*2.2. NO2 Fumigation Treatment*

The gas fumigation box was a custom-made open-top automatic monitoring gas concentration device [31]. The NO2 cylinder (Dalian Date Gas, Dalian, China) was connected to an electromagnetic valve and axial flow fan as the air supply source. Solenoid valve controls regulated the test gas, while the axial flow of the fan is used to send the test gas into the air chamber. A gas pressure reducer and a gas flowmeter in the middle of the gas delivery system control the gas flow. A solenoid valve associated with the micro-flux switch valve controls the average flow in low-frequency pulse width modulation (PWM) mode. The gas chamber was composed of organic glass in the shape of a six-sided prism, with a cross section diagonal length of 1.16 m and height of 1.85 m. Vent holes were set on the top, with a gas grid plate 0.30 m from the bottom of the chamber. The NO2 gas first enters the bottom space under the gas grid, and then passes through the gas chamber from the bottom of the 1200 holes, each with a diameter of 2 mm and evenly distributed across the grid plate. A fan was installed at the top of the fume chamber to ensure that the conditions were evenly distributed among the plants. The concentration of NO2 used in the experiment was 4 <sup>μ</sup>L·L−1, and the concentration was selected according to our pre-experiment results. We used 0.5, 1, 2, 4, 6, 8 <sup>μ</sup>L·L−<sup>1</sup> NO2 to fumigate mulberry leaves. The results showed that the net photosynthetic rate in leaves of mulberry was the highest as NO2 concentration 4 <sup>μ</sup>L·L<sup>−</sup>1. The fumigation time is 4 hours/day, from 7:30 to 11:30; the temperature in the fumigation chamber was maintained at 28 ◦C. Samples were taken before the fumigation began (0 h) and at 4 and 8 h after fumigation. The indicators of N metabolism and photosynthetic gas exchange parameters as well as chlorophyll *a* fluorescence parameters were determined.

#### *2.3. Measurement of NO3* −*-N Content*

An analysis of nitrate nitrogen was completed by using the salicylic acid method [32]. Standard solutions of 500 mg/L nitrate nitrogen with deionized water to create a 20, 40, 60, 80, and 100 mg/L series of standard solutions. Then, 0.5 g of plant material was placed into each test tube, to which 10 mL of deionized water was added before being placed into a boiling water bath for 30 min. Afterwards, test tubes were cooled with tap water, and extracts were filtered into volumetric flasks. The residues were rinsed, and the samples were allowed to finally settle to 25 mL. Then, 0.1 mL aliquots of the above series of standard solutions and sample extracts were respectively transferred into new test tubes, and the standard solution was replaced with 0.1 mL of distilled water.

Then, 0.4 mL of 5% salicylic acid solution was added prior to each sample being shaken well with a 20 min of room temperature incubation. After incubation, 9.5 mL of 8% NaOH solution was added, and samples were shaken and allowed to cool to room temperature. The total volume of the sample was 10 mL. The absorbance was measured at 410 nm with a blank as a reference. A standard curve was drawn, and a regression equation was fitted to the standard by using the nitrate concentration as the abscissa and the absorbance as the ordinate. The concentration of nitrate nitrogen was calculated using the inferred regression equation, and content was calculated by using the formula NO3 −-N content = (*C* × *V*/1000)/*W*, where *C* is the regression equation calculated NO3 −-N concentration, *V* is the extract total mL of sample liquid, and *W* is the sample fresh weight.

#### *2.4. Measurement of Amino Acid Content*

A colorimetric analysis was done on hydrated ninhydrin for amino acids [33]. To assess amino acid content, 200 μg/mL amino acid standard solutions were measured into volumes of 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL, respectively, and transferred into 25 mL volumetric flasks, to which water was added until a volume of 4.0 mL was reached. Then, 1 mL of ninhydrin solution (20 g/L) and 1 mL of phosphate buffer (pH 8.04) was added to each flask, followed by mixing and incubation in a 90 ◦C water bath until the color became constant. At this point, samples were removed rapidly and cooled to room temperature, and water was added until a final volume was reached. Samples were then shaken well and allowed to stand for 15 min. The absorbance A of the remaining solutions was then determined relative to a reagent blank as a reference solution at a wavelength of 570 nm. In the standard curve, the micrograms of amino acids represented the abscissa, while absorbance A was the ordinate. The standard curve was drawn based on these data, and the regression equation was then inferred.

Then, 0.5 g plant samples were added to 5 mL of 10% acetic acid and ground in a mortar. These ground samples were then washed into 100 mL volumetric flasks, diluted to a set volume in water, and filtered into triangle bottles for determination of the filtrate. Then, 4 mL of the clarified sample solution was subjected to the standard curve inference procedure outlined above, in which the absorbance A value was measured under the same conditions and the microgram content of amino acids was calculated using a regression equation of the following form: amino acid content (μg/100 g) = *C*/(*m* × 1000) × 100, where *C* represents the mass number of amino acids and the *m* represented the mass of the sample.

#### *2.5. Measurement of Nitrate Reductase Activity*

Nitrate reductase (NR) activity was determined using the kit produced by Suzhou Keming Company (Suzhou, China). This kit functions by assaying how NR catalyzes the reduction of nitrate to nitrite according to the reaction NO3 <sup>−</sup> + NADH + H<sup>+</sup> → NO2 <sup>−</sup> + NAD<sup>+</sup> + H2O. The resulting nitrite quantitatively produces red azo compounds under acidic conditions with p-aminobenzenesulfonic acid and α-naphthylamine. The generated red azo compound has a maximum absorption peak at 540 nm and was measured by spectrophotometry.

#### *2.6. Measurement of Nitrite Reductase Activity*

Nitrite reductase (NiR) activity was determined using the kit produced by Suzhou Keming Company. This kit functions by assaying how nitrite reductase reduces NO2 − to NO, and the sample is subjected to the diazotization reaction to generate a purple-red compound that indicates a NO2 − decrease; that is, a change in absorbance at 540 nm reflects the activity of nitrite reductase.

#### *2.7. Measurement of Gas Exchange Parameters*

Gas exchange was measured with a portable photosynthesis system (LICOR-6400; LI-COR, Lincoln, NE, USA). All the photosynthetic measurements were taken at a constant airflow rate of <sup>400</sup> <sup>μ</sup>mol·s<sup>−</sup>1, and the temperature was 28 ± <sup>2</sup> ◦C. While using the liquefied CO2 cylinders were used to provide different CO2 concentrations, across a CO2 concentration gradient of 50, 100, 200, 300, 400, 600, 800, 1000, and 1200 <sup>μ</sup>mol·mol−1. The photosynthetically active radiation (PAR) was 1000 <sup>μ</sup>mol <sup>m</sup>−2·s−<sup>1</sup> in order to avoid light limitation of photosynthesis. The photosynthetic rate (*Pn*) and stomatal conductance of H2O (*G*s) were measured. Photosynthesis curves plotted against intercellular CO2 concentrations (*Pn*-*C*i curve) was analyzed to estimate the maximum carboxylation rate (*V*cmax) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and dark respiration rate (*R*d) [34].

#### *2.8. Measurement of Chlorophyll A Fluorescence Transient and Light Absorbance at 820 nm*

According to the method developed by Schansker et al. [35], leaves were first dark-adapted for 20 min, and then the rapid chlorophyll fluorescence-induced kinetic curve (OJIP curve) and 820 nm light absorption curve were measured using a plant efficiency analyzer (M-PEA, Hansatech, King's Lynn, UK). The OJIP curve was induced under 3500 <sup>μ</sup>mol·m−2·s−<sup>1</sup> pulsed light, and the fluorescence signal was recorded from 10 μs to the end of 2 s, with an initial recording rate of 105 data per second. The relative value of the difference between the maximum (*I*o) and minimum (*I*m) absorbance at 820 nm, i.e., Δ*I*/*I*<sup>o</sup> = (*I*<sup>o</sup> − *I*m)/*I*o, was used as an index for measuring photosystem

I (PSI) activity. The OJIP fluorescence induction curve was analyzed with reference to the JIP-test used by Strasser et al. [36] to measure the initial fluorescence (*F*o), the maximum fluorescence (*F*m), the light energy absorbed by the unit reaction center (*ABS*/*RC*), the unit reaction center captured for the reducing energy of *Q*<sup>A</sup> (*TR*o/*RC*), energy captured by the unit reaction center for electron transfer (*ET*o/*RC*), and energy dissipated in the unit reaction center (*DI*o/*RC*). The potential activity of photosystem II (PSII) was then calculated as *F*v/*F*<sup>o</sup> = (*F*m/*F*o) − 1. The maximum photochemical efficiency of PSII was calculated as *F*v/*F*<sup>m</sup> = 1 − (*F*o/*F*m). Lastly, the number of active reaction centers per unit area was calculated as *RC*/*CS*<sup>m</sup> = (*F*v/*F*m) (*V*J/*M*o) (*ABS*/*CS*m).
