*2.9. Measurement of Photochemical Quenching, Electron Transfer Rate, and Absorbed Energy of the PSII Reaction Center*

After dark adaptation of the leaves for 30 min, chlorophyll fluorescence quenching analysis was performed using a pulse modulation fluorimeter (FMS-2, Hansatech, King's Lynn, UK). In this procedure, the leaf adapts to the light for the first 30 s, and the light intensity is the same as the ambient light intensity before the measurement of the leaf (1000 <sup>μ</sup>mol·m−2·s−1). The steady-state fluorescence parameter (*F*s) and electron transfer rate (ETR) under the light-adapted condition was measured, and the saturated pulsed light (8000 <sup>μ</sup>mol·m−2·s−1) was used to determine the photochemical quenching (qP), the maximum fluorescence value (*F*m ). Then, the method of Hendrickson et al. [37] was followed to determine the light energy absorbed by the PSII reaction center into each of four parts [38]. That is, the following parameters were inferred: quantum yield (*Φ*PSII) for the photochemical reaction, quantum yield (*Φ*NPQ) dependent on the proton gradient lutein cycle on both sides of the thylakoid membrane, quantum yield of fluorescence and heat energy dissipation (*Φ*f,D), and the thermal dissipation of the quantum yield (*Φ*NF) of the deactivated PSII reaction center. These parameters were inferred using the following formulae:

> *Φ*PSII = [1 − (*F*s/*F*m )] [(*F*v/*F*m)/(*F*v/*F*mM)] *Φ*NPQ = [(*F*s/*F*m ) − (*F*s/*F*m)] [(*F*v/*F*m)/(*F*v/*F*mM)] *Φ*f,D = (*F*s/*F*m) [(*F*v/*F*m)/(*F*v/*F*mM)] *Φ*NF = 1 − [(*F*v/*F*m)/(*F*v/*F*mM).

The sum of each of these parameters is 1, namely *Φ*PSII + *Φ*NPQ + *Φ*f,D + *Φ*NF = 1 (Figure 1).

**Figure 1.** Energy allocation pathways of photosystem II (PSII): proportion of light energy used in the quantum yield of PSII photochemistry (*Φ*PSII), xanthophyll-mediated thermal dissipation (*Φ*NPQ), quantum yield used in thermal dissipation in non-functional PSII (*Φ*NF), and quantum yield of fluorescent and heat energy dissipation (*Φ*f,D).

#### *2.10. Statistical Analysis*

Excel and SPSS software were used to conduct statistical analyses on the measured data. The data in the figure shows means of four plants ± standard deviation. Tukey Multiple Comparisons test was adopted to compare the differences between treatments. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### **3. Results**

#### *3.1. Effects of N Metabolism Indicators*

NO2 fumigation affected nitrogen metabolism in mulberry. The nitrate nitrogen content of mulberry leaves fumigated with NO2 increased by 56.10% (*p* < 0.05) at 4 h after the start of the treatment, and when the fumigation time lasted for 8 h, it was nearly doubled (*p* < 0.05) compared to the control. The amino acid content in mulberry was not significantly increased (*p* > 0.05) when NO2 was fumigated for 4 hours; however, it increased by 35.17% (*p* < 0.05) after 8 hours of fumigation.

Nitrate reductase and nitrite reductase activities increased significantly (*p* < 0.05) by 4 h after the start of the treatment, and when the fumigation time lasted for 8 h, the activities enhanced more (Figure 2a–d).

**Figure 2.** Effects of N Metabolism Indicators. (**a**) NO3 −-N content, (**b**) amino acid content, (**c**) nitrate reductase activity, and (**d**) nitrite reductase activity in leaves of mulberry seedlings exposure to 4 <sup>μ</sup>L·L−<sup>1</sup> NO2 for 0 h, 4 h (4 h·d−1, for one day), and 8 h (4 h·d−1, for 2 days). Date represent means of four plants ± standard deviations. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### *3.2. Effects of Gas Exchange Parameters*

NO2 fumigation changed the *Pn*-*C*i curve of mulberry leaves, and *Pn* showed an upward trend with the increase of CO2 concentration. When the CO2 concentration was lower than 400 <sup>μ</sup>mol·mol<sup>−</sup>1, *Pn* increases approximately linearly with the increase of CO2 concentration. Subsequently, as the CO2 concentration continued to increase, the rate of *Pn* increase slowed. When the CO2 concentration reached 1200 <sup>μ</sup>mol·mol<sup>−</sup>1, the *Pn*-*C*i gradually flattened (Figure 3a). Under the same CO2 concentration, the *Pn* of mulberry leaves with NO2 fumigation was significantly higher than that of the control, indicating that NO2 fumigation improved the carbon assimilation capability of mulberry leaves. Stomatal conductance (*G*s), the maximum carboxylation rate (*V*cmax), and dark respiration rate (*R*d) of the mulberry leaves increased significantly (*p* < 0.05) after 4 h and 8 h of fumigation compared with the control (Figure 3b–d).

**Figure 3.** Effects of Gas Exchange Parameters. (**a**) *Pn*-Ci curve, (**b**) conductance of H2O, (**c**) the maximum carboxylation rate, and (**d**) dark respiration rate in leaves of mulberry seedlings exposure to 4 <sup>μ</sup>L·L−<sup>1</sup> NO2 for 0 h, 4 h (4 h·d−1, for one day), and 8 h (4 h·d−1, for 2 days). Date represent means of four plants ± standard deviations. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### *3.3. Effects of Distribution of Light Absorbed by PSII*

Of the light energy absorbed by PSII, the fraction allocated to photochemical conversion (*Φ*PSII), increased slightly, while the fraction dissipated nonphotochemically in a manner dependent on the trans-thylakoid proton-gradient and the xanthophyll cycle (*Φ*NPQ) increased after NO2 fumigation. By contrast, the proportion of basic fluorescence quantum yields and heat-dissipated quantum yields (*Φ*f,D) and heat-dissipation quantum yields of inactive PSII reaction centers (*Φ*NF) declined after NO2 fumigation. The proportions of *Φ*NPQ and *Φ*PSII increased by 2.49% (*p* < 0.05), 36.23% (*p* < 0.05) at 4h and increased by 5.67% (*p* < 0.05), 41.07% (*p* < 0.05) at 8h. By contrast, the proportions of *Φ*f,D and *Φ*NF decreased by 5.01% (*p* < 0.05), 60.41% (*p* < 0.05) at 4h and decreased by 10.34% (*p* < 0.05) and 75.74% (*p* < 0.05) at 8 h (Figure 4). The quantum yield for heat dissipation in mulberry leaves fumigated with <sup>4</sup> <sup>μ</sup>L·L−<sup>1</sup> NO2 was reduced to increase the photosynthetic energy used for photochemical reaction. As shown in Figure 7, the partial energy from total energy absorbed by mulberry leaves fumigated by NO2 was used for NO2 metabolism; whether this affects the activity of PSII and PSI is discussed in the following sections.

**Figure 4.** Light energy used for quantum yield of PSII photochemistry (*Φ*PSII), xanthophyll-mediated thermal dissipation (*Φ*NPQ), basic fluorescence quantum yield and heat dissipation quantum yield (*Φ*f,D), and quantum yield used in thermal dissipation in non-functional PSII (*Φ*NF) in leaves of mulberry seedlings exposure to 4 <sup>μ</sup>L·L−<sup>1</sup> NO2 for 0 h, 4 h (4 h·d−1, for one day), and 8 h (4 h·d−1, for 2 days). Date represent means of four plants ± standard deviations. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### *3.4. Effects of Chlorophyll A Fluorescence Transient*

NO2 fumigation significantly changed the shape of OJIP curves. The fluorescence intensity at points O and J (*F*<sup>o</sup> and *F*J, respectively) decreased, and the fluorescence intensity at points I and P (*F*<sup>I</sup> and *F*p, respectively) increased significantly. The OJIP curves of the leaves were normalized to the span O-P. The variable fluorescence at point J on the OJIP curve of leaves was significantly increased by NO2. The difference between the normalized O-P curve and the control was assessed, and the difference between ΔVt and the control at point J was most significant at 2 ms (Figure 5a–d).

**Figure 5.** Effects of Chlorophyll A Fluorescence Transient. (**a**) OJIP transmission, (**b**) fluorescence intensity at points O, J, I, and P, (**c**) standardized OJIP transmission, (**d**) standardized OJIP transmission and CK in leaves of mulberry seedlings exposure to 4 <sup>μ</sup>L·L−<sup>1</sup> NO2 for 0 h, 4 h (4 h·d<sup>−</sup>1, for one day), and 8 h (4 h·d−1, for 2 days). Date represent means of four plants <sup>±</sup> standard deviations. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### *3.5. Effects of PSII activity*

NO2 fumigation had a significant effect on PSII potential photochemical activity (*F*v/*F*o), PSII maximum photochemical efficiency (*F*v/*F*m), photochemical quenching (qP), electron transport rate (ETR), and number of active reaction centers per unit area (*RC*/*CS*m), which is shown in Table 1. *F*v/*F*o, *F*v/*F*m, qP, ETR, and *RC*/*CS*m increased by 33.44% (*p* < 0.05), 1.22% (*p* < 0.05), 30% (*p* < 0.05), 33.64% (*p* < 0.05), and 27.07% (*p* < 0.05) at 4 h, respectively, and increased by 39.38% (*p* < 0.05), 2.44% (*p* < 0.05), 35% (*p* < 0.05), 37.67% (*p* < 0.05), and 33.37% (*p* < 0.05) at 8 h, respectively.

**Table 1.** PSII potential photochemical activities (*F*v/*F*o), PSII photochemical efficiency (*F*v/*F*m), photochemical quenching (qP), electron transport rate (ETR), and the number of reactive centers per unit area (*RC*/*CS*m) in leaves of mulberry seedlings exposure to 4 <sup>μ</sup>L·L−<sup>1</sup> NO2 for 0 h, 4 h (4 h·d<sup>−</sup>1, for one day), and 8 h (4 h·d<sup>−</sup>1, for 2 days). Date represent means of four plants <sup>±</sup> standard deviations. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.


#### *3.6. Effects of PSI Activity*

The relative drop in 820 nm light signals during far-red light illumination reflects the activity of PSI Δ*I*/*I*o. NO2 fumigation increased the drop in the 820 nm optical signal, and the difference increased significantly at 8 h, indicating that NO2 fumigation increased the PSI activity of mulberry leaves (Figure 6a). PSI activity was not significantly raised (*p* > 0.05) for 4 h fumigation, but when fumigated for 8 h, the PSI activity was improved by 20.22% (*p* < 0.05) compared with the control (Figure 6b).

**Figure 6.** Effects of PSI activity. (**a**) Changes in light transmission at 820 nm, (**b**) Relative values of Δ*I/I*<sup>o</sup> in leaves of mulberry seedlings exposure to 4 <sup>μ</sup>L·L−<sup>1</sup> NO2 for 0 h, 4 h (4 h·d<sup>−</sup>1, for one day), and 8 h (4 h·d<sup>−</sup>1, for 2 days), where the <sup>Δ</sup>*I*/*I*<sup>o</sup> values at 8 h were taken as 100%. Date represent means of four plants ± standard deviations. Different lowercase letters for the same parameter indicate significant differences among different treatments at *p* < 0.05 levels.

#### **4. Discussion**

The present study revealed that the absorption of NO2 by mulberry leaves is not only involved in nitrogen metabolism in vivo, but also increases the leaf content of nitrate nitrogen and amino acids, enhances the activity of nitrate reductase (NR) and nitrite reductase (NiR), and improves the nitrogen metabolism capacity (Figure 2). Zeevaart et al. [39] fumigated pea seedlings with ammonium nitrogen as the only nitrogen source condition and found that NO2 induces NR activity, in perhaps the earliest study on NO2 and nitrogen metabolism. Subsequent studies have since found that atmospheric NO2 has a significant effect on NR. Lower concentrations of NO2 increase NR activity in barley (*Hordeum vulgre*) [40], Scots pine (*Pinus sylvestris*) [41], and red spruce (Picea rubens) [42]. Similarly, when Hisamatsu et al. [43] fumigated squash (*Cucurbita maxima*) seedlings with NO2, NR activity in cotyledon was significantly reduced. In the present study, NR activity had exhibited a small increase at 4 h but increased sharply at 8 h. Enhanced NR activity promotes the improvement of nitrogen metabolism. It not only increases the consumption of excess light energy, but also provides necessary enzymes for carbon metabolism and accelerates carbon metabolism.

The 4 <sup>μ</sup>L·L−<sup>1</sup> NO2 fumigation treatment significantly increased the net photosynthetic rate of mulberry leaves (Figure 3a), accelerating photosynthetic electron transport and enhancing phosphorylation activity in leaves. Additionally, the increase of stomatal conductance promoted the degree of CO2 acquisition and transportation (Figure 3b), thereby accelerating the production and accumulation of organic matter in plants [44]. At the same time, the maximum carboxylation rate, which is an important parameter for characterizing photosynthetic capacity of plants, also increased (Figure 3b), indicating that NO2 fumigation increased Rubisco activity of mulberry leaves, thereby enhancing the fixation ability of CO2 [45]. Moreover, the dark respiration rate was significantly increased (Figure 3d), indicating that its fumigated leaves consumed excess light energy through respiration to protect the photosynthetic system and increase the photosynthetic rate.

Accordingly, the quantitative study of the final destination of light energy absorbed by plant leaves is an important part of research on photosynthesis. Earlier studies on light energy are more directly expressed by actual photochemical efficiency (*Φ*PSII) and non-photochemical quenching (NPQ) [46,47]. However, in higher plants, NPQ is only determined by the establishment of proton gradients on both sides of the thylakoid membrane and the xanthophyll cycle [48]. Thus, NPQ does not represent all non-photochemical quenching processes. In addition, plant leaves absorb light energy via mechanisms other than photochemical reactions, including physiological processes such as photorespiration, the water–water cycle, and xanthophyll cycle, which can be areas in which light energy is distributed as the final destination [49–51]. The theory developed by Hendrickson et al. [50] clarifies these processes. In this framework, excitation energy can be divided into the light energy absorbed by the PSII reaction centers and used for the quantum yield of photochemical reaction (*Φ*PSII), the quantum production dependent on the trans-thylakoid proton-gradient for xanthophyll cycle quantum yield (*Φ*NPQ), fluorescence quantum yield and heat dissipation (*Φ*f,D), and heat dissipation quantum yield in inactive PSII reaction centers (*Φ*NF) [51]. In our experiment, mulberry leaves fumigated with NO2 showed proportional increases in *Φ*PSII and *Φ*NPQ, indicating that NO2 fumigation promoted the establishment of light-induced proton absorption in the chloroplast H+ concentration gradients on both sides of the thylakoid. In other words, the presence of ΔpH on both sides of the thylakoid membrane increased the power of ATP synthesis in chloroplasts and promoted the photoprotective mechanism based on the xanthophyll cycle. Additionally, *Φ*f,D and *Φ*NF proportionally decreased, indicating that the proportion of heat dissipation and inactivation reaction centers decreased and that the absorbed light energy was mostly used for photosynthetic carbon assimilation, thereby improving the photochemical efficiency of the mulberry leaf (Figure 4).

In this experiment, the changes in PSII of mulberry leaves were analyzed using the JIP test. NO2 changed the structure and function of PSII and the photosynthetic primary reaction process in mulberry leaves. However, because the OJIP curve is greatly influenced by the environment, its relative fluorescence intensity is affected by various environmental factors. Therefore, the OJIP curve was often normalized to the span O-P. The relative variable fluorescence of *V*<sup>J</sup> and *V*<sup>I</sup> at points J (2 ms) and I point (30 ms) for NO2-fumigated leaves was significantly decreased (Figure 5), indicating that the PSII reaction center receptor-side electronic primary quinone receptors (*Q*A) to the secondary quinone receptor (*Q*B) transmission capacity and plastoquinone (PQ) accept electronic ability were enhanced (Figure 7). The potential photochemical activity of PSII (*F*v/*F*o), PSII maximum photochemical efficiency (*F*v/*F*m), photochemical quenching (qP), electron transfer rate (ETR), and the number of active reaction centers per unit area (*RC*/*CS*m) were significantly higher under fumigation than in the control treatment (Table 1), indicating that NO2 fumigation enhances the activity of PSII reaction centers and the degree of openness of reaction centers, which promotes the electron transport of leaves, photosynthetic primary reaction process, and the rate of light photons received by PSII reaction centers. The proportion of light energy used in photochemical reactions increased, thus accelerating the synthesis of NADPH and ATP, as well as the conversion efficiency of light energy and carbon assimilation processes, which increased the photochemical efficiency. However, Hu et al. [31] quantified the photosynthetic responses of hybrid poplar cuttings to 4 <sup>μ</sup>L·L−<sup>1</sup> NO2. It was found that significant declines in *<sup>F</sup>*v/*F*m, indicating inhibition of and even damage to photosynthetic apparatus. The study of the maximum redox capacity (Δ*I*/*I*o) of PSI

was also increased (Figure 6) as well as the ability to receive electrons; the ability of PSII to supply electrons in the photosynthetic apparatus was matched by the ability of PSI to receive electrons. Thus, not only can electron transfer be promoted efficiently, but electrons can also be transferred to ferredoxin, which distributes electrons to the nitrogen metabolism, photorespiration, and NO2 metabolic pathways (Figure 7).

**Figure 7.** Photosynthetic electron flow distribution. The electrons produced by water splitting in photosynthesis pass through photosystem II (PSII), primary quinone receptor (*Q*A), secondary quinone receptor (*Q*B), plastoquinone (PQ), cytochrome *b6f* (Cyt *b6f*), plastid blue pigment (PC), and photosystem I (PSI) to ferredoxin (Fd) were assigned to four pathways: CO2 assimilation, nitrogen metabolism, photorespiration, and NO2 metabolic pathways (new pathway).

#### **5. Conclusions**

The metabolism of atmospheric NO2 utilized and consumed light energy and photosynthetic electrons absorbed by leaves of mulberry fumigated with NO2. However, this part of the photosynthetic energy consumption did not reduce the photosynthetic capacity of the mulberry leaves, but instead increased the photosynthetic efficiency of the plant leaves. This is because the mulberry leaves absorbed NO2 and conducted nitrogen metabolism and respiration, which consumed excess light energy, and thus protected the photosynthetic apparatus. However, the light energy absorbed by the PSII reaction center in the form of heat dissipation in mulberry plants fumigated with 4 <sup>μ</sup>L·L−<sup>1</sup> NO2 was also reduced, such that the absorbed light energy was more effectively used in photosynthetic carbon assimilation. Therefore, in the case where the concentration of the atmospheric pollutant NO2 is lower than the concentration that can damage the mulberry, NO2 can be absorbed by the mulberry to reduce the haze in the air.

**Author Contributions:** Y.W. and G.S. conceived and designed the study. Y.W., W.J., Y.C., and D.H. performed the experiments. Y.W., J.W., and M.Z. contributed to the sample measurement and data analysis. Y.W. and G.S. wrote the paper.

**Funding:** This research was funded by the National Natural Science Foundation of China, grant number 31870373, and the Natural Science Foundation of Heilongjiang Province, grant number ZD201105 and the Applicant and Developmental Project for Agriculture of Heilongjiang Province, grant number GZ13B004.

**Acknowledgments:** The authors want to appreciate Wah Soon Chow from the Australian National University for revising the manuscript and our colleague Yanbo Hu for his advice and great comments to improve the paper.

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

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