylene levels in *Arabidopsis thaliana* through increasing the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase 2 (*ACS2*) and *ACS6*, which are involved in the biosynthesis of the ethylene precursor ACC [14]. Furthermore, Cd was shown to enhance ethylene and jasmonic acid (JA) concentrations in *Pisum sativum* [15] and abscisic acid (ABA), salicylic acid (SA) and JA levels in *Oryza sativa* [16]. It also interfered with auxin (AUX) homeostasis in *A. thaliana* seedlings, where it significantly altered AUX concentrations and distribution in primary root tips and cotyledons. It decreased indole-3-acetic acid (IAA) content, enhanced IAA oxidase activity and affected transcript levels of putative AUX biosynthetic and catabolic genes [17]. Similarly, a negative effect of Cd exposure on AUX biosynthesis, transport and distribution was also reported in *O. sativa* [18]. Interestingly, extensive cross-talk exists between ROS and phytohormones in plant development and stress tolerance, as was recently reviewed by Xia and colleagues (2015) [19].

In order to improve plant growth on Cd-polluted soils, for example for phytoremediation purposes [20], it is of crucial importance to increase our knowledge on the mechanisms underlying the negative impact of Cd exposure on plant development. Therefore, the main aim of this work is to provide an overview of Cd-induced effects on plant growth and development, specifically addressing (1) seed dormancy and germination, (2) vegetative plant growth and (3) reproductive plant growth. Important underlying mechanisms are discussed, highlighting the involvement of ROS and phytohormones. Furthermore, perspectives for future research are proposed, focusing on the investigation of transgenerational effects of Cd exposure and the involvement of the plant microbiome in response to Cd stress.

### **2. Seed Germination**

Seed germination, accompanied with a release from seed dormancy, is one of the most important events in a plant's life cycle. It determines whether a plant is willing to take the risk of environmental exposure in order to reach reproductive maturity and produce seeds of its own. In the past, this trait has been exploited in cereals to obtain seeds with a low dormancy level, so that when the seeds were sown onto the field, they would germinate quickly and evenly. This led to the problem known as pre-harvest sprouting, during which seeds already germinate when they are still attached to the mother plant, resulting in a significant reduction of yield and quality of the seeds [21–23].

Abscisic acid and gibberellic acid (GA) are the major phytohormones regulating germination. They act antagonistically, with high levels of ABA causing the preservation of a dormant seed and high levels of GA initiating germination [24,25]. The ABA/GA ratio thereby acts as a central hub integrating environmental signals [26]. However, recent studies also indicate a role for other plant hormones. Auxin, previously thought not to have an important role in seed germination, acts alongside ABA in keeping dormancy high [27]. Furthermore, AUX and ABA are dependent on each other in this process, as AUX operates by keeping the expression of a major ABA signaling downstream regulator, *ABSCISIC ACID INSENSITIVE 3* (*ABI3*), high [28]. In addition, ABA represses the elongation of the embryonic axis through AUX-regulated signaling [29]. Another hormone, ethylene, works in the opposite way. Ethylene is able to stop the inhibitory effect of ABA on endosperm weakening, thereby facilitating seed germination [30,31]. Several other phytohormones including brassinosteroids, cytokinins (CK), JA, strigolactones and SA, have been shown to either stimulate or inhibit germination. However, their mutual interactions and importance in this process are still unclear [27].

Next to the hormonal regulation of seed germination, the event is also characterized by an increase in ROS levels, especially hydrogen peroxide (H2O2) [32]. Reactive oxygen species are assumed to function upstream of the hormonal interactions by stimulating GA biosynthesis and metabolism and inducing ABA catabolism [33,34]. However, Bahin and colleagues (2011) stated that ROS mainly function through GA signaling since H2O2 did not influence ABA metabolism and signaling in *Hordeum vulgare* seeds [35]. On the other hand, ABA was shown to prevent the accumulation of ROS within the embryonic axis of the seeds [33]. Currently, the interactions between ROS and ABA/GA are still under debate [36]. The role of ROS in seed germination is dual and is defined as the oxidative window of germination [37]. Too low concentrations of ROS within the seed will fail to induce seed germination, whereas excessive ROS levels will lead to irreversible seed damage. In dry seeds, ROS levels tend to be rather low [37]. This is accomplished by maintaining a high level of antioxidant capacity scavenging ROS molecules. Depletion of antioxidants leads to a ROS increase, which initiates germination under favorable conditions [38]. Once H2O2 is elevated, it has the capacity to selectively oxidize mRNAs and proteins [39]. In *A. thaliana* seeds, ROS-induced carboxylation of 12S cruciferins, the major storage proteins, occurs. Upon seed imbibition, these oxidized molecules disappear rapidly, indicating their role in early seedling establishment [40]. A similar mechanism of proteome oxidation was found in sunflower seeds [41]. Furthermore, upon exposure to methylviologen, a ROS-inducing agent, genes involved in Ca and redox signaling were differentially expressed [33]. Hou and colleagues (2019) found that three major events were important in releasing *Leymus chinensis* seeds from dormancy [42]. A decrease in proteins related to AsA and aldarate metabolism accompanied with an increase of thioredoxins (Trx) changed the antioxidant system and led to an increase in ROS. This was followed by the oxidation of stored mRNAs and proteins. Furthermore, an increase in β-tubulin led to cytoskeleton changes and resulted in physical dormancy release by transporting substances related to germination and cell wall loosening. Thirdly, these cytoskeleton changes in turn affected chromatin remodeling and proteins [42].

Surrounded by a rigid seed coat, the plant embryo is well protected from environmental stresses [43,44]. Germination is initiated with the uptake of water followed by embryonic expansion [45]. Metabolic reactivation accompanied with metabolic respiration is characterized by a steep increase in oxygen consumption just after imbibition of the seed. Furthermore, protein synthesis, DNA repair and remobilization of stored reserves are essential processes in the successful germination of the seeds [46]. Physical constraints are imposed by outer seed tissue, and seed germination involves rupture of the testa and the endosperm [47]. Upon imbibition of the seed, the testa becomes more permeable over time and Cd content begins to increase in inner seed tissues [48]. Thereby, genotypic variations of seed coat permeability might be an important factor contributing to the effects of metals on seed germination [49,50]. Subsequently, Cd-inhibited seed germination occurs in a dose-dependent manner [51–53]. Seeds of *Trigonella foenum-graecum* exposed to solutions of chromium (Cr), lead (Pb) and Cd showed that Cd had the strongest germination inhibition effect at 10 mg L−1, which was the highest concentration tested [54]. Likewise, in *Triticum aestivum*, less Cd than Pb was needed to inhibit the seed germination process [55]. Nevertheless, large differences can occur between plant species. Ahsan and colleagues (2007) reported that adding 1 mM of CdCl2 to the solution completely inhibited seed germination of the *O. sativa* cultivar Hwayeong [56]. Seeds of *H. vulgare* were apparently

more tolerant to Cd and their germination rate was only fully inhibited around 9.5 mM CdCl2 [57]. In addition, Cd tolerance of germination might differ largely within one plant species, as was shown within both *T. aestivum* and *O. sativa* cultivars [49,51]. Some cultivars still germinate vigorously with high Cd concentrations, while others will fail. Interestingly, under the threshold of 0.5 mM CdCl2, most tested *O. sativa* cultivars showed an increased germination compared to the control seeds [49]. A similar observation was made by Lefèvre and colleagues (2009) with *Dorycnium pentaphyllum* Scop. seeds [58]. Here, adding 10 μM CdCl2 also significantly increased germination compared to control seeds, while after exposure to 1 mM CdCl2 for 17 days, no more than 40% germination was obtained. These last findings could be indicative for seed germination stimulation at low Cd concentrations, i.e., hormesis, which are undoubtedly dependent on the plant species examined.

Cadmium is known to inhibit seed germination through different mechanisms (Figure 1). In *Vigna unguiculata* seeds, the inhibitory effect of Cd was proposed to be due to an impairment of water uptake, thereby limiting the water availability for the developing embryo [59]. A limited water supply is not the only problem for proper germination. An inhibition of starch mobilization from the endosperm accompanied with an impaired translocation of soluble sugars to the embryonic axis can lead to further starvation of this embryonic axis [50]. A reduction of hydrolyzing enzymes, such as α-amylase, proteases and acid phosphatases, in *Sorghum bicolor* seeds was suggested to be responsible for this reduced storage mobilization. A decrease in α-amylase activity has been reported multiple times in relation to a decrease in starch release from cotyledons [50,60,61]. He and colleagues (2008) pointed out that Ca is a vital element for amylase activity and the replacement of the chemically similar Cd ion could disrupt normal enzyme functioning [62]. Furthermore, in radish seeds, a direct competition for Ca-calmodulin binding sites occurred between Ca and Cd ions [63]. The interaction between Ca and calmodulin is suggested to serve a role in metabolic activation during the early phases of seed germination [64]. An alteration in the remobilization process is also observed in *Vicia faba* seeds by the leakage of soluble sugars and amino acids into the imbibition medium, which is probably related to the loss of membrane integrity [65]. An increase of malondialdehyde content is observed in Cd-exposed *P. sativum* embryos, which might indicate membrane lipid peroxidation [66].

Cadmium is a known inducer of oxidative stress resulting in elevated ROS levels [9,15,67]. Cadmium-induced oxidative stress was able to oxidize Trx isoforms in *P. sativum* seeds [68]. These proteins are potentially involved in monitoring the redox state of storage proteins in both cereals and dicotyledons [69]. Furthermore, the GSH levels were twofold lower, accompanied with a decrease in glutathione reductase (GR) activity, which suggests that intracellular oxidative stress occurred in seeds under Cd exposure [70]. This depletion of the reduced GSH pool might be partially compensated by a higher level of glutaredoxin (Grx) level, which is able to bind Cd at its active site. Peroxiredoxin (Prx) expression was elevated under Cd exposure in both cotyledons and embryonic axes of *P. sativum* seeds. Cadmium could bind to the cysteine residues of Prx, which could serve as a Cd sink [71]. This mechanism might protect the seed from Cd-induced oxidative stress.

Studies that link the interaction between Cd and phytohormones during seed germination are still scarce. Treatment with ethylene is shown to have an alleviating role on Cd-inhibited germination in *Cajanus cajan* [72]. In *O. sativa*, the α-amylase activity is enhanced when Cd-exposed seeds are pre-treated with SA and seedlings show a reduced Cd uptake [73]. In conclusion, seed germination comes forward as a tightly regulated orchestra between hormones and the ROS balance. Within the oxidative window of seed germination hypothesis, one might assume that a small amount of Cd could stimulate the initiation of germination as long as the adverse effects of Cd are not impossible to overcome by the plant's antioxidant defense mechanisms.

**Figure 1.** Possible interference mechanisms of cadmium on the process of seed germination. Cadmium (Cd) negatively affects metabolic reactivation by reducing levels of hydrolyzing enzymes, starch mobilization and seed imbibition. Furthermore, it can alter redox signaling via calcium (Ca), mitogen-activated protein kinases (MAPKs) and transcription factors (TFs) and the level of phytohormones such as abscisic acid (ABA), auxin (AUX), giberrellic acid (GA) and ethylene (ET). Both are of major importance in the seed germination process. One-way arrows: indicate a stimulating effect, whereas T-shaped arrows represent an inhibitory effect. Two-way arrows signify an interaction and dashed lines indicate effects which are still uncertain.

### **3. Vegetative Plant Growth**

Once germination has occurred, two major processes drive plant growth, namely cell division and cell expansion, which is limited by the cell wall during vegetative growth. In the following sections, the effect of Cd on these processes is uncovered in detail (Figure 2).

### *3.1. The DNA Damage Response*

Stress-induced effects on cell cycle progression often result from the activation of the DNA damage response (DDR). Upon perceiving DNA damage, cells trigger this response, which includes the activation of DNA repair pathways. When the extent of DNA damage is low, cell cycle progression is transiently inhibited in order to repair the DNA before DNA replication or cell division take place. When the damaged DNA cannot be repaired, cells undergo terminal differentiation or programmed cell death (PCD) [74].

The induction of cell cycle arrest upon the perception of DNA damage requires the activation of one of two phosphatidylinositol-3-OH-kinase-like kinases: ataxia telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR). Whereas ATM is mainly activated by the presence of DNA double-strand breaks (DSBs), ATR is involved in responses to stalled replication forks. However, both types of DNA damage often occur simultaneously, causing the activation of both kinases [74]. Whereas cell cycle regulation in response to DNA damage depends on p53 in animals, plants lack a p53 orthologue. Instead, SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) is considered as the plant counterpart of p53. This transcription factor belongs to the NO APICAL MERISTEM/*ARABIDOPSIS* TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON (NAC) domain family and is activated through phosphorylation by ATM. Once active, SOG1 induces the expression of a multitude of genes involved in DNA repair, cell cycle progression and cell death [74,75]. In addition to SOG1, also WEE1 plays a key role in the plant DDR. This kinase can be activated by both ATM and ATR and mainly controls S phase progression [74].

**Figure 2.** Schematic overview of important players affecting vegetative plant growth upon Cd exposure. Cadmium exposure is well known to affect concentrations of reactive oxygen species (ROS) and phytohormones, which are closely intertwined. Cadmium induces DNA damage, thereby activating the DNA damage response, which can either induce programmed cell death (PCD) or affect cell cycle progression, depending on the extent of DNA damage. In addition, Cd exposure induces cell wall modifications as a strategy to reduce Cd entry into cells. This in turn limits cell expansion, which is intertwined with the cell cycle and specifically endoreduplication. Cadmium-induced DNA damage and cell wall modifications could either result from its effects on ROS and phytohormone levels or arise through an alternative pathway. Together, Cd-induced PCD, cell cycle alterations and inhibition of cell expansion contribute to its negative effect on vegetative growth. One-way arrows indicate stimulating effects, whereas T-shaped arrows represent an inhibitory effect. Two-way arrows signify an interaction.

### 3.1.1. DNA Damage

Cadmium exposure is well known to induce DNA damage in mammalian cells, as reviewed by Bertin and Averbeck (2006) and Filipic (2012) [76,77]. The mechanisms responsible for this Cd-induced DNA damage include ROS-induced formation of 8-hydroxyguanosine and the inhibition of DNA repair systems. Furthermore, Cd is able to interfere with proteins containing a zinc finger motif, implicated in the maintenance of genome stability, DNA repair and DNA damage signaling [76,77].

At present, in-depth knowledge on Cd-induced DNA damage and its underlying mechanisms in plants is lacking. However, many studies have demonstrated that Cd exposure induces DNA damage in multiple plant species. Table 1 provides an overview of recently published research (since 2014) demonstrating different types of Cd-induced DNA damage in a broad range of plant species. Several authors reported that Cd exposure increases the percentage tail length, tail intensity and tail moment determined through single cell gel electrophoresis, also known as the comet assay (Table 1). The detection of DNA damage using this method relies on the fact that DNA strand breaks facilitate DNA migration from the nucleoids towards the anode during gel electrophoresis, thereby forming a "comet tail". Whereas the alkaline comet assay (performed at pH > 13) detects both DNA single-stranded breaks (SSBs) and DSBs, only DSBs are detected through the neutral comet assay [78,79]. Taken together, the results obtained through this method suggest that Cd exposure induces DNA strand breaks in different plant species including *V. faba* [80–82], *Nicotiana tabacum* [83,84], *Allium sativum* [82], *Solanum tuberosum* [85], *Allium cepa* [80,86,87], *Lemna minor* [88,89], *Lactuca sativa* [86,90], *H. vulgare* [91], *Brassica oleracea* and *Trifolium repens* [92]. In *S. tuberosum*, the Cd-induced increase in percentage tail DNA was more pronounced in roots as compared to leaves and appeared later in the latter organ [85]. Similarly, Cd exposure for 24 and 72 h caused significant increases in the percentage tail DNA and the tail moment in *N. tabacum* roots, whereas no effects were observed in leaves [83]. This is likely due to the fact that roots form the entry route for Cd into the plant and are therefore exposed earlier than leaves. Furthermore, roots of both plant species were shown to accumulate higher Cd levels than leaves [83,85].

In addition to inducing DNA strand breaks, Cd is also shown to induce micronucleus formation and chromosomal aberrations, which are both detected through microscopic analysis (Table 1). Micronuclei arise when chromosome fragments or entire chromosomes fail to be included in the nuclei of the daughter cells at the end of mitosis, as they do not manage to properly attach to the mitotic spindle during anaphase. They eventually become enclosed by a nuclear membrane and appear as small nuclei after conventional nuclear staining. Micronuclei are generally considered as biomarkers for genotoxicity [93,94]. Chromosomal aberrations frequently detected during mitosis in Cd-exposed plants include anaphase and telophase bridges, sticky chromosomes, chromosome breaks, non-oriented chromosomes and laggards chromosomes [80,86,95,96]].

Another approach frequently used to determine Cd-induced genotoxicity in plants is the investigation of random amplified polymorphic DNA (RAPD) profiles. This method consists of a PCR amplification of genomic DNA using multiple short (approximately 10 nucleotides) random primers, followed by gel electrophoresis and visualization of the amplified PCR products. Mutations and other types of DNA damage induced by exposure to stress factors such as Cd can result in the disappearance as well as *de novo* creation of primer annealing sites, thereby yielding an altered RAPD profile [97]. This subsequently allows for calculation of the genomic template stability (GTS) using the following formula: GTS (%) = (1 – a/n) × 100, where "a" represents the number of polymorphic bands in the treated samples and "n" identifies the total number of bands in the control samples [98,99]. As indicated in Table 1, Cd exposure was shown to alter the RAPD profile in both roots and leaves of a broad range of plant species, resulting in a reduced GTS.

**Table 1.** Overview of recent research articles (published since 2014) demonstrating Cd-induced DNA damage, arranged by plant species. Cadmium is shown to induce different types of DNA damage, including DNA strand breaks, chromosomal aberrations and micronuclei in different plant species. Furthermore, it alters the expression of DNA repair genes and changes amplified fragment length polymorphism (AFLP), inter-simple sequence repeat (ISSR), random amplified polymorphic DNA (RAPD), sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) profiles, thereby reducing the genomic template stability (GTS).↑and ↓ symbols indicate increases and decreases, respectively.


Other markers used to assess Cd-induced DNA damage are the full peak coefficient of variation determined via flow cytometric analysis [113] and DNA polymorphisms determined by PCR-based methods (besides RAPD) such as amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) [103,104,108,109]]. However, in comparison to the other DNA damage indicators described, the use of these markers in studies evaluating Cd genotoxicity in plants is currently limited.

Despite the large number of studies indicating Cd-induced genotoxicity in plants, its underlying mechanisms are still largely unknown. However, as Cd exposure is well known to induce ROS production, it is likely that DNA oxidation contributes to the observed damage. Therefore, it would be of interest to study the extent of oxidative DNA damage in Cd-exposed plants. Furthermore, it would be interesting to assess the extent of DNA repair in Cd-exposed plants to gain an insight into plant responses to the observed Cd-induced damage. Although methods for the assessment of oxidative DNA damage and DNA repair are widely adopted in animal samples, these methods have so far received little attention in plant research. However, their optimization might significantly enhance our knowledge on DNA damage and repair induced by Cd and other stress factors in plants.

### 3.1.2. The Cell Cycle

As a consequence of inducing DNA damage, Cd exposure can affect cell cycle progression. The formation of new cells through cell division is the primary driving force for organ growth in plants. Similar to that in other eukaryotes, the classical plant cell cycle consists of four phases: gap 1 (G1) phase, DNA synthesis (S) phase, gap 2 (G2) phase and mitotic (M) phase [114]. The gap phases enable the control of accurate and full completion of previous phases. As a consequence, many important regulatory mechanisms controlling cell cycle progression operate at the G1/S and G2/M transitions. During the S phase, nuclear DNA is replicated, whereas the replicated sister chromatids are divided over the two daughter cells arising through cytokinesis during the M phase. The cell cycle is regulated by the activity of cyclin-dependent kinases (CDKs), which are serine/threonine protein kinases that phosphorylate target proteins crucial for cell cycle progression. As their name implies, CDKs form heterodimers with regulatory cyclins in order to become activated [115]. During progression throughout the cell cycle, CDK activity shows a typical pattern, reaching two thresholds: one for DNA replication (S phase) and one for cell division (M phase) [116].

In addition to dividing through the classical cell cycle, plant cells can also undergo endoreduplication. During this alternative cell cycle mode, plant cells replicate their nuclear DNA (S phase) without intermittent cell division (M phase), resulting in endopolyploidy (*i.e*., the existence of different ploidy levels in adjacent cells of a species) [117].

Similar to the classical cell cycle, endoreduplication is also regulated by the action of CDK-cyclin complexes. During an endocycle, CDK activity only reaches the threshold for DNA replication but not for cell division. Endoreduplication is important for normal plant growth and development, as it is tightly related to cell differentiation and expansion [116]. The importance of endoreduplication in plant development and its connection to cell size become apparent during trichome development. Trichomes are large, single epidermal cells that develop on most aerial parts of *A. thaliana* plants and typically contain three to four branches. These highly specialized structures—involved in plant protection against external stress factors such as herbivory, frost and ultraviolet radiation—require endoreduplication for their development and reach a final DNA content of 32C, with C representing the haploid DNA content [118,119]. Interestingly, trichomes were previously shown to accumulate Cd, possibly to prevent Cd-induced damage at more sensitive sites within the plant [120,121]. In addition to its involvement in plant growth and development, endoreduplication could also serve as a strategy in plant defense against biotic and abiotic stress factors, as reviewed by Scholes and Paige (2015) [122]. Under stress conditions, an increased ploidy level and therefore a higher number of DNA templates could help to sustain genome integrity and stimulate genetic pathways responsible for plant defense [122].

Cadmium-induced disturbances of root and leaf growth were shown to coincide with effects on cell division in a wide range of plant species in a multitude of experimental set-ups. An overview of recently published research (since 2014) demonstrating Cd-induced effects on multiple cell cycle-related parameters in several plant species is provided in Table 2. In general, Cd negatively affects cell cycle progression, as becomes apparent from a decreased mitotic index (i.e., the ratio between the number of cells undergoing mitosis and the total cell number), determined via microscopic analysis. As suggested by Monteiro et al. (2012), Cd might bind to the sulfhydryl groups of cysteine residues present in tubulins, thereby affecting microtubule formation and disturbing cell division [90]. Although many studies have addressed the influence of Cd exposure on the cell cycle in roots, knowledge regarding its effects on this process in leaves is scarce. However, Baryla et al. (2001) demonstrated that leaves of Cd-exposed *Brassica napus* plants contained a smaller number of stomatal guard cells and were characterized by a larger mesophyll cell size as compared to their control counterparts, suggesting that Cd also affects the cell cycle in leaves [123]. Similarly, Cd exposure inhibited cell division and increased cell size in both the upper and lower cell layers of young *Elodea canadensis* leaves. Interestingly, this response coincided with a strong Cd-induced disturbance of the typical cell wall structure, which was likely the consequence of the accumulation of large amounts of Cd in the apoplast and the binding of Cd ions to cell wall components [124] (*cfr. infra*). Furthermore, Cd exposure was shown to reduce both adaxial pavement cell number and surface area in different leaves of *A. thaliana* in a time-dependent manner. The decreased cell surface area might be related to a lower extent of endoreduplication in leaves of Cd-exposed plants, as indicated by a significantly decreased endoreduplication factor (i.e., the average number of endocycles that has taken place per cell) [105]. In contrast, other studies reported a decreased percentage of cells with a 2C nuclear DNA content and an increased percentage of cells with a higher nuclear DNA content in roots of *A. thaliana* [101,102] and *P. sativum* [125–127]. Although an increased proportion of 4C cells might indicate a cell cycle arrest in G2 phase, an increased level of 8C cells points towards an elevated extent of endoreduplication. These data suggest that Cd exposure stimulates this alternative cell cycle variant in roots, whereas inhibiting it in leaves. However, in young *A. thaliana* leaves, an increased extent of endoreduplication was observed shortly after the start of Cd exposure [Hendrix et al., personal communication]. It could be hypothesized that increased ploidy levels confer tolerance to Cd stress, since Talukdar (2014) demonstrated that roots of tetraploid and triploid *Lathyrus sativus* plants were less sensitive to Cd as compared to their diploid counterparts [95]. Furthermore, it is tempting to speculate that the increased extent of endoreduplication is related to a stimulation of trichome development, as these structures are characterized by a high nuclear DNA content [118,119] and provide sites for Cd sequestration [120,121]. However, this hypothesis requires further investigation.

Cadmium-induced effects on the cell cycle in roots and leaves often coincide with alterations in the expression of cell-cycle related genes, such as those encoding cyclins and CDKs (Table 2). Indeed, *CYCB1* expression decreased upon Cd exposure in *Glycine max* cell suspension cultures, possibly affecting G2/M progression [128,129]. Exposure to 100 μM Cd for 15 days affected the expression of a large number of cell cycle-related genes in roots of *O. sativa*. Interestingly, Cd-induced effects on a number of these genes were altered by simultaneous treatment with 2,3,5-triiodobenzoic acid (an inhibitor of polar AUX transport), indole-3-butytric acid (an AUX hormone), Tiron (a superoxide (O2 •−) scavenger) or sodium diethyldithiocarbamate (an SOD inhibitor) [130]. In another study, the authors demonstrated that treatment with ABA or tungstate (an ABA inhibitor) also influenced Cd-induced effects on transcript levels of certain genes involved in cell cycle regulation [131]. These data emphasize the involvement of plant hormones and ROS in Cd-induced effects on the cell cycle. The significance of various phytohormones and their complex interactions in regulating cell division and endoreduplication was extensively reviewed by Tank et al. (2014) [132]. Although the precise role of ROS in cell cycle regulation is still unclear, their importance in this process is illustrated by the fact that oxidative and reductive signals are required for certain cell cycle transitions. In addition, transcript levels and activities of cyclins and CDKs were demonstrated to be altered upon redox perturbations. The antioxidative metabolite GSH might constitute an important redox-related cell cycle regulator, as it was reported to be translocated into the nucleus during cell division [133] and the severely GSH-deficient *root meristemless 1* (*rml1*) *A. thaliana* mutant is unable to form a root apical meristem due to a lack of cell division [134]. Furthermore, ROS and redox homeostasis are also crucial mediators of cytokinesis, as indicated by a disturbed cell division in mutants characterized by impaired ROS production or signal transduction [135,136]. The involvement of ROS and redox regulators in the cell cycle is discussed in more detail in a recent review by Mhamdi and Van Breusegem (2018) [137].

Additional evidence for Cd-induced effects on transcript levels of cell cycle-related genes comes from *A. thaliana*. In roots of this species, Cd exposure caused a downregulation of several G1/S marker genes such as *HISTONE H4* and *E2Fa* and G2/M marker genes, including *CYCB1;1* and *CYCB1;2*. This response coincided with an altered expression of DNA repair genes and was less pronounced in mutants with an impaired DNA mismatch repair pathway, suggesting that DNA damage contributes to the observed cell cycle arrest [101,102]. In agreement with this hypothesis, Hendrix et al. (2018) demonstrated significant increases in the expression of several DNA repair genes and genes encoding CDK inhibitors of the SIAMESE-related (SMR) family in leaves of *A. thaliana* exposed to 5 μM Cd for 72 h [105]. Interestingly, these *SMR* genes were previously reported to be transcriptionally upregulated in response to ROS-induced DNA damage in plants exposed to hydroxyurea [138], further pointing towards the involvement of DNA damage in the Cd-induced cell cycle inhibition.


*Int. J. Mol. Sci.* **2019** , *20*, 3971

**Table 2.** Cadmium exposure is shown to reduce the mitotic index (i.e., the ratio between the number of cells undergoing

Overview of recent research articles (published since 2014)

demonstrating

 Cd-induced

 effects on cell

cycle-related

 mitosis and the total cell number), alter nuclear

 parameters,

 arranged by plant species.

### 3.1.3. Cell Death

In case DNA damage is severe and cannot be repaired, the DDR is responsible for activating PCD [74]. Plant PCD is defined as a genetically encoded and actively controlled form of cellular suicide and is generally subdivided into developmental PCD and environmentally induced PCD. Developmental PCD plays a crucial role in normal plant growth and development, being involved in seed development and germination, as well as vegetative development. During the latter, PCD is for example involved in the differentiation of xylem tracheary elements, the emergence of lateral and adventitious roots and in senescence [140]. It is well-known that ROS and NO are important regulators of both developmental and environmentally induced plant PCD, as was recently reviewed by Locato et al. (2016) [140].

Interestingly, Cd-induced DNA damage and cell cycle effects observed in different plant species often coincide with a decrease in cell viability. For example, Kuthanova et al. (2008) demonstrated that exposure to 50 μM Cd affected cell cycle progression in synchronized tobacco BY-2 cell cultures and significantly reduced cell viability [141]. The type of cell death induced upon Cd exposure strongly depends on the cell cycle phase at which Cd exposure started. Whereas Cd application during S and G2 phase resulted in an apoptosis-like PCD type, characterized by DNA fragmentation, no such effect was observed in cells exposed to Cd during the other cell cycle phases. Instead, cells exposed during M phase displayed a rapid cell death, coinciding with fragmented late telophasic nuclei, while the decrease in viability of cells treated with Cd at G1 phase took place at a slower rate [141]. Similarly, the decreased mitotic index and chromosomal aberrations observed in *A. cepa* root tips upon exposure to a range of Cd concentrations was accompanied by a strong increase in Evans blue staining, indicative of cell death [80]. However, no effect on cell viability was observed in root tips of *T. aestivum* exposed to a range of Cd concentrations for 48 h, although these conditions induced oxidative modifications of cell cycle-related proteins and cell cycle arrest [142]. These data emphasize that the occurrence of Cd-induced cell death depends on many factors including the plant species, Cd concentration and exposure duration.

The Cd-induced PCD response in tobacco BY-2 was shown to depend on three ROS waves: (1) an initial transient NADPH oxidase-dependent increase in H2O2 levels, (2) the accumulation of mitochondrial O2 •− and (3) fatty acid hydroperoxide accumulation, which coincides with the occurrence of cell death [143]. The importance of ROS in Cd-induced PCD is also supported by the work from Tamás et al. (2017) [144], who showed that cell death in roots of Cd-exposed *H. vulgare* is mostly pronounced at locations where O2 •− generation was observed (i.e., the transition and distal elongation zones). However, Cd-induced cell death was only observed in roots of plants exposed to 60 μM Cd, whereas no effect was observed upon exposure to lower concentrations, suggesting that cell death only occurs when ROS concentrations exceed a certain threshold [144]. In an *Arabidopsis* cell suspension culture, the H2O2 production required for PCD was shown to depend on NO production, as inhibition of NO synthesis partially prevented H2O2 accumulation and cell death [145]. As summarized by Locato et al. (2016) [140], different underlying mechanisms have been proposed for the observed NO-dependency of Cd-induced PCD. First, NO-dependent nitrosylation of PCs could reduce their ability to chelate Cd, thereby increasing free Cd levels and enhancing toxicity symptoms [145,146]. Furthermore, NO could increase Cd accumulation by affecting transcript levels of genes involved in Cd uptake and detoxification [147,148]. A final mechanism proposed is the Cd-induced activation of MPK6, which subsequently activates caspase-3-like, a PCD executor [149].

It is likely that phytohormones are also involved in regulating Cd-induced PCD, possibly through interactions with ROS. Simultaneous exposure of a tomato cell suspension culture to Cd and ethylene caused a stronger Cd-induced PCD response as compared to treatment with Cd alone. Furthermore, Cd-induced PCD was mitigated by application of the ethylene biosynthesis inhibitor 2-aminoethoxyvinyl glycine and the ethylene receptor blocker silver thiosulfate [150], clearly indicating the key role of ethylene signaling in Cd-induced PCD. Furthermore, a Cd-induced increase in cellular SA concentrations of tobacco cells was shown to induce a MAPK signaling pathway involved in mediating

PCD [151]. Similarly, exogenously applied SA was able to alleviate Cd-induced ROS production, photosynthetic damage and cell death in Cd-exposed *A. thaliana* [152]. The cell death observed in this study was likely a consequence of a strong Cd-induced activation of autophagy. This process involves the vacuolar degradation and recycling of cellular macromolecules or entire organelles and was previously reported to be induced upon Cd exposure in various plant species including *A. thaliana* [152], *G. max* [153], *T. aestivum* [154] and *Theobroma cacao* [155]. Although their interplay has not yet been studied in Cd-exposed plants, crosstalk between autophagy and several phytohormones exists in plants exposed to other environmental stimuli or during normal plant development [156]. Furthermore, an interplay between autophagy and ROS was also shown, with ROS contributing to the establishment of autophagy and autophagy contributing to ROS scavenging. A comprehensive overview of the involvement of ROS and phytohormones and their interplay in autophagy in stress-exposed plants was recently published by Signorelli et al. (2019) [157]. However, the involvement and interplay of ROS and phytohormones in Cd-induced autophagy remains largely unknown. Interestingly, autophagy contributes to nutrient recycling and remobilization during senescence [157], a process which was shown to be prematurely induced upon Cd exposure in different plant species, as indicated by increases in senescence-related parameters such as protease activity, lipid peroxidation and the expression of senescence-associated genes (SAGs) [9].

Taken together, these data suggest that Cd-induced DNA damage is an important trigger for effects on plant growth either through the induction of cell death or through influences on the cell cycle. In order to prevent Cd entry into cells and subsequent DNA damage, cell wall structure can be altered to increase the number of Cd binding sites. This often coincides with an increased cell wall rigidity, which can in turn limit cell expansion and hence plant growth.

### *3.2. The Cell Wall*

The primary cell wall consists of structural proteins that are embedded in a matrix of polysaccharides which includes cellulose, hemicellulose and pectin [158–160]. These components ensure that inner structures have sufficient support, but at the same time remain adjustable for the expanding cell. Furthermore, these polysaccharides contain functional groups such as hydroxyl, thiol and carboxyl groups that enable the cell wall to bind large amounts of divalent and trivalent metals including Cd [161]. The homogalacturonan (HG) domain of pectin is created by the golgi apparatus and secreted into the cell wall in a highly methylesterified form. Using a Ca ion, two HG domains can be linked together by their carboxylic groups [162]. Calcium can be replaced within this structure by other metals such as Pb, Cu, Cd and Zn that show greater affinity [161].

### 3.2.1. The Cell Wall as Major Storage Compartment for Cadmium

The cell wall is the primary defense structure of a plant's cell against pathogen attacks or unfavorable environmental conditions such as drought and metals [163–165]. By keeping excess Cd out of the cytoplasm, it prevents damage to macromolecules, proteins and DNA caused directly or upon Cd-induced oxidative stress. Since roots are the organs in direct contact with Cd from the soil, their cell walls play an important role in this process. Many studies show that most Cd is stored within the cell walls of roots by various plant species [166,167]. For example, in the plant *Coptis chinensis*, the roots and rhizomes retained between 62 and 77% of all Cd [168]. When the capacity of the cell wall is exceeded, Cd might additionally form complexes with PCs and is subsequently sequestered within the vacuole [167,169,170]. However, Dong and colleagues (2016) reported that upon exposure to 200 μM CdCl2, most Cd in the leaves was present in the soluble fraction in *Arachis hypogaea* and compartmentalization in vacuoles might be of greater importance here [171].

### 3.2.2. Cadmium-Induced Cell Wall Modifications

In addition to the formation of a rigid barrier, the cell wall might also be actively modified under Cd exposure [172–174]. By increasing the pectin methylesterase (PME) activity, the amount of de-esterified pectin increases, thereby creating more negative charges to bind Cd [175,176]. Moreover, an increase in pectin and hemicellulose content can also contribute to the latter [177,178]. Recently, in *O. sativa* seedlings it was shown that root aeration increases pectin content and PME activity resulting in Cd toxicity alleviation [179]. Since pectin is especially synthesized in young, expanding cells, a delay in root maturation due to aeration might enhance pectin deposit. Next to cell wall modifications related to primary components, another effect that occurs under Cd exposure, is the activation of lignin biosynthesis [180,181]. Lignin is incorporated in the secondary cell walls of specialized cells such as tracheids and vessel elements of the xylem and is created by the oxidative polymerization of monolignol subunits by class III peroxidases (PODs) and laccases [182]. Adding lignin to the cells' walls decreases the permeability to Cd, but at the same time restricts cell elongation, thereby inhibiting growth. An increase of POD activity in response to Cd has been observed in various plant species [180,181,183]. It is of particular interest that POD uses H2O2 as a substrate, but at the same time H2O2 comes forward as a major signaling molecule within the redox network, which is strongly disturbed by Cd exposure. These close interactions between lignification and redox regulation have been extensively reviewed by Loix and colleagues (2017) [173]. Cell wall expansion is largely regulated by ROS homeostasis in the apoplast [184]. Whereas H2O2 contributes to cell wall stiffening, hydroxyl radicals (•OH) lead to cell wall loosening due to pectin and xyloglucan cleavage [185,186].

The role of cellulose under Cd exposure is still a matter of debate. The cell wall cellulose content was shown to decrease in *O. sativa* and *Zea mays* under Cd exposure, but an increase was observed in *Linum usitatissimum*. Therefore, it is proposed that different defense strategies are present between monocots and dicots [175,177,187]. Recently, it was shown that Cd accumulates preferably within cellulose in the Cd-sensitive *G. max* BX10 variety, but contrarily, the Cd-tolerant *G. max* HX3 accumulated more Cd in cell wall-related pectin [188]. This might even suggest intraspecies differences in sequestration of Cd in different cell wall components. Cellulose microfibrils are cable-like structures composed of many β-1,4-linked glucose molecules, which are added together by cellulose synthase using uridine diphosphate glucose as a donor molecule [189]. Cadmium is known to reduce the amount of cytosolic sucrose, which could have detrimental effects on cellulose biosynthesis, as UDP-glucose is thought to be mainly produced by sucrose synthase (SUSY) from sucrose [174,190]. In a recent proteomic study by Gutsch and colleagues (2018), SUSY was shown to be upregulated in response to long-term Cd exposure in *Medicago sativa* [191]. The photosynthetic capacity of these plants was inhibited, as indicated by a lower abundance of photosynthetic proteins, which cuts off glucose supply for cell wall biosynthesis. However, a higher SUSY activity might suggest that the plants still invested in cellulose synthesis. In *Miscanthus sacchariflorus*, several genes involved in cellulose biosynthesis were upregulated under Cd exposure as well. These included cellulose synthase A, cellulose synthase-like protein D4 and cellulose synthase-like protein H1 [192].

In addition to these polysaccharides and lignin, two protein families, i.e., xyloglucan endotransglucosylases/hydrolases (XTH) and expansins (EXP), were demonstrated to affect cell wall expansion as well [193]. Expansins have a function in breaking the non-covalent binding of polysaccharides resulting in cell wall loosening [158,194]. In *N. tabacum*, *NtEXP1*, *NtEXP4* and *NtEXP5* transcripts were abundant in the shoot apices and young leaves, but not in roots and mature leaves, supporting their function in cell wall extension [195]. Furthermore, these genes were induced by growth-stimulating hormones such as AUX, GA and CK, but Cd exposure inhibited their transcription. This was supported by a study in *Brassica juncea* by Sun and colleagues (2011) who found that overexpression of *BjEXPA1* led to a higher Cd sensitivity [196]. However, the opposite was found for the *TaEXP2* gene in *T. aestivum* [197]. This gene was upregulated under Cd exposure and its overexpression led to an increase in biomass and root elongation. This improved plant performance was stated to be due to an enhanced translocation of Cd to the vacuoles, a higher antioxidant capacity and a higher water retention. Secondly, XTHs have the ability to cut and rejoin xyloglucan, which locks cellulose microfibrils and thereby contributes to cell wall loosening [198]. In *A. thaliana*, *XTH33* was required for Cd accumulation within the roots [199]. In addition, *XTH33* was shown to be a

direct target of EIN3 which acts as a master transcription factor in ethylene-mediated Cd-induced root growth inhibition. When *PeXTH* from *Populus euphratica* was introduced in *N. tabacum*, a decrease of xyloglucan was observed in the cell wall of the roots, leading to a reduced number of Cd binding sites, thereby reducing Cd influx into the roots and limiting Cd toxicity [200]. Lastly, in a proteomic study with two cultivars of *A. hypogaea*, it was hypothesized that XTHs and α-expansins might be important in keeping cell wall extensibility under Cd exposure in the Cd-sensitive cultivar, which showed a higher capacity of cell wall modification [201]. Both XTH and EXP proteins are activated by AUX, inducing acidic growth of the cell wall [202].

### 3.2.3. The Role of The Cell Wall in Cadmium Hyperaccumulators

Plants that are able to withstand very high metal concentrations in the soil and show an enhanced metal accumulation within the aboveground organs are defined as hyperaccumulators [169]. These remarkable plant species account for less than 0.2% of all angiosperms, although additional heavy metal-accumulating plants are likely to be identified [203]. The trait is expected to have evolved multiple times but is of very high occurrence within the Brassicaceae family [169]. It is a nice coincidence that frequently studied hyperaccumulators such as *Arabidopsis halleri* and *Noccea caerulescens*, previously known as *Thlaspi caerulescens*, are phylogenetic closely related to the model plant *A. thaliana*, which does not accumulate metals. As shown by Benzarti et al. (2008), the Cd hyperacummulator *N. caerulescens* has a more than tenfold higher EC50 value for Cd-induced root growth inhibition in comparison to several non-accumulator plants [204]. However, large differences in the ability to accumulate Cd also exist between populations of hyperaccumulating species [203]. In *A. halleri*, some individuals of metallicolous populations were able to survive in the presence of 450 μM CdSO4, whereas concentrations as low as 100 μM CdSO4 caused mortality within non-metallicolous populations [205]. Tolerance mechanisms to cope with high soil metal concentrations include enhanced metal uptake and xylem loading, followed by detoxification in the shoot, as reviewed by Verbruggen and colleagues (2009) [206].

The role of Cd storage in the cell wall of hyperaccumulators might not be that straightforward. In a comparative study between a non-hyperaccumulating and a hyperaccumulating ecotype of *Sedum alfredii*, it was shown that for the latter more cell wall-bound Cd was available for xylem loading [207]. Furthermore, no increase of pectin or hemicellulose 2 was detected in the hyperaccumulating ecotype, which was accompanied with a lower PME activity. This led the authors to the conclusion that Cd translocation could at least partly be due to a difference in cell wall modification regulation [207]. In addition, cell wall modifications in the shoot cell walls of *A. halleri* under Cd exposure were more pronounced in less tolerant populations [205]. A Cd tolerance mechanism might already be present under controlled conditions in *A. halleri* in contrast to *A. thaliana* [208]. Gene expression related to redox balance, Ca signaling, and cell wall remodeling was more affected in *A. thaliana*, yet *PME* and *CESA* transcripts (encoding for cellulose synthases) were more upregulated in *A. halleri*. This could result in a larger extent of Cd binding to the cell wall and cell wall stiffening, which in turn leads to a greater barrier for cytosolic entering [208]. Moreover, Peng and colleagues (2017) showed that the cell wall of a newly discovered hyperaccumulator *Sedum plumbizincicola* plays a crucial role in Cd tolerance [209]. In contrast to a non-hyperaccumulating ecotype of *S. alfredii*, Fourier transform infrared (FT-IR) spectroscopy displayed a higher absorbance ratio of –COO− against –COOR, indicating a lower esterification of pectin and a more efficient binding of metal ions for *S. plumbizincicola*.

### **4. Reproductive Growth**

In order for a plant to complete its life cycle, it must commence the reproductive phase, which in higher plants involves the setting of flowers, pollination and fertilization, followed by the production of seeds. In *Brassica campestris*, the GSH and AsA content dropped the most when plants were exposed to Cd at their flowering stage, indicating that the reproductive phase is highly susceptible [210]. In *L. usitatissimum* plants grown in Cd-contaminated soils, a decrease in fitness was noted due to 31.8% fewer seeds and 25.6% fewer fruits [211].

Although *A. thaliana* plants showed a reduction in silique counts under long-term Cd exposure, they were still able to complete their life cycle, thereby producing seeds with equal germination capacity as control plants [212]. The transition from the vegetative to the reproductive phase under 10 μM Cd was unaffected, which was in agreement with the results from Maistri and colleagues (2011), however an accelerated emergence of inflorescence was observed under 5 μM Cd [213]. Limited research has been done on the later stages of plant development in relation to Cd exposure. Nevertheless, accumulation of large amounts of Cd in the seeds, even without visible toxic effects to the plant, can be a great threat to human health [214]. Cadmium uptake by roots and translocation to aboveground parts is a process that continues throughout the plant's life cycle. In *O. sativa*, most of the Cd accumulated in the grains during the early phases of grain development either directly via the xylem or through remobilization through the phloem [215]. However, in *Solanum lycopersicum* the majority of Cd was taken up in the final stage of fruit development [216]. This co-occurred with a disturbance in nutritional status, as potassium (K), Fe and Zn contents in fruits decreased, while Ca and magnesium (Mg) increased. Exposure to 100 μM Cd resulted in an absence of fruit setting. Furthermore, an interesting finding was reported in the monoecious plant *Crocus sativus*, where a shift to more male flowers was apparent upon Cd exposure [217]. This was also observed in a study with *Cannabis sativa*, where treatment with Pb resulted likewise in male flowers and was demonstrated to be due to a hormonal shift with increasing GA levels in these plants, while zeatine (a specific form of CK) decreased [218].

Cadmium has been shown to negatively affect pollen germination accompanied with a disruption of pollen tube morphology in multiple plant species [219,220]. Even very low Cd concentrations of 0.01 μg mL−<sup>1</sup> were able to inhibit either pollen germination or tube growth in *Vicia angustifolia* and *Vicia tetrasperma*, indicating that this process is very sensitive to Cd [221]. The impaired cell elongation of the pollen tube by Cd is a consequence of its interference with the anionic content of secretory vesicles and its interaction with the cell wall, which contains large quantities of pectin and callose [222]. Cell wall thickening, an increase in cell diameter and abnormal pollen tube growth were observed in all *Prunus avium* cultivars tested in vitro under Cd exposure [223]. Pollen tubes of *Picea wilsonii* showed swelling of the tips accompanied by cytoplasmic vacuolization [224]. Furthermore, the importance of ROS/Ca signaling in pollen tube formation has been well documented [137,225]. Exogenously applied •OH to pollen of *N. tabacum* caused loosening of the intine (i.e., the inner layer of the pollen tube cell wall), leading to a disrupted polar growth, while H2O2 stiffened the cell wall [226]. Both treatments resulted in a reduced pollen germination, since only the region of the germinating pore should be weakened through •OH-dependent reactions. In conclusion, Cd negatively affects plant fitness by interfering with various processes including pollen tube formation and pollen germination resulting in smaller numbers of seeds that show a reduced germination as was described in the section on seed germination. In conclusion, knowledge on the effects of Cd on the reproductive phase of plant development is still relatively scarce in contrast to information on its effects on vegetative growth and deserves more attention in the future.

### **5. Conclusion and Future Perspectives**

Cadmium exposure interferes with all stages of plant development, inhibiting seed germination, vegetative growth and reproductive growth. Important players in the Cd-induced disturbance of root and leaf growth are DNA damage, which subsequently affects cell cycle progression or even causes cell death, and structural alterations of the cell wall. Furthermore, Cd alters pollen tube morphology, inhibits pollen germination and can be transported into seeds and fruits. The type and extent of Cd-induced effects strongly depends on many factors, including the plant species, organ, cell type, Cd concentration and exposure duration.

Additional research on the mechanisms underlying these Cd-induced disturbances of plant growth and development is required to develop and further improve strategies to enhance plant growth on Cd-polluted soils. In this context, it would be interesting to also focus on transgenerational effects involved in plant adaptation to Cd exposure. Although research in this field is currently limited, a recent study showed that Cd-induced alterations of RAPD profiles in *Urtica pilulifera* parent plants are transmitted to the next generation [112]. Similarly, Carvalho et al. (2018) demonstrated that exposure of *S. lycopersicum* to Cd resulted in Cd accumulation in seeds, which altered their nutrient profile and caused a decreased mitotic index in root tips of the offspring [227]. Although the mechanisms underlying these transgenerational effects are currently unknown, epigenetic changes might be involved, as Cd exposure was previously shown to alter DNA methylation patterns in *A. thaliana* [104]. In addition, the possible involvement of the microbiome in plant adaptation to Cd exposure should also be taken into account, as transgenerational exposure of *A. thaliana* to Cd significantly altered the seed endophytic community [228,229]. Furthermore, inoculation with specific plant-associated bacteria improved root growth of Cd-exposed *A. thaliana* plants [230], emphasizing the importance of the plant microbiome in coping with environmental stress factors and optimizing plant growth and development under suboptimal conditions. Finally, the use of soil amendments such as biochar and silicon-based fertilizers is promising for future applications to alleviate Cd toxicity, thereby improving crop growth and quality [231,232].

**Author Contributions:** All authors participated in the conception of the topic. M.H., A.C. and S.H. wrote the manuscript. M.H. and S.H. made the figures and tables. All authors read and approved the final manuscript after critically revising it for important intellectual content.

**Funding:** This work was supported by the Research Foundation Flanders (FWO) by project funding for M.H. [G0B6716], S.H. [G0C7518CUY], M.J. [SBOS000119N], J.D. [FWO PhD grant], and bijzonder onderzoeksfonds (BOF) from Hasselt University to V.I. [BOF-18D02CUYA] and S.V. [PhD grant].

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

### **Abbreviations**



### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Advances in the Uptake and Transport Mechanisms and QTLs Mapping of Cadmium in Rice**

**Jingguang Chen 1,2, Wenli Zou 1, Lijun Meng 1,\*, Xiaorong Fan 2,\*, Guohua Xu <sup>2</sup> and Guoyou Ye 1,3**


Received: 28 June 2019; Accepted: 11 July 2019; Published: 11 July 2019

**Abstract:** Cadmium (Cd), as a heavy metal, presents substantial biological toxicity and has harmful effects on human health. To lower the ingress levels of human Cd, it is necessary for Cd content in food crops to be reduced, which is of considerable significance for ensuring food safety. This review will summarize the genetic traits of Cd accumulation in rice and examine the mechanism of Cd uptake and translocation in rice. The status of genes related to Cd stress and Cd accumulation in rice in recent years will be summarized, and the genes related to Cd accumulation in rice will be classified according to their functions. In addition, an overview of quantitative trait loci (QTLs) mapping populations in rice will be introduced, aiming to provide a theoretical reference for the breeding of rice varieties with low Cd accumulation. Finally, existing problems and prospects will be put forward.

**Keywords:** cadmium accumulation; absorption and transport; QTL location; mapping population; rice (*Oryza sativa* L.)

### **1. Introduction**

Cadmium (Cd) is a soil contaminant and with a high mobility in living organisms, and is characterized as a toxic heavy metal [1,2]. In China, about 2.786 <sup>×</sup> 10<sup>9</sup> m2 of agricultural land is contaminated by Cd [3]. Frequent applications of nitrogen fertilizer in the agricultural land of many areas of China have resulted in more acidic soil, and acidic soil means that cadmium is more easily absorbed by plants [4]. Rice (*Oryza sativa* L.) is the main food for more than half of the world's population. Cd is easily transferred from soil to rice and accumulates in rice plants and grains [2,3], and is then enriched in the human body through the food chain, thereby threatening human health [5–7], and causing effects such as anemia, cancer, heart failure, hypertension, cerebral infarction, proteinuria, severe lung damage, eye cataract formation, osteoporosis, emphysema, and renal insufficiency [8,9]. It is worth mentioning that Itai-itai disease, which occurred in Japan in the 1950s, was caused by the long-term intake of cadmium-contaminated rice [10]. On average, weekly Cd accumulation was as high as 3–4 mg kg−<sup>1</sup> body weight in Japan at that time [11]. Between 1990 and 2015, the average dietary Cd intake of the general population more than doubled in China [12,13]. Therefore, reducing Cd uptake by crops, especially rice, is of great significance to food safety and human health.

The purpose of this review is to explore the mechanism of cadmium uptake and transport and the genetic characteristics of Cd accumulation in rice, and to summarize the research status of genes and QTLs related to cadmium stress and cadmium accumulation in rice. It has important guiding significance for breeding high-quality rice varieties with a low accumulation of Cd in grain and the safe production of rice in mild and moderate Cd-contaminated soil.

### **2. Toxic E**ff**ects of Cadmium Exposure on Rice**

Cd stress seriously affects rice germination and growth [2,3,14–18], and it was found that excessive Cd exposure can not only significantly decrease the rice seed germination rate [14], but also cause chlorosis and necrosis in rice plants during the vegetative stage [19,20]. Cd stress causes severe physical and physiological changes in rice plants as it causes a reduction in the length; width; and number of roots, shoots, and leaves. Furthermore, chlorophyll contents, stomatal conductance, and the water use efficiency of rice are also significantly affected [3,17,18,21–23]. Cd also affects the absorption and transport of essential nutrients in rice [15,16,18–20]. Additionally, Cd can be transported to rice grains, reducing their yield, quality, and nutrients [15,16,24–27]. In general, Cd stress inhibits rice growth [18,28–30].

Rice possesses some tolerance mechanisms to cadmium at physiological and molecular levels [31–35]. As root cell walls of the outermost layer have direct contact with the soil solution, this protects the protoplasts against Cd toxicity [36–38]. Furthermore, plants reduce Cd translocation to the shoots by immobilizing Cd in the cell walls and vacuoles of root cells, thus reducing their sensitivity and the harm of Cd to another cellular organelle [39–42]. Several adenosine triphosphate (ATP)-binding cassette (ABC) proteins have been reported to mediate vacuolar compartmentation of Cd-glutathione and/or phytochelatin (PC) conjugates in *Arabidopsis thaliana* [43,44]. Rice *OsPDR5*/*ABCG43* is likely to encode ABC-type protein functions in Cd extrusion from the cytoplasm [45]. Overexpression of Cd transporter OsHMA3 located in vacuole membranes in rice roots can increase the tolerance of rice to Cd and reduce the accumulation of Cd in grains [46,47]. Exudates of roots contain metal chelators which play a role in the adjustment of the rhizosphere pH and the metal chelating process [48]. Most of the chelated toxic metals inside plants target vacuoles through metal detoxification processes [38,49]. Organic acids secreted from roots, e.g., malate, citrate, etc., are involved in metal uptake, the long-distance transport of metal, and the transport of metal into vacuoles [50,51]. It was found that chelators play a crucial role in keeping Cd in the rice roots and form a barrier in Cd translocation [52].

Cd stress can induce plants to enhance their antioxidant defense system and regulate ion homeostasis to improve their tolerance to Cd [32,53–58]. For example, Cd stress can induce plants to increase the production of glutathione (GSH), abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and nitric oxide (NO) [59–65]. Mitogen-activated protein kinase OsWJUMK1, OsMSRMK2, OsMSRMK3, and OsMAPK2 can affect rice root growth under Cd stress by regulating auxin signal changes [66–69]. Auxin transporter *OsAUX1* has been reported to be involved in root development and the Cd stress response in rice [34]. In addition, the concentration of iron and cadmium was positively correlated during rice seedling growth [70]. It has been reported that increasing the supply of boron, iron, zinc, silicon, or magnesium can reduce the accumulation and toxicity of cadmium in rice [71–76].

In addition, some genes related to Cd stress have been reported in rice (Table 1). *OsHMA9* is a copper efflux protein located in the plasma membrane, which may have a cadmium efflux function to excrete Cd from root cells and reduce Cd accumulation in rice [77]. Knock-out of the low cadmium gene (*LCD*) reduced the accumulation of cadmium and increased the growth of rice under the condition of an excessive cadmium supply, and LCD may be a protein related to cadmium homeostasis [78]. Overexpression of *OsCDT1* can increase the growth of *Arabidopsis thaliana* under cadmium treatment; the cysteine-rich peptide encoded by *OsCDT1* is possibly involved in rice Cd tolerance [79]. *OsCLT1* probably mediates the export of γ-glutamylcysteine and glutathione from plastids to the cytoplasm, which in turn affects As and Cd detoxification in rice [80].





### **3. Uptake and Transport Pathway of Cd in Rice**

Cadmium is transported from the roots to shoots and then to grains through four steps: (i) uptake by roots; (ii) transportation to shoots through loading to the xylem; (iii) distribution and transportation through nodes; and (iv) transportation to grains through the phloem from leaf blades (Figure 1).

**Figure 1.** A schematic of cadmium transport from the soil to grains in rice. Cadmium is absorbed from the soil by the roots, and *OsNramp1*, *OsNramp5*, and *OsCd1* mediate this process. *OsHMA3* plays a key role in cadmium segregation to vacuoles in root cells and thus negatively regulates cadmium xylem loading. *OsHMA2*, *OsCCX2*, and *CAL1* regulate cadmium transport to the xylem. *OsLCT1* contributes to cadmium remobilization from leaf blades via the phloem and is likely to play a part in intervascular cadmium transfer at nodes.

### *3.1. Functional Analysis of Related Genes*

Cd can enter rice plants through the uptake mechanism of essential elements such as Mn, Zn, and Fe, etc. [106,107,119]. Fe2<sup>+</sup> transporters *OsIRT1* and *OsIRT2* display Cd2<sup>+</sup> influx activity in yeast, which indicates that *OsIRT1* and *OsIRT2* may play a role in cadmium uptake in the root system [95,120]. Overexpression of *OsIRT1* significantly increased the accumulation of Cd in roots and shoots in Murashige & Skoog (MS) medium containing excess Cd, but no obvious phenotype was observed under field conditions, suggesting that *OsIRT1* may be involved in cadmium uptake in roots, but its contribution is largely affected by environmental conditions [96]. Oryza sativa Natural Resistance-Associated Macrophage Protein 5 (OsNramp5), located at the plasma membrane of root cells, was found to be the major transporter of Cd uptake in rice roots, responsible for the transport of Cd from the soil solution to the root cells [106,107]. OsNramp5 is also an Mn transporter, and the knock-out of *OsNramp5* can significantly reduce the uptake and accumulation of cadmium in grains, but also lead to the decrease of growth and yield due to manganese deficiency [107–109]. Recently, Liu et al. [121] located a major QTL, *qGMN7.1*, according to the Mn concentration in the grains of a recombinant inbred line (RILS) crossed between 93–11 (low grain Mn) and PA64s (high grain Mn). Fine mapping delimited *qGMN7.1* to a 49.3 kb region containing *OsNRAMP5*, and sequence variations in

the *OsNRAMP5* promoter caused changes in its transcript level and in grain Mn levels. Tang et al. [110] reported that a series of new indica rice lines with low cadmium accumulation were developed by knocking out the metal transporter *OsNramp5* using the CRISPR/CAS9 system. OsNRAMP1, located on the plasma membrane, also exhibits the activity of Cd transport, and participates in the uptake and transport of Cd in root cells [111,112]. OsZIP1, a zinc-regulated/iron-regulated transporter-like protein, expression in yeast can enhance its sensitivity to Cd [84], and the overexpression of *OsZIP6* can increase the Cd uptake in *X. laevis oocytes* [98].

After root absorption, xylem-mediated Cd translocation from the roots to shoots is the main factor determining the cadmium accumulation in shoots [122]. *OsHMA2* and *OsHMA3* were reported to play a role in this process [46,103,123,124]. *OsHMA2* participates in the transport of Cd from the roots to shoots and plays an important role in controlling the distribution of Cd through the phloem to developing tissues [103,104,123]. Compared with wild-type (WT) samples, the Cd concentration in the shoots of an *oshma2* mutant was significantly lower [104]. OsHMA3 plays a role in the vacuolar sequestration of Cd in root cells, the overexpression of *OsHMA3* reduces the Cd load in the xylem and Cd accumulation in shoots, and the functional deficiency of *OsHMA3* results in very high root-to-shoot Cd translocation in rice [46,47,105,125]. Recent reports showed that OsCCX2, a putative cation/calcium (Ca) exchanger, was localized in the plasma membrane and plays an important role in Cd transport by impacting Cd root-to-shoot translocation and the Cd distribution in the shoot tissues, and the knock-out of *OsCCX2* resulted in a significant Cd reduction in the grains [94]. Tan et al. [99] reported that *OsZIP7* plays a key role in xylem-loading in roots and inter-vascular transfer in nodes to deliver Zn and Cd upward in rice.

Nodes are the central organ of Cd transport from the xylem to phloem, and play an important role in Cd transport to grains [126–128]. OsLCT1 is a Cd-efflux transporter on the plasma-membrane involved in phloem Cd transport [101]. *OsLCT1* expression was higher in leaf blades and nodes during the reproductive stage, especially in node I. Compared with wild-type (WT), the Cd concentration in phloem exudates and in grains of *OsLCT1* RNAi plants decreased significantly, although the Cd concentration in xylem sap did not differ. These results suggest that *OsLCT1* in leaf blades functions in Cd remobilization by the phloem, and in node I, *OsLCT1* is likely to play a part in intervascular Cd transfer from enlarged large vascular bundles to diffused vascular bundles, which connect to the panicle [101,102]). The positions of cloned cadmium stress-related genes in rice chromosomes are shown in Figure 2.

**Figure 2.** Positions of cloned cadmium stress-related genes in rice chromosomes.

### *3.2. Location of Related QTLs*

Rice varieties show obvious genetic variation in terms of their cadmium accumulation ability, which is a valuable resource for dissecting functional alleles and genetic improvement [19,20,25]. However, only a few quantitative trait loci (QTLs) related to cadmium accumulation in rice have been reported. *OsHMA3*, *CAL1* (Cd Accumulation in Leaf 1), and *OsCd1* are the only Cd-related QTLs cloned so far. OsHMA3 encodes a cadmium transporter located in the vacuole membrane, which transports cadmium into vacuoles for sequestration [105]. Loss of OsHMA3 function significantly increased cadmium transport to rice shoots and grains [101,129]. On the other hand, the overexpression of *OsHMA3* can increase the tolerance of rice to Cd and reduce Cd accumulation in grains [46,105,119]. *CAL1* (cadmium accumulation in leaf 1) was identified and cloned by Luo et al. [88] as a quantitative trait locus (QTL) in rice, which explained 13% of the variation in leaf cadmium concentration in a doubled haploid population. *CAL1* regulates the root-to-shoot translocation of cadmium via the xylem vessels, and knockout mutants of *CAL1* significantly reduced the concentration of cadmium in rice leaves [88]. Yan et al. [90] discovered that the gene *OsCd1* belongs to the major facilitator superfamily through genome-wide association studies (GWAS), which was associated with divergence in rice grain Cd accumulation. Interestingly, the natural variation *OsCd1V449* in *Japonica*, which is associated with a reduced Cd transport ability and decreased grain Cd accumulation, shows a potential value in low-Cd rice selection [90].

A series of QTLs related to rice varieties that control the Cd concentration in rice have been reported (Table 2). Ishikawa et al. [130] obtained a mapping population consisting of 85 back-cross inbred lines (BIL) from hybridization between a low-cadmium-accumulation variety of *Japonica* rice (Sasanishiki) and a high-cadmium-accumulation variety of *Indica* rice (Habataki). Two QTLs were located on chromosomes 2 and 7, separately, with an increased cadmium concentration in grains. *qGCd7* plays an important role in increasing the cadmium concentration in grains, which can explain 35.5% of phenotypic variation [130]. Kashiwagi et al. [131] identified two QTLs, known as *qcd4–1* and *qcd4–2*, affecting the cadmium concentration in shoots. Sato et al. [132] reported two QTLs controlling the cadmium concentration in brown rice: *qLCdG11* explained 9.4%–12.9% of phenotypic variation and *qLCdG3* explained 8.3%–13.9% of phenotypic variation. Yan et al. [133] constructed an recombinant inbred lines (RIL) population of F7 to identify Cd accumulation and distribution. A total of five main effect QTLs (*scc10* was correlated with Cd accumulation in shoots; *gcc3, gcc9,* and *gcc11* with Cd accumulation in grains; and *sgr5* with the Cd distribution ratio in shoots and roots) were detected. Among them, *sgr5* had the greatest effect on the distribution of Cd in grains. Abe et al. [134] used a population consisting of 46 chromosome segment substitution lines (CSSL) to identify eight QTLs related to the grain cadmium content by single-label analysis using ANOVA. The result showed that *qlGCd3* had a high F-test value. A recombinant inbred population derived from Xiang 743/Katy was grown in Cd-polluted fields and used to map the QTLs for Cd accumulation in rice grains, and two QTLs, *qCd-2* and *qCd-7*, were identified in 2014 and 2015 [135]. Liu et al. [136] used 276 accessions with 416 K single nucleotide polymorphisms (SNPs) and performed a genome-wide association analysis of grain Cd concentrations in rice grown in heavily multi-contaminated farmlands, and 17 QTLs were found to be responsible for the grain Cd concentration.



#### *Int. J. Mol. Sci.* **2019** , *20*, 3417

### **4. Future Perspectives**

Cadmium is a kind of heavy metal that presents extreme biological toxicity. Cd accumulated in rice can enter the food chain, thereby threatening human health [5–7]. Cadmium in rice can be reduced by agronomic practices (including soil amendments, fertilizer management, water management, and tillage management) and bioremediation (including phytoremediation and microbial remediation) [18,145–150]. In addition, understanding the mechanism of cadmium translocation and the factors affecting cadmium accumulation in rice are also important for formulating effective strategies to reduce cadmium accumulation in rice. In recent years, some genes related to cadmium transport in rice have been studied, and significant progress has been made in understanding the mechanism of cadmium uptake and transport. In order to understand the mechanism of cadmium transport in rice, it is necessary to identify more unknown transporters or other molecules.

Biotechnology offers a promising approach to reducing the Cd content in rice grains. Mutations of the *OsNramp5* gene result in obvious decreases in Cd uptake in roots and Cd accumulation in rice grains [106,108,151]. Using the CRISPR/Cas9 gene editing technology to knock out *OsNramp5* in both parental lines, Tang et al. [110] generated a hybrid rice cultivar that accumulated very low levels of Cd in the grain. Another target for gene editing is *OsLCT1*, which is involved in the phloem transport of Cd from the vegetative tissues to the grains [101]. Knockdown of this gene by RNAi reduced the grain Cd concentration by 30%–50% [101]. Overexpression of functional *OsHMA3* in *Nipponbare* decreased Cd translocation and Cd accumulation in rice grains [46,105]. Overexpression of *OsHMA3* is a highly effective method for reducing Cd accumulation in *Indica* rice, and rice grains produced using this approach are almost Cd-free, with little effect on the grain yield or essential micronutrient concentrations [152].

However, commercial transgenic rice is not commonly accepted by the general public and prohibited in many countries. Ishikawa et al. [151] produced three rice mutants by carbon ion-beam irradiation, where cadmium was hardly detected in mutant seeds when planted in cadmium-contaminated paddy fields and there was no significant difference between the mutant and wild-type (WT) in agronomic traits, which could be directly applied to breeding projects. Another possible strategy is marker-assisted breeding, which uses molecular markers to track the genetic composition of rice and bred rice varieties. For example, identifying a low-cadmium-related QTL and then introducing it into high-cadmium cultivars might be a viable approach [122]. However, only a few of QTLs related to cadmium accumulation in rice have been cloned [90,105], and the natural allele variation of grain cadmium accumulation differences among rice varieties has not been fully explored. Further research is necessary to clone more QTLs for controlling grain Cd accumulation, thus providing tools for the marker-assisted molecular breeding of rice cultivars with a low accumulation of Cd in grains.

**Author Contributions:** J.C. and W.Z. wrote the first draft of the manuscript and organized the tables and figures; L.M and X.F. conceived and supervised their ideas; G.X. and G.Y. reviewed the manuscript. All authors read and approved the final manuscript.

**Acknowledgments:** This research was financially supported by the National Natural Science Foundation of China (No. 31601286 to L.M.); the CAAS Innovative Team Award to GY's team, Shenzhen Science and Technology Projects (No. JSGG20160608160725473 to Q.Q.); the China Postdoctoral Science Foundation (No. 2016M590160 to L.M.); the Fundamental Research Funds for Central Non-profit Scientific Institution (to G.Y.); the Jiangsu Science Fund for Distinguished Young Scholars (Grant BK20160030 to X.F.); and Dapeng district industry development special funds (KY20180218).

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

### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

International Journal of *Molecular Sciences*
