**1. Introduction**

Heavy metal contamination in soils has become a major threat to global agriculture due to both direct toxicity to crops and the subsequent impacts on human health [1–3].

**Citation:** Ma, C.; Hao, Y.; Zhao, J.; Zuverza-Mena, N.; Meselhy, A.G.; Dhankher, O.P.; Rui, Y.; White, J.C.; Xing, B. Graphitic Carbon Nitride (C3N4) Reduces Cadmium and Arsenic Phytotoxicity and Accumulation in Rice (*Oryza sativa* L.). *Nanomaterials* **2021**, *11*, 839. https:// doi.org/10.3390/nano11040839

Academic Editor: Eleonore Fröhlich

Received: 28 February 2021 Accepted: 22 March 2021 Published: 25 March 2021

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Heavy metals in agricultural soils can be derived from both geogenic (soils) and anthropogenic (mining, smelting, solid waste, etc.) sources [4–6]. Cadmium (Cd) and arsenic (As) are two heavy metals that are commonly found in soils [7]. Due to its elemental properties, Cd can replace Ca and cause chronic Cd poisoning, called itai-itai disease [8]. Inorganic As has been classified as a human carcinogen by the United States Environmental Protection Agency (USEPA) [9] and can also cause a series of human diseases [10]. In addition, heavy metals have caused ecotoxicological effects on soil organisms (microorganisms, plants, and animals) due to their toxicity, bioaccumulation, and persistence in environments [11–14]. According to Tóth et al., the average topsoil concentrations of Cd and As in the European Union were 0.09 ± 0.11 and 3.72 ± 2.92 mg/kg, respectively [15]. Similarly, Cd and As concentrations in soils in the United States were 0.2–2 and 0.4–40 mg/kg [16,17]. Heavy metal-induced phytotoxicity to crops has been extensively investigated, with detailed studies addressing metal speciation and accumulation, bioavailability, physiological responses, crop yield, and quality, as well as plant defense mechanisms [18–22]. In addition, efforts have been made to stabilize heavy metal contaminants in agricultural soils using different types of amendments (minerals, organic matters, biofertilizers, rhizosphere microbial community), which have subsequently reduced metal accumulation in crops and risk of human exposure [23–25]. Thus, it is important to not only explore novel, sustainable, and efficient strategies to reduce heavy metal uptake but also reveal the underlying interaction and uptake mechanisms to maximize benefits.

Nano-enabled techniques have been widely used in agriculture for the purposes of monitoring plant health, enhancing crop yield, and suppressing abiotic and biotic stresses [26–29]. A number of recent studies have demonstrated positive impacts of both metal- and carbon-based nanomaterials on alleviating contaminant-induced abiotic stress and toxicity. For example, Ma et al. (2020) reported that zinc oxide (ZnO) nanoparticles (NPs) could significantly reduce the Cd and As accumulation in rice tissues when grown in metal co-contaminated rice paddies, including reduced grain contamination [7]. Similar findings of ZnO NPs alleviating heavy metal toxicity to *Leucaena leucocephala* seedlings were also reported [30]. Other metal-based NPs, such as TiO2 [31,32] and CuO [33], also exhibited positive impacts by alleviating heavy metal phytotoxicity and enhancing crop growth. With regard to carbon-based nanomaterials, most of the studies have been conducted to facilitate understanding of the interactions between nanomaterials and contaminants [34–37]. Only a small number of studies have evaluated the impacts of carbon-based nanomaterials on alleviating contaminant-induced toxicity to crops. For example, nanoscale biochar reduced Cd accumulation in rice and subsequently ameliorated Cd-induced phytotoxicity as measured by plant growth, pigment production, and lipid peroxidation [38]. In addition, Jia et al. (2020) reported that magnetic carbon nanotubes altered phenanthrene and associated metabolite accumulation in lettuce, suggesting this approach as a novel strategy for soil remediation [39]. Additional investigations exploring the potential of sustainable carbon-based nanomaterials to reduce heavy metal accumulation and phytotoxicity to crops are needed.

Graphitic carbon nitride nanosheets (C3N4) have attracted attention in recent years due to their unique structure and excellent catalytic properties. Containing only carbon and nitrogen, C3N4 can be easily synthesized using low-cost nitrogen-enriched compounds such as urea and melamine under heat condensation [40–42]. Xiao et al. (2019) reported the superior adsorption performance of C3N4 for heavy metal removal from wastewater; the maximum adsorption capacities of Cd, lead (Pb), and chromium (Cr) were approximately 123, 37, and 684 mg/g, respectively [43]. Similar results were demonstrated for C3N4 quantum dots (QD) removal of mercury chloride (HgCl2), with a binding efficiency of 24.63 mg HgCl2/10 mg C3N4 [44]. However, biotic and in vivo experiments investigating C3N4 potential for reducing heavy metal accumulation in crops are very limited. Hao et al. (2021) reported that C3N4 not only significantly reduced the Cd content of rice tissues but also increased the nitrogen content to offset the Cd-induced nitrogen deficiency [45]. However, a mechanistic understanding of C3N4 regulation of heavy metal transporters at the molecular level remains elusive.

Rice, a semiaquatic annual grass species, is the most important cereal crop in developing countries and the most consumed staple food all over the world [46]. In the present study, rice (*Oryza sativa* L.) seedlings were hydroponically exposed to C3N4 and Cd- or As-amended nutrient solutions under greenhouse conditions for 14 days. At harvest, physiological parameters and elemental content of rice tissues were measured across all treatments. In addition, the relative expression of Cd- and As-related transporters was analyzed as affected by C3N4 and both Cd and As. The findings provide important information on the role of C3N4 in reducing Cd and As bioavailability and subsequent phytotoxicity to crops. More importantly, the work further demonstrates the use of sustainable nano-enabled techniques as a novel strategy for soil remediation to ensure a safe food supply.
