*3.4. Macro- and Micronutrient Content Analysis in Rice Tissues*

Both macro- and micronutrients are involved in critical metabolism and development processes throughout the plant life cycle [61]. Upon exposure to abiotic stressors, such as heavy metals, displacement of nutrient uptake and distribution in rice could result in phytotoxicity and may trigger plant defense mechanisms. Consequently, the macro- and micronutrient contents of rice tissues upon coexposure to Cd or As and C3N4 were measured. Exposure to both Cd and As significantly altered rice root and shoot macronutrients, although the observed changes were metal specific (Figure 4 and Figure S2). For example,

exposure to Cd increased shoot P content by more than 90% as compared with controls, regardless of the C3N4 presence (Figure 4B), whereas no difference was found with As treatments (Figure 4B). Similarly, the shoot S content was increased by more than 40% and 60% upon Cd treatments as compared with the control and the As treatments, respectively (Figure 4D). Exposure to As led to decreases in the tissue macronutrient content relative to the Cd treatments. For example, the lowest K contents in both roots and shoots were evident in the As-treated tissues, regardless of the presence of C3N4 (Figure S2C,D). Similar results were also observed for the shoot S, Ca, and Mg contents (Figure 4D,F and Figure S2B). Importantly, the addition of C3N4 had little impact on macronutrient content, the exception being shoot S and Ca in the 250 mg/L C3N4 treatment, where the contents were significantly higher than those in the control (Figure 4D,F).

**Figure 4.** The contents of macronutrients in rice roots and shoots upon exposure to As and Cd with or without the addition of C3N4. (**A**,**C**,**E)** represent the P, S, and Ca contents in rice roots across all treatments, respectively. (**B**,**D**,**F)** represent the P, S, and Ca contents in rice shoots across all treatments, respectively. Values of each nutrient content in shoots followed by different letters are significantly different at *p* < 0.05.

In general, changes to the tissue micronutrient content were of a lesser magnitude than those to the macronutrient content, although some statistically significant changes were evident (Figure 5). For example, the Cu contents in roots cotreated with As and C3N4 were significantly lower than those in the control (Figure 5A). Similarly, root Fe content in the Cd treatments with or without C3N4 was increased by more than 100% relative to the control (Figure 5C); additionally, 50–80% increases in the shoot Zn contents were found in the Cd treatments, regardless of C3N4 exposure (Figure 5G). In comparison with Cd, As exerted less impact on micronutrient displacement in rice, the exception being shoot Fe, where the Fe content was decreased by 20% relative to the control (Figure 5D). Overall, the nutrient analysis demonstrates that heavy metal exposure can induce nutrient displacement in rice and that C3N4 amendment has little impact on these alterations.

**Figure 5.** The contents of micronutrients in rice roots and shoots upon exposure to As and Cd with or without the addition of C3N4. (**A**,**C**,**E**,**G**) represent the Cu, Fe, Mn, and Zn contents in rice roots across all treatments, respectively. (**B**,**D**,**F**,**H**) represent the Cu, Fe, Mn, and Zn contents in rice shoots across all treatments, respectively. Values of each nutrient content in roots followed by different letters are significantly different at *p* < 0.05.

Nutrient displacement and dysfunction induced by exposure to heavy metals or other contaminants can severely compromise crop health. For example, tetracycline reduced the macronutrient (K, P, and S) contents of rice by approximately 20% relative to the control [58]. High doses of metal-based NPs, such as Ag, CeO2, and In2O3, can significantly decrease Fe content and subsequently compromise plant metabolism [49,62]. No published studies have reported on the levels of mineral nutrients in crops affected by C3N4 exposure. It is worth noting that the addition of C3N4 had little impact on nutrient accumulation in rice, suggesting that the mode of action may be through stabilization of heavy metals in soils.

### *3.5. Plant Molecular Response*
