## 3 Results and Discussion
### Effects of CuO NPs on Nitrate Reduction in Soybeans  
As the main nitrogen source for plant uptake and utilization, nitrate nitrogen (NO\({}_{3}\)\({}^{-}\)-N) is not only a nutrient but also a signaling molecule, which plays a critical regulatory role in breaking seed dormancy and leaf growth [25]. The NO\({}_{3}\)\({}^{-}\)-N in the soil is taken up by the root cells via a specific nitrate transporter, where it is eventually reduced to ammonia nitrogen under the action of NR and NiR.  
In comparison with the control, both 20 nm and 50 nm CuO NPs at 1 mg/kg increased the content of NO\({}_{3}\)\({}^{-}\)-N in shoots by 17.1% and 28.5% (Figure 2A), respectively. At 10 mg/kg, the addition of 50 nm CuO NPs increased the NO\({}_{3}\)\({}^{-}\)-N content in shoots by 31.4%, while 20 nm CuO NPs significantly reduced the content of NO\({}_{3}\)\({}^{-}\)-N in shoots (Figure 2A). In roots, the addition of 20 nm CuO NPs decreased the NO\({}_{3}\)\({}^{-}\)-N content with increasing the use doses, while 50 nm CuO NPs significantly increased the NO\({}_{3}\)\({}^{-}\)-N content with increasing the use doses (Figure 2B); particularly, root application of 50 nm CuO NPs at 10 mg/kg increased the root NO\({}_{3}\)\({}^{-}\)-N content by 23.5% relative to the control (Figure 2B). The possible explanations that both sizes of CuO NPs altered the NO\({}_{3}\)\({}^{-}\)-N content in roots can be ascribed to either the increases in the activities of NR and NiR enzymes or the timing to activate the NR and NiR enzymes not being coordinated with that of NO\({}_{3}\)\({}^{-}\)-N production.  
NR is the first enzyme involved in N assimilation and is also considered a limiting factor for plant growth. In shoots, the NR enzyme activity was increased by 88.2% and 75.6% in the 20 nm and 50 nm CuO NP treatments at 1 mg/kg and increased by 98% and 110.9% at 10 mg/kg, respectively, relative to the control (Figure 2C). In roots, only 20 nm CuO NP treatments with 1 and 10 mg/kg increased the activity of the roots' NR enzyme by 59.7% and 41.5%, respectively, relative to the control, while 50 nm CuO NP treatments had no impacts regardless of the use doses (Figure 2D). The increase in NR enzyme activity enhanced the NO\({}_{3}\)\({}^{-}\)-N to NO\({}_{2}\)\({}^{-}\)-N conversion in shoots and roots as a function of CuO NPs. The 20 nm CuO NP treatments increased the NR enzyme activities more than the 50 nm NP treatments in both shoots and roots, which could be the reason for the decreases in the NO\({}_{3}\)\({}^{-}\)-N content in 20 nm CuO NP-treated tissues. The NiR enzyme reduces the nitrate reduction product of the previous step from NO\({}_{2}\)\({}^{-}\)-N to NH\({}_{4}\)\({}^{+}\)-N [41]. All the CuO NP treatments decreased the NiR enzyme activities in shoots (Figure 2E); however, the NiR enzyme activity in roots was increased by 41-48% and 54-55% as affected by 20 and 50 nm CuO NPs, respectively, relative to the control.  
In general, 50 nm CuO NPs accumulated more NO\({}_{3}\)\({}^{-}\)-N in soybean tissues than 20 nm NPs, and the increase in NO\({}_{3}\)\({}^{-}\)-N contents suggests that under the catalysis of NR and NiR enzymes, more NO\({}_{3}\)\({}^{-}\)-N was reduced to NO\({}_{2}\)\({}^{-}\)-N, NH\({}_{4}\)\({}^{+}\)-N, and other nitrogen compounds, all of which could boost the nitrogen assimilation pathway in soybeans.  
Figure 1: Phenotypic images (**A**), fresh shoot weight (**B**), root weight (**C**), and NBT staining and DAB staining (**D**) of soybean plants treated with 20 and 50 nm CuO NPs at 1 and 10 mg/kg for three weeks. Values are means of 8 plants \(\pm\) SD, and different letters in each panel represent significantly different values at \(p<\) 0.05. Scale = 15 cm.