3.5.1. RAPD Analysis upon Exposure to C3N4 and Heavy Metals

RAPD was employed to assess the potential of both C3N4 and heavy metals to induce DNA damages in rice. In shoot tissues, the amplicon sizes of all bands were between 500–1500 bp, and there were no significant changes in the total number of DNA bands across all C3N4 and heavy metal treatments relative to the control. In root tissues, the presence of Cd significantly altered the total number of DNA bands. Two additional DNA bands (>1500 bp) were evident in the Cd alone treatment, and one band (>1500 bp) was found in the cotreatment with Cd and C3N4. Conversely, the total number of DNA bands was unchanged upon As treatment (Figure S3), indicating that Cd had more negative impact on rice DNA than As. C3N4 had no significant impact on the total number of DNA bands in rice tissues. These findings align with those of Venkatachalam et al., who reported that 50 mg/L Cd caused an additional band at 1100 bp in exposed *Leucaena leucocephala* seedlings; conversely, ZnO NPs alone had no impact relative to the control [30]. Mosa et al. (2018) used three different primers (OPA7, OPA8, and OPA9) in cucumber to demonstrate that copper NPs induced additional bands as compared with the corresponding control, suggesting that copper NPs can also cause genomic alteration [63].

### 3.5.2. Relative Expression of Cd and As Transporters

In order to explore the underlying mechanisms by which C3N4 altered heavy metal accumulation in rice, the relative expressions of Cd- and As-related transporters in root and shoot tissues were evaluated (Figure 6, Figure 7, Figure S4 and S5). The relative expression of rice iron-regulated transporter 1, *OsIRT1,* in roots in the Cd alone treatment was upregulated by approximately threefold of the control; however, the addition of C3N4 reduced this expression by 25% (Figure 6A). No difference was noted in the expression of the other *IRT* gene (*OsIRT2*) in the roots with Cd alone or with coexposure to C3N4 (Figure 6B). However, the addition of C3N4 significantly reduced the Cd-related transporter expression of rice heavy metal P-type ATPases (*OsHMA2* and *OsHMA3*) and natural resistance-associated macrophage protein 5 (*OsNramp5*) in metal-treated roots (Figure 6C,D,F). The expression of *OsNramp1* was rather insensitive to Cd exposure, with an expression increase less than 50% of the control. Although C3N4 slightly increased the *OsNramp1* expression, the increase was also less than 50% as compared with the control and the Cd alone treatment (Figure 6E). In the shoots, the relative expression of Cd-related transporters was not significantly upregulated upon exposure relative to the control (Figure S4A–E), with the exception being *OsNramp5*, whose level was approximately 50% higher than that of the control (Figure S4F).

**Figure 6.** The relative expression of Cd transport-associated genes in rice roots upon exposure to Cd with or without the addition of C3N4. (**A**,**B**) represent the relative expression of Fe-regulated transporters *IRT1* and *IRT2*, respectively, in roots. (**C**,**D**) represent the relative expression of heavy metal ATPases *HMA2* and *HMA3*, which mediate the Cd loading and translocation from roots to shoots, in roots. (**E**,**F**) show the relative expression of the natural resistance-associated macrophage proteins *Nramp1* and *Nramp5*, respectively, in rice roots affected by As and C3N4. Single asterisk "\*" indicates the significant difference between control and each treatment at *p* < 0.05; double asterisks "\*\*" indicate the significant difference between control and each treatment at *p* < 0.01 using a Student's *t*-test.

With regard to As-related transporters (both arsenite and arsenate), exposure to As induced upregulation of rice nodulin 26-like intrinsic proteins, *OsNIP1;1*, and phosphate transporter, *OsPT4*, in the roots relative to the control (Figure 7D,E). Either the expression of the remaining genes was increased by less than 50% of the control, or no change was evident upon exposure to As and C3N4 (Figure 7A–C). Downregulation of the *OsPT8* expression was evident in both the As alone and cotreatment with C3N4 (Figure 7F). Similarly, in shoots the regulation of As-related transporters in the As treatments was similar to the control (<50% change) (Figure S5). However, the addition of C3N4 downregulated the expression of low silica transporters, *Lsi1* and *Lsi2*, and *OsPT4* in shoot tissues (Figure S5A,B,E).

**Figure 7.** The relative expression of As transport-associated genes in rice roots upon exposure to As with or without the addition of C3N4. (**A**–**C**) represent the relative expression of the Si transport-related genes (*Lsi1*, *2*, and *6*), which have a demonstrated association with arsenite transport in roots. (**D**) shows the relative expression of nodulin 26-like intrinsic proteins (*NIPs1;1*) associated with arsenite uptake in roots. (**E**,**F)** show the relative expression of the Pht1 family genes, *OsPT1* and *OsPT8*, involving arsenate uptake, respectively, in rice roots as affected by As and C3N4. Single asterisk "\*" indicates the significant difference between control and each treatment at *p* < 0.05; double asterisks "\*\*" indicate the significant difference between control and each treatment at *p* < 0.01 using a Student's *t*-test.

The expression and function of genes involved in Cd and As transport in rice have been extensively studied. In the present work, exposure to Cd upregulated both *OsIRTs* in rice, which is consistent with the amounts of Fe and Cd detected in rice tissues. Similar results were reported by Jiang et al. (2020), who demonstrated that Cd transporter-related genes were elevated in rice upon exposure to Cd, while the presence of glutamate lowered their expression and consequently reduced the Cd uptake [52]. Ma et al. (2016) also demonstrated that the expression of Fe-related transporters in *Arabidopsis* was downregulated upon CeO2 NP treatment, which could explain the reduced Fe content as compared with the control [49]. Regarding As, aquaporin-related genes in wheat and tomato were notably upregulated upon exposure to graphene and As; additionally, coexposure to these two analytes could result in relatively higher expression of these genes relative to the single analyte treatments [64].

### **4. Conclusions**

In summary, C3N4 significantly alleviated Cd- and As-induced phytotoxicity to rice without exerting any additional or unique negative impact on plant growth as determined by phenotype and biomass. In addition, C3N4 modulated the expression of Cd and As transporter genes and subsequently reduced contaminant accumulation or bioavailability, offering one of the mechanistic insights into the observed effects. Further investigation evaluating grain yield and quality in rice coexposed to heavy metals and C3N4 is warranted. Overall, the present work demonstrates that C3N4 nanosheets are able to alleviate the phytotoxicity and reduce the accumulation of Cd and As in rice. Therefore, the use of C3N4 is a promising material to be studied as a sustainable and safe nano-enabled strategy for reducing heavy metal accumulation in important food crops grown in contaminated soils.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2079-499 1/11//839/s1, Figure S1: Phenotypic image of rice treated with 50 and 250 mg/L C3N4 for 14 days; Figure S2: The content of macronutrients (Mg and K) in rice roots and shoots upon exposure to

As, Cd with or without the addition of C3N4; Figure S3: Random Amplified Polymorphic DNA (RAPD) analysis with random oligonucleotide primer OPC20; Figure S4: The relative expression of Cd transport-associated genes in rice shoots upon exposure to Cd with or without the addition of C3N4; Figure S5: The relative expression of As transport-associated genes in rice shoots upon exposure to As with or without the addition of C3N4; Table S1: A list of used primers.

**Author Contributions:** Conceptualization, C.M., J.C.W. and B.X.; methodology, C.M. and Y.H.; formal analysis, C.M., Y.H. and J.C.W.; investigation, C.M., Y.H., J.Z., N.Z.-M. and A.G.M.; resources, J.C.W. and B.X.; data curation, C.M. and J.C.W.; writing—original draft preparation, C.M.; writing—review and editing, C.M., O.P.D., Y.R., J.C.W. and B.X.; supervision, J.C.W. and B.X.; project administration, J.C.W. and B.X.; funding acquisition, J.C.W. and B.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2019ZT08L213) and USDA NIFA and Hatch Programs (MAS 00549 and CONH00147).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article or supplementary material.

**Acknowledgments:** This work is supported by the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2019ZT08L213) and USDA NIFA and Hatch Programs (MAS 00549 and CONH00147).

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

### **References**

