Heavy Metals Accumulation, Toxicity and Detoxification in Plants Printed Edition of the Special Issue Published in International Journal of Molecular Sciences www.mdpi.com/journal/ijms Luigi De Bellis and Alessio Aprile Edited by Heavy Metals Accumulation, Toxicity and Detoxification in Plants Heavy Metals Accumulation, Toxicity and Detoxification in Plants Editors Luigi De Bellis Alessio Aprile MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editors Luigi De Bellis University of Salento Italy Alessio Aprile University of Salento Italy Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal International Journal of Molecular Sciences (ISSN 1422-0067) (available at: https://www.mdpi.com/ journal/ijms/special issues/plant heavy metals). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Article Number , Page Range. ISBN 978-3-03936-633-0 ( H bk) ISBN 978-3-03943-478-7 (PDF) Cover image courtesy of Alessio Aprile. c © 2020 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. Contents About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Alessio Aprile and Luigi De Bellis Editorial for Special Issue “Heavy Metals Accumulation, Toxicity, and Detoxification in Plants” Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 4103, doi:10.3390/ijms21114103 . . . . . . . . . . . . . . 1 Giovanni DalCorso, Elisa Fasani, Anna Manara, Giovanna Visioli and Antonella Furini Heavy Metal Pollutions: State of the Art and Innovation in Phytoremediation Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 3412, doi:10.3390/ijms20143412 . . . . . . . . . . . . . . 7 Yanchun Cui, Manling Wang, Xuming Yin, Guoyun Xu, Shufeng Song, Mingjuan Li, Kai Liu and Xinjie Xia OsMSR3, a Small Heat Shock Protein, Confers Enhanced Tolerance to Copper Stress in Arabidopsis thaliana Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 6096, doi:10.3390/ijms20236096 . . . . . . . . . . . . . . 25 Michiel Huybrechts, Ann Cuypers, Jana Deckers, Verena Iven, St ́ ephanie Vandionant, Marijke Jozefczak and Sophie Hendrix Cadmium and Plant Development: An Agony from Seed to Seed Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 3971, doi:10.3390/ijms20163971 . . . . . . . . . . . . . . 39 Jingguang Chen, Wenli Zou, Lijun Meng, Xiaorong Fan, Guohua Xu and Guoyou Ye Advances in the Uptake and Transport Mechanisms and QTLs Mapping of Cadmium in Rice Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 3417, doi:10.3390/ijms20143417 . . . . . . . . . . . . . . 69 Eugeniusz Małkowski, Krzysztof Sitko, Michał Szopi ́ nski, ̇ Zaneta Giero ́ n, Marta Pogrzeba, Hazem M. Kalaji and Paulina Ziele ́ znik-Rusinowska Hormesis in Plants: The Role of Oxidative Stress, Auxins and Photosynthesis in Corn Treated with Cd or Pb Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 2099, doi:10.3390/ijms21062099 . . . . . . . . . . . . . . 87 Zhaoyang Hu, Yufei Zhang, Yue He, Qingqing Cao, Ting Zhang, Laiqing Lou and Qingsheng Cai Full-Length Transcriptome Assembly of Italian Ryegrass Root Integrated with RNA-Seq to Identify Genes in Response to Plant Cadmium Stress Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 1067, doi:10.3390/ijms21031067 . . . . . . . . . . . . . . 109 Yaping Sun, Qianyun Lu, Yushen Cao, Menghua Wang, Xiyu Cheng and Qiong Yan Comparative Transcriptome Analysis of the Molecular Mechanism of the Hairy Roots of Brassica campestris L. in Response to Cadmium Stress Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 180, doi:10.3390/ijms21010180 . . . . . . . . . . . . . . 129 Antonio Z ́ u ̃ niga, Daniel Laporte, Alberto Gonz ́ alez, Melissa G ́ omez, Claudio A. S ́ aez and Alejandra Moenne Isolation and Characterization of Copper- and Zinc- Binding Metallothioneins from the Marine Alga Ulva compressa (Chlorophyta) Reprinted from: Int. J. Mol. Sci. 2020 , 21 , 153, doi:10.3390/ijms21010153 . . . . . . . . . . . . . . . 145 Jifu Li, Yidan Jia, Rongshu Dong, Rui Huang, Pandao Liu, Xinyong Li, Zhiyong Wang, Guodao Liu and Zhijian Chen Advances in the Mechanisms of Plant Tolerance to Manganese Toxicity Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 5096, doi:10.3390/ijms20205096 . . . . . . . . . . . . . . 161 v Alessio Aprile, Erika Sabella, Enrico Francia, Justyna Milc, Domenico Ronga, Nicola Pecchioni, Erika Ferrari, Andrea Luvisi, Marzia Vergine and Luigi De Bellis Combined Effect of Cadmium and Lead on Durum Wheat Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 5891, doi:10.3390/ijms20235891 . . . . . . . . . . . . . . 177 Sarfraz Shafiq, Qudsia Zeb, Asim Ali, Yasar Sajjad, Rashid Nazir, Emilie Widemann and Liangyu Liu Lead, Cadmium and Zinc Phytotoxicity Alter DNA Methylation Levels to Confer Heavy Metal Tolerance in Wheat Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 4676, doi:10.3390/ijms20194676 . . . . . . . . . . . . . . 195 Paula S. M. Celis-Pl ́ a, Fernanda Rodr ́ ıguez-Rojas, Lorena M ́ endez, Fabiola Moenne, Pamela T. Mu ̃ noz, M. Gabriela Lobos, Patricia D ́ ıaz, Jos ́ e Luis S ́ anchez-Lizaso, Murray T. Brown, Alejandra Moenne and Claudio A. S ́ aez MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Influence on Metal Exclusion/Extrusion Mechanisms and Photosynthesis Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 4547, doi:10.3390/ijms20184547 . . . . . . . . . . . . . . 213 Fernanda Rodr ́ ıguez-Rojas, Paula S. M. Celis-Pl ́ a, Lorena M ́ endez, Fabiola Moenne, Pamela T. Mu ̃ noz, M. Gabriela Lobos, Patricia D ́ ıaz, Jos ́ e Luis S ́ anchez-Lizaso, Murray T. Brown, Alejandra Moenne and Claudio A. S ́ aez MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Involvement in the Regulation of Detoxification Mechanisms Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 4546, doi:10.3390/ijms20184546 . . . . . . . . . . . . . . 229 Arleta Małecka, Agnieszka Konkolewska, Anetta Han ́ c, Danuta Barałkiewicz, Liliana Ciszewska, Ewelina Ratajczak, Aleksandra Maria Staszak, Hanna Kmita and Wiesława Jarmuszkiewicz Insight into the Phytoremediation Capability of Brassica juncea (v. Malopolska): Metal Accumulation and Antioxidant Enzyme Activity Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 4355, doi:10.3390/ijms20184355 . . . . . . . . . . . . . . 249 Yong Luo, Yuewei Wei, Shuguang Sun, Jian Wang, Weifeng Wang, Dan Han, Huifang Shao, Hongfang Jia and Yunpeng Fu Selenium Modulates the Level of Auxin to Alleviate the Toxicity of Cadmium in Tobacco Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 3772, doi:10.3390/ijms20153772 . . . . . . . . . . . . . . 267 Huihong Wang, Yuanyuan Liu, Zaihui Peng, Jianchun Li, Weipeng Huang, Yan Liu, Xuening Wang, Shengli Xie, Liping Sun, Erqin Han, Nengbiao Wu, Keming Luo and Bangjun Wang Ectopic Expression of Poplar ABC Transporter PtoABCG36 Confers Cd Tolerance in Arabidopsis thaliana Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 3293, doi:10.3390/ijms20133293 . . . . . . . . . . . . . . 281 Haoyue Shu, Jun Zhang, Fuye Liu, Chao Bian, Jieliang Liang, Jiaqi Liang, Weihe Liang, Zhiliang Lin, Wensheng Shu, Jintian Li, Qiong Shi and Bin Liao Comparative Transcriptomic Studies on a Cadmium Hyperaccumulator Viola baoshanensis and Its Non-Tolerant Counterpart V. inconspicua Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 1906, doi:10.3390/ijms20081906 . . . . . . . . . . . . . . 295 Xiaoyan He, Marvin E.A. Richmond, Darron V. Williams, Weite Zheng and Feibo Wu Exogenous Glycinebetaine Reduces Cadmium Uptake and Mitigates Cadmium Toxicity in Two Tobacco Genotypes Differing in Cadmium Tolerance Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 1612, doi:10.3390/ijms20071612 . . . . . . . . . . . . . . 315 vi Mingge Han, Xuke Lu, John Yu, Xiugui Chen, Xiaoge Wang, Waqar Afzal Malik, Junjuan Wang, Delong Wang, Shuai Wang, Lixue Guo, Chao Chen, Ruifeng Cui, Xiaoming Yang and Wuwei Ye Transcriptome Analysis Reveals Cotton ( Gossypium hirsutum ) Genes That Are Differentially Expressed in Cadmium Stress Tolerance Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 1479, doi:10.3390/ijms20061479 . . . . . . . . . . . . . . 331 vii 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 2 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. ix International Journal of Molecular Sciences 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 3 [ 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 e ff ects 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. Int. J. Mol. Sci. 2020 , 21 , 4103; doi:10.3390 / ijms21114103 www.mdpi.com / journal / ijms 1 Int. J. Mol. Sci. 2020 , 21 , 4103 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 e ff ects 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 e ff ect 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]. 2 Int. J. Mol. Sci. 2020 , 21 , 4103 Table 1. Contributors to the special issue “Heavy Metals Accumulation, Toxicity, and Detoxification in Plants”. ABC: ATP-binding cassette. Authors Title Heavy Metals Type Małkowski et al. [7] Hormesis in Plants: The Role of Oxidative Stress, Auxins and Photosynthesis in Corn Treated with Cd or Pb Cadmium Lead Original Research Hu et al. [8] Full-Length Transcriptome Assembly of Italian Ryegrass Root Integrated with RNA-Seq to Identify Genes in Response to Plant Cadmium Stress Cadmium Original Research Sun et al. [9] Comparative Transcriptome Analysis of the Molecular Mechanism of the Hairy Roots of Brassica campestris L. in Response to Cadmium Stress Cadmium Original Research Z ú ñiga et al. [10] Isolation and Characterization of Copper- and Zinc-Binding Metallothioneins from the Marine Alga Ulva compressa (Chlorophyta) Copper, Zinc Original Research Cui et al. [11] OsMSR3, a Small Heat Shock Protein, Confers Enhanced Tolerance to Copper Stress in Arabidopsis thaliana Copper Original Research Aprile et al. [12] Combined E ff ect of Cadmium and Lead on Durum Wheat Cadmium, Lead Original Research Shafiq et al. [13] Lead, Cadmium and Zinc Phytotoxicity Alter DNA Methylation Levels to Confer Heavy Metal Tolerance in Wheat Cadmium, Lead, Zinc Original Research Celis-Pl á et al. [14] MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Influence on Metal Exclusion / Extrusion Mechanisms and Photosynthesis Copper Original Research Rodr í guez-Rojas et al. [15] MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Involvement in the Regulation of Detoxification Mechanisms Copper Original Research Małecka et al. [16] Insight into the Phytoremediation Capability of Brassica juncea (v. Malopolska): Metal Accumulation and Antioxidant Enzyme Activity Cadmium, Copper, Lead, Zinc Original Research Luo et al. [17] Selenium Modulates the Level of Auxin to Alleviate the Toxicity of Cadmium in Tobacco Cadmium Original Research Wang et al. [18] Ectopic Expression of Poplar ABC Transporter PtoABCG36 Confers Cd Tolerance in Arabidopsis thaliana Cadmium Original Research Shu et al. [19] Comparative Transcriptomic Studies on a Cadmium Hyperaccumulator Viola baoshanensis and Its Non-Tolerant Counterpart V. inconspicua Cadmium Original Research He et al. [20] Exogenous Glycinebetaine Reduces Cadmium Uptake and Mitigates Cadmium Toxicity in Two Tobacco Genotypes Di ff ering in Cadmium Tolerance Cadmium Original Research Han et al. [21] Transcriptome Analysis Reveals Cotton ( Gossypium hirsutum ) Genes That Are Di ff erentially Expressed in Cadmium Stress Tolerance Cadmium Original Research Li et al. [4] Advances in the Mechanisms of Plant Tolerance to Manganese Toxicity Manganese Review Huybrechts et al. [5] Cadmium and Plant Development: An Agony from Seed to Seed Cadmium Review Chen et al. [6] Advances in the Uptake and Transport Mechanisms and QTLs Mapping of Cadmium in Rice Cadmium Review Dal Corso et al. [3] Heavy Metal Pollutions: State of the Art and Innovation in Phytoremediation All Review 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 di ff erent 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 3 Int. J. Mol. Sci. 2020 , 21 , 4103 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 di ff erent 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 e ffi ciently 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 QTLs Quantitative trait loci sHSP small heat shock protein GST glutathione s -transferase HMA heavy metal ATPase ABC ATP-binding cassette References 1. Hawkes, J.S. Heavy metals. J. Chem. Educ. 1997 , 74 , 1369–1374. [CrossRef] 2. European Environment Agency (EEA). Environmental Pressures of Heavy Metal Releases from Europe’s Industry. Available online: https: // www.eea.europa.eu / themes / industry / industrial-pollution-in-europe (accessed on 27 May 2020). 3. DalCorso, G.; Fasani, E.; Manara, A.; Visioli, G.; Furini, A. Heavy Metal Pollutions: State of the Art and Innovation in Phytoremediation. Int. J. Mol. Sci. 2019 , 20 , 3412. [CrossRef] [PubMed] 4. Li, J.; Jia, Y.; Dong, R.; Huang, R.; Liu, P.; Li, X.; Wang, Z.; Liu, G.; Chen, Z. Advances in the Mechanisms of Plant Tolerance to Manganese Toxicity. Int. J. Mol. Sci. 2019 , 20 , 5096. [CrossRef] [PubMed] 5. Huybrechts, M.; Cuypers, A.; Deckers, J.; Iven, V.; Vandionant, S.; Jozefczak, M.; Hendrix, S. Cadmium and Plant Development: An Agony from Seed to Seed. Int. J. Mol. Sci. 2019 , 20 , 3971. [CrossRef] [PubMed] 6. Chen, J.; Zou, W.; Meng, L.; Fan, X.; Xu, G.; Ye, G. Advances in the Uptake and Transport Mechanisms and QTLs Mapping of Cadmium in Rice. Int. J. Mol. Sci. 2019 , 20 , 3417. [CrossRef] [PubMed] 7. Małkowski, E.; Sitko, K.; Szopi ́ nski, M.; Giero ́ n, ̇ Z.; Pogrzeba, M.; Kalaji, H.M.; Ziele ́ znik-Rusinowska, P. Hormesis in Plants: The Role of Oxidative Stress, Auxins and Photosynthesis in Corn Treated with Cd or Pb. Int. J. Mol. Sci. 2020 , 21 , 2099. [CrossRef] [PubMed] 8. Hu, Z.; Zhang, Y.; He, Y.; Cao, Q.; Zhang, T.; Lou, L.; Cai, Q. Full-Length Transcriptome Assembly of Italian Ryegrass Root Integrated with RNA-Seq to Identify Genes in Response to Plant Cadmium Stress. Int. J. Mol. Sci. 2020 , 21 , 1067. [CrossRef] [PubMed] 9. Sun, Y.; Lu, Q.; Cao, Y.; Wang, M.; Cheng, X.; Yan, Q. Comparative Transcriptome Analysis of the Molecular Mechanism of the Hairy Roots of Brassica campestris L. in Response to Cadmium Stress. Int. J. Mol. Sci. 2020 , 21 , 180. [CrossRef] [PubMed] 10. Z ú ñiga, A.; Laporte, D.; Gonz á lez, A.; G ó mez, M.; S á ez, C.A.; Moenne, A. Isolation and Characterization of Copper- and Zinc- Binding Metallothioneins from the Marine Alga Ulva compressa (Chlorophyta). Int. J. Mol. Sci. 2020 , 21 , 153. [CrossRef] [PubMed] 11. Cui, Y.; Wang, M.; Yin, X.; Xu, G.; Song, S.; Li, M.; Liu, K.; Xia, X. OsMSR3, a Small Heat Shock Protein, Confers Enhanced Tolerance to Copper Stress in Arabidopsis thaliana Int. J. Mol. Sci. 2019 , 20 , 6096. [CrossRef] [PubMed] 4 Int. J. Mol. Sci. 2020 , 21 , 4103 12. Aprile, A.; Sabella, E.; Francia, E.; Milc, J.; Ronga, D.; Pecchioni, N.; Ferrari, E.; Luvisi, A.; Vergine, M.; De Bellis, L. Combined E ff ect of Cadmium and Lead on Durum Wheat. Int. J. Mol. Sci. 2019 , 20 , 5891. [CrossRef] [PubMed] 13. Shafiq, S.; Zeb, Q.; Ali, A.; Sajjad, Y.; Nazir, R.; Widemann, E.; Liu, L. Lead, Cadmium and Zinc Phytotoxicity Alter DNA Methylation Levels to Confer Heavy Metal Tolerance in Wheat. Int. J. Mol. Sci. 2019 , 20 , 4676. [CrossRef] [PubMed] 14. Celis-Pl á , P.S.M.; Rodr í guez-Rojas, F.; M é ndez, L.; Moenne, F.; Muñoz, P.T.; Lobos, M.G.; D í az, P.; S á nchez-Lizaso, J.L.; Brown, M.T.; Moenne, A.; et al. MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Influence on Metal Exclusion / Extrusion Mechanisms and Photosynthesis. Int. J. Mol. Sci. 2019 , 20 , 4547. [CrossRef] 15. Rodr í guez-Rojas, F.; Celis-Pl á , P.S.M.; M é ndez, L.; Moenne, F.; Muñoz, P.T.; Lobos, M.G.; D í az, P.; S á nchez-Lizaso, J.L.; Brown, M.T.; Moenne, A.; et al. MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Involvement in the Regulation of Detoxification Mechanisms. Int. J. Mol. Sci. 2019 , 20 , 4546. [CrossRef] 16. Małecka, A.; Konkolewska, A.; Han ́ c, A.; Barałkiewicz, D.; Ciszewska, L.; Ratajczak, E.; Staszak, A.M.; Kmita, H.; Jarmuszkiewicz, W. Insight into the Phytoremediation Capability of Brassica juncea (v. Malopolska): Metal Accumulation and Antioxidant Enzyme Activity. Int. J. Mol. Sci. 2019 , 20 , 4355. [CrossRef] 17. Luo, Y.; Wei, Y.; Sun, S.; Wang, J.; Wang, W.; Han, D.; Shao, H.; Jia, H.; Fu, Y. Selenium Modulates the Level of Auxin to Alleviate the Toxicity of Cadmium in Tobacco. Int. J. Mol. Sci. 2019 , 20 , 3772. [CrossRef] [PubMed] 18. Wang, H.; Liu, Y.; Peng, Z.; Li, J.; Huang, W.; Liu, Y.; Wang, X.; Xie, S.; Sun, L.; Han, E.; et al. Ectopic Expression of Poplar ABC Transporter PtoABCG36 Confers Cd Tolerance in Arabidopsis thaliana Int. J. Mol. Sci. 2019 , 20 , 3293. [CrossRef] [PubMed] 19. Shu, H.; Zhang, J.; Liu, F.; Bian, C.; Liang, J.; Liang, J.; Liang, W.; Lin, Z.; Shu, W.; Li, J.; et al. Comparative Transcriptomic Studies on a Cadmium Hyperaccumulator Viola baoshanensis and Its Non-Tolerant Counterpart V. inconspicua Int. J. Mol. Sci. 2019 , 20 , 1906. [CrossRef] [PubMed] 20. He, X.; Richmond, M.E.; Williams, D.V.; Zheng, W.; Wu, F. 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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 / ). 5 International Journal of Molecular Sciences Review Heavy Metal Pollutions: State of the Art and Innovation in Phytoremediation Giovanni DalCorso 1, *, Elisa Fasani 1 , Anna Manara 1 , Giovanna Visioli 2 and Antonella Furini 1, * 1 Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy 2 Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area delle Scienze, 11 / A, 43124 Parma, Italy * Correspondence: giovanni.dalcorso@univr.it (G.D.); antonella.furini@univr.it (A.F.); Tel.: + 0039-045-802-7950 (G.D. & A.F.) 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 e ffi cient 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 di ff erent organs, cell types, and tissues through tight homeostasis mechanisms to ensure metal requirement, storage, and re-mobilization under di ff erent 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 Int. J. Mol. Sci. 2019 , 20 , 3412; doi:10.3390 / ijms20143412 www.mdpi.com / journal / ijms 7 Int. J. Mol. Sci. 2019 , 20 , 3412 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 e ff ects of non-essential heavy metals, while maintaining the uptake of essential elements, plants have evolved a homeostatic network that controls metal uptake, tra ffi cking, storage, and detoxification. Although a basal metal tolerance is usual, to guarantee the correct concentration of essential metal nutrients in di ff erent cell types at di ff erent 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 su ffi cient 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 di ff erent cell types and delivery inside cells. Membrane proteins are able to transport di ff erent metals across cellular membranes, playing a pivotal role in each influx-e ffl ux step of the translocation from roots to shoots. The function of several transporters involved in import, tra ffi cking, 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-a ffi nity 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 e ffl ux 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 di ff usion 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 2 + / H + antiporter involved in Zn tolerance, which in case of Zn excess accumulates Zn into the vacuole [16]. In addition to metal tra ffi cking, 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 di ff erent heavy metals although Cd seems to be the most e ff ective stimulator [ 20 ]. 8 Int. J. Mol. Sci. 2019 , 20 , 3412 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 insu ffi cient 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 su ff er from two main disadvantages, being expensive and frequently ine ffi cient 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, phyt