## 4 Conclusions  
The results showed that the use of 20 and 50 nm CuO NP treatments at low doses could increase the growth and nitrogen assimilation of soybeans in terms of N-associated enzyme activities and nitrogenous compounds (Figure 7). Moreover, the use doses of CuO NPs did not significantly alter the Cu contents in soybean tissues, although a dose-dependent fashion of Cu contents in soil samples was evident. More importantly, our findings demonstrated that 20 and 50 nm CuO NPs were beneficial to the early growth and increased the nitrogen assimilation abilities of soybeans at low doses without causing any oxidative stresses. At the same use dose, the 50 nm CuO NP treatments outperformed the 20 nm CuO NP treatments, as determined by crop growth and nitrogen assimilation. Overall, nanoscale Cu-based agrochemicals have exhibited great potential for application in sustainable agriculture, particularly in legume crops in this case, and could also potentially reduce the risk of excessive use of nitrogen fertilizer in the agricultural environment.  
Figure 6: Cu content in shoots (**A**), roots (**B**), and soil samples (**C**) in the 20 and 50 nm CuO NP treatments at 1 and 10 mg/kg for three weeks. Error bars represent the standard error of 5 replicates, and the different letters in each panel represent significantly different values at \(p<0.05\).  
Figure 7: A schematic diagram of the size effects of CuO NPs on nitrogen fixation in a soybean plant. The numbers in the figure show the percentage change of NP treatments relative to the control (numbers with blue color indicate an increase; red color indicates a decrease).  
**Supplementary Materials:** The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/nano14090746/s1](https://www.mdpi.com/article/10.3390/nano14090746/s1)]([https://www.mdpi.com/article/10.3390/nano14090746/s1](https://www.mdpi.com/article/10.3390/nano14090746/s1)), Detailed information, including three experiments, four descriptions, five figures, and five tables, is provided in the Supplementary Materials file. Figure S1: Scanning electron microscopy (SEM) scan of different sizes of CuO NPs in deionized water; Figure S2: DCFH-DA staining of soybean blades under different sizes of CuO NPs at 1 and 10 mg/kg; Figure S3: DAB and NBT staining of root tips under different sizes of CuO NPs at 1 and 10 mg/kg; Figure S4: DCFH-DA staining of soybean root under different sizes of CuO NPs at 1 and 10 mg/kg; Figure S5: GSH content in soybean shoot and root under different sizes of CuO NPs at 1 and 10 mg/kg; Table S1: length and biomass of soybean plants under the treatment of 20 nm CuO NPs in pre-experiment; Table S2: length and biomass of soybean plants under the treatment of 50 nm CuO NPs in pre-experiment: Table S3: NO\({}_{3}\) -N content in shoot and root of soybean plants in pre-experiment; Table S4: length of soybean plants; Table S5: Element content in soybean shoot, root and soil. (See Refs. [50;54;55]).  
**Author Contributions:** Conceptualization, C.M., W.J. and Y.G.; investigation, Y.G., H.L., X.X., A.L. and Y.H.; formal analysis, Y.G., H.S. and C.L.; writing--original draft preparation, Y.G.; writing--review and editing, C.M., W.J. and H.L.; supervision, C.M.; project administration, C.M.; funding acquisition, C.M. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This work was funded by the National Natural Science Foundation of China (42177191) and the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2019 ZT08L213).  
**Data Availability Statement:** Data are contained within the article and Supplementary Materials.  
**Conflicts of Interest:** The authors declare no conflict of interest.