**Yue Wang, Weiwei Jin, Yanhui Che, Dan Huang, Jiechen Wang, Meichun Zhao and Guangyu Sun \***

School of Life Sciences, Northeast Forestry University, Harbin 150040, Heilongjiang, China; wangyue@nefu.edu.cn (Y.W.); jinwei6677066@163.com (W.J.); carcar@nefu.edu.cn (Y.C.); 18804501876@163.com (D.H.); 15771398049@163.com (J.W.); 15846070751@163.com (M.Z.) **\*** Correspondence: sungy@nefu.edu.cn; Tel.: +86-0451-8219-1507

Received: 10 February 2019; Accepted: 30 March 2019; Published: 5 April 2019

**Abstract:** Nitrogen dioxide (NO2) is recognized as a toxic gaseous air pollutant. However, atmospheric NO2 can be absorbed by plant leaves and subsequently participate in plant nitrogen metabolism. The metabolism of atmospheric NO2 utilizes and consumes the light energy that leaves absorb. As such, it remains unclear whether the consumption of photosynthetic energy through nitrogen metabolism can decrease the photosynthetic capacity of plant leaves or not. In this study, we fumigated mulberry (*Morus alba* L.) plants with 4 <sup>μ</sup>L·L−<sup>1</sup> NO2 and analyzed the distribution of light energy absorbed by plants in NO2 metabolism using gas exchange and chlorophyll a fluorescence technology, as well as biochemical methods. NO2 fumigation enhanced the nitrogen metabolism of mulberry leaves, improved the photorespiration rate, and consumed excess light energy to protect the photosynthetic apparatus. Additionally, the excess light energy absorbed by the photosystem II reaction center in leaves of mulberry was dissipated in the form of heat dissipation. Thus, light energy was absorbed more efficiently in photosynthetic carbon assimilation in mulberry plants fumigated with 4 <sup>μ</sup>L·L−<sup>1</sup> NO2, which in turn increased the photosynthetic efficiency of mulberry leaves.

**Keywords:** nitrogen dioxide; nitrogen metabolism; photorespiration; heat dissipation; excess absorbed light energy; electron transfer; photochemical efficiency

## **1. Introduction**

Atmospheric nitrogen oxides (NOx) mainly include nitric oxide (NO), nitrogen dioxide (NO2), dinitrogen trioxide (N2O3), dinitrogen monoxide (N2O), and dinitrogen pentoxide (N2O5). Nitrogen oxides other than NO2 are extremely unstable and can be converted to NO2 in the presence of light, humidity, or heat [1]. NO2 sources are divided into natural and man-made sources. Natural sources mainly include lightning, stratospheric photochemistry, and microbiological processes in ecosystems. NO2 formed in nature is generally in ecological balance at a natural point of equilibrium, which is low relative to man-made air pollution [2]. The emission of NO2 from man-made sources is indeed the main component of atmospheric pollutants, which forms aerosol particles of nitric acid with particulate matter in the air. These aerosols form secondary pollution with pollutants from sources that include fossil fuel and biomass combustion as well as various electroplating, carving, welding, and other industrial emissions [3,4].

NO2 not only causes acid rain, but also changes the competition and species composition among wetland and terrestrial plant taxa, reduces atmospheric visibility, increases acidification and eutrophication of surface water, and increases the toxin content of fish and other aquatic organisms [5]. Cheng et al [6] found that tiny particles of water in the air act as incubators during hazy conditions and trap NO2 to interact and form sulfate. Additionally, stationary polluted weather systems accelerate chemical reactions, trapping near-surface NO2, leading to NO2 concentrations that are more than

three times higher than that found in sunny weather. This increase in aerosol mass concentration leads to an increase in water content, accelerating the accumulation of sulfate and causing severe haze. In addition, NO2 is also a respiratory system irritant. After being inhaled, NO2 first affects the respiratory organs, the lungs in particular. The combination of nitrite and nitric acid that occurs when NO2 encounters mucus membranes is a strong irritant with corrosive effects [7].

NO2 affects the normal growth of plants. When NO2 concentrations are higher than the annual average NO2 concentration limit of 53 ppb in the United States [8], NO2 can damage the leaves of plants, causing chlorosis in angiosperms, needle burns in conifers [9,10], reduced leaf area [11], and lower stem weights [12]. However, when the concentration of NO2 is lower than the average annual NO2 concentration of 53 ppb in the United States, the total leaf area, nutrient intake, and aboveground biomass were more than doubled [13,14]. Similar results have been found in different plant species, including *Arabidopsis thaliana* [15,16], tobacco (*Nicotiana plumbaginifolia* L.) [17], and crops such as lettuce (*Lactuca sativa* L.), sunflower (*Helianthus annuus* L.), cucumber (*Cucumis sativus* L.), and squash (*Cucurbita moschata* L.) [9]. In addition, atmospheric NO2 can shorten flowering periods in tomato (*Solanum lycopersicum* L. 'Micro-Tom'), increasing the number of flowers and the yield of the fruit [18].

In China, the concentration of NO2 emission, which caused formation of fine particulate matter (PM2.5), is not enough to injure tree plants. As a result, trees have been used to absorb atmospheric nitrogen dioxide to reduce atmospheric PM2.5 [19].

Photosynthesis is the foundation of plant growth and development, and the primary source of photosynthetic energy is light [20]. When the absorption of light energy is excessive, the excess excitation energy can harm the photosynthetic systems, causing photosynthetic inhibition, and even photooxidation and photodamage. Plants have multiple photoprotective mechanisms that reduce the potential harm of excess light energy to the photosynthetic apparatus under strong light. Reducing excess energy in addition to heat dissipation dissipates energy by other means [21]. Nitrogen metabolism and photorespiration also use and consume light energy or photosynthetic electrons absorbed by leaves [22,23]. However, it is unclear whether this consumption of photosynthetic energy will reduce the photosynthetic capacity of plant leaves and thereby hinder the growth and development of plants.

Mulberry, which has strong adaptability to the environment, has been an important economic tree species in China since ancient times. Nowadays, mulberry can be used for sericulture, new high-protein forage grass, fruit tree, and greening trees in northeast China [24–26]. We have studied the response of mulberry to atmospheric pollutant SO2 and found that mulberry is very sensitive to it [24]. We also studied the response characteristics of mulberry to nitrogen and obtained significant results in the absorption and metabolism of nitrogen by mulberry trees [27–30]. We think atmospheric NO2 also affects the nitrogen metabolism of mulberry trees. In the present study, we fumigated mulberry leaves with NO2 and assessed the impact on nitrogen metabolism, photorespiration, photosynthetic energy distribution, and electron flow distributions. Our assays characterized the response of photosynthetic efficiency to light energy used and consumed by mulberry plants, including the absorption of NO2 via nitrogen metabolism in vivo.

#### **2. Materials and Methods**

#### *2.1. Plant Material and Growth Conditions*

Seedlings of *Morus alba* L. were selected as experimental materials. Mulberry seeds were provided by the Heilongjiang Sericulture Institute of Heilongjiang Province in China. Seeds with strong, full, and uniform size were selected, disinfected with 75% ethanol for 3min, rinsed with distilled water for 5–6 times, and soaked with distilled water at 25 ◦C for 24 h. The seeds were blotted with sterile filler paper and sown into the seedling tray. Two seeds were sown in each hole. After germination and cultivation, the test seedlings were grown to a height of 10 cm and then transplanted into pots with a diameter of 12 cm and a height of 15 cm. Experimental seedlings were cultured in a seedling greenhouse with an average temperature of 28/25 ◦C (light/dark), light intensity of 400 <sup>μ</sup>mol m−2·s<sup>−</sup>1, photoperiod of 12 h/12 h (light/dark), and relative humidity of about 75%. The culture substrate

was uniformly mixed with peat and vermiculite, and irrigated with 800 mL of tap water every 2 days. In order to ensure relative consistency of the experimental materials, the branches and leaves of the mulberry seedlings were removed at the time of transplantation, such that only 5 cm of the main root and the main stem were preserved. Two plants were planted into each pot, with 80 plants in total cultivated. When the seedlings had grown to a height of 30–40 cm, 12 mulberry seedlings with uniform growth were selected for NO2 fumigation treatment.
