Nitrogen Application Alleviates the Adverse Effects of Defoliation Stress on Lolium perenne L. by Enhancing the Antioxidant System and Promoting Photosynthesis

: Perennial grasses undergo compensatory growth after defoliation. Nitrate is the main nitrogen source for the growth of perennial ryegrass and plays a signiﬁcant role in plant resistance to stress. The aim of the study was to understand the physiological mechanism of ryegrass in response to defoliation stress under different nitrate supplies and to explore possible ways to alleviate defoliation stress. We performed pot experiments where 12-week-old ryegrass plants grown in low (0.05 mM KNO 3 ) or moderate nitrate (5 mM KNO 3 ) conditions were defoliated and subsequently supplied with different concentrations of nitrate following defoliation treatments. During the regrowth stage, the regrowth rate, biomass, photosynthetic parameters, and the response of the antioxidant system to low or moderate nitrate supply of ryegrass were investigated. The results showed that moderate nitrate supply after defoliation increased the content of photosynthetic pigments in ryegrass and improved its photosynthetic efﬁciency. In addition, adding moderate nitrate after defoliation increased the activity of antioxidant enzymes and the accumulation of osmotic regulating substances, thereby enhancing plant resistance, effectively reducing the damage to plants caused by defoliation stress, and promoting plant regrowth, especially for plants grown in a low nitrate environment before defoliation. Therefore, this study showed that the addition of exogenous nitrate could counteract some of the adverse effects of defoliation stress on the growth and development of ryegrass.


Introduction
Perennial ryegrass (Lolium perenne) is one of the most important grasses in temperate regions of the world, which is widely used in pastures due to its easy establishment and high nutritional value [1]. Defoliation and grazing are important management methods for perennial grassland ecosystems, affecting grassland productivity, forage quality, and livestock growth and production. Forage grass undergoes compensatory growth after grazing or mowing to adapt to severe and frequent defoliation [2]. Since defoliation causes mechanical stress to plant leaves, triggering extravasation of cytosol from the defoliation part and an imbalance in the water balance of the plant, plants produce a series of morphophysiological adjustments after defoliation in response to the removal of leaf and stem tissues [3]. Studies have shown that perennial forage grasses improve stress resistance by activating their own antioxidant enzyme defense system and osmotic regulation [4]. However, in perennial ryegrass, little is known about whether leaf defoliation elicits an antioxidant response of the defense system in perennial ryegrass.
Defoliation leads to the loss of meristem of grass plants, reduces the photosynthesis of plants, and makes the carbon required by plant growth and regrowth unable to be satisfied [5]. To provide the necessary energy, ryegrass mobilizes carbohydrate reserves from the sheath and leaf base to maintain leaf regrowth [6][7][8]. During early regrowth, the conductivity of 1300 µS cm −1 of 0.05 mM (low nitrogen, LN) or 5 mM KNO 3 (moderate nitrogen, MN) as sole N source on the fourth day, and KCl was added to the low N solution to equalize K + concentrations thereby maintaining intracellular osmotic pressure balance between treatments. Four-day watering was one nitrogen treatment cycle. After the plants were grown for 12 weeks, plants grown in low-nitrogen (LN) and moderate-nitrogen (MN) conditions were randomly divided into 3 groups with 24 pots in each group, and different treatments were performed respectively ( Figure 1). For plants grown in the LN condition, we randomly selected 2 groups of them and defoliated the ryegrass 4.5 cm above the soil surface. One group was supplemented with 30 mL of half-strength Hoagland solution of 0.05 mM KNO 3 every two days (LN-Defoliation-LN), and the other group was administered 30 mL of half-strength Hoagland solution of 5 mM KNO 3 every two days (LN-Defoliation-MN). The remaining non-defoliated group of plants continued to receive 30 mL of the same nutrient solution as before (LN-Intact-LN). For plants grown in the MN condition, we also randomly selected 2 groups of them for defoliation treatment. After defoliation, we selected 1 group to supplement 30 mL of half-strength Hoagland solution of 0.05 mM KNO 3 every 2 days (MN-defoliation-LN), the other group to supplement 30 mL of half-strength Hoagland solution of 5 mM KNO 3 every 2 days (MN-defoliation-MN), and for the remaining non-defoliated plant group, we continued to irrigate 30 mL of the same nutrient solution as before (MN-Intact-MN). The defoliation time is defined as the time 0 of this experiment. Subsequently, perennial ryegrass root, stubble (0-4.5 cm above the ground), and leaf (4.5 cm above the ground) tissues were harvested in liquid nitrogen before and 2, 5, 10, and 17 days after defoliation, rapidly frozen and stored at −80 • C. Six independent plants subjected to each of the treatments were pooled as 1 biological replicate for further analysis. At least 3 biological replicates were performed.
Agronomy 2022, 12, x FOR PEER REVIEW 3 of 17 N treatment was started 5 days after perennial ryegrass had germinated. During the treatment period, perennial ryegrass was watered with 30 mL of water on the second day and 30 mL of half-strength Hoagland solution with a pH of 6.0 and an electrical conductivity of 1300 µS cm -1 of 0.05 mM (low nitrogen, LN) or 5 mM KNO3 (moderate nitrogen, MN) as sole N source on the fourth day, and KCl was added to the low N solution to equalize K + concentrations thereby maintaining intracellular osmotic pressure balance between treatments. Four-day watering was one nitrogen treatment cycle. After the plants were grown for 12 weeks, plants grown in low-nitrogen (LN) and moderate-nitrogen (MN) conditions were randomly divided into 3 groups with 24 pots in each group, and different treatments were performed respectively ( Figure 1). For plants grown in the LN condition, we randomly selected 2 groups of them and defoliated the ryegrass 4.5 cm above the soil surface. One group was supplemented with 30 mL of half-strength Hoagland solution of 0.05 mM KNO3 every two days (LN-Defoliation-LN), and the other group was administered 30 mL of half-strength Hoagland solution of 5 mM KNO3 every two days (LN-Defoliation-MN). The remaining non-defoliated group of plants continued to receive 30 mL of the same nutrient solution as before (LN-Intact-LN). For plants grown in the MN condition, we also randomly selected 2 groups of them for defoliation treatment. After defoliation, we selected 1 group to supplement 30 mL of half-strength Hoagland solution of 0.05 mM KNO3 every 2 days (MN-defoliation-LN), the other group to supplement 30 mL of half-strength Hoagland solution of 5 mM KNO3 every 2 days (MNdefoliation-MN), and for the remaining non-defoliated plant group, we continued to irrigate 30 mL of the same nutrient solution as before (MN-Intact-MN). The defoliation time is defined as the time 0 of this experiment. Subsequently, perennial ryegrass root, stubble (0-4.5 cm above the ground), and leaf (4.5 cm above the ground) tissues were harvested in liquid nitrogen before and 2, 5, 10, and 17 days after defoliation, rapidly frozen and stored at −80 °C. Six independent plants subjected to each of the treatments were pooled as 1 biological replicate for further analysis. At least 3 biological replicates were performed.

Plant Regrowth, Biomass, Nitrate Uptake Rate and Nitrogen Contents
Plant height, defined as the distance from the soil surface to the top of the uppermost leaf blade, was measured every day after defoliation. The growth rate of the plant was defined as the height of the plant regrowth per hour from the time 0 of the experiment after defoliation. The leaf width and tiller number were measured 1 day before and 17 days after defoliation. The aboveground and belowground biomass of individual plants was harvested on the 17th day. Then we brought them to the laboratory for oven drying at 70 °C to a constant weight, with three biological replicates of 6 independent plants in each treatment. The rate of nitrate uptake by roots was determined by 15 N labeling. At 2 days after defoliation, the plant was gently sucked onto a paper towel and then immediately washed with 0.1 mM CaSO4 for 1 min to remove any compounds adsorbed on the

Plant Regrowth, Biomass, Nitrate Uptake Rate and Nitrogen Contents
Plant height, defined as the distance from the soil surface to the top of the uppermost leaf blade, was measured every day after defoliation. The growth rate of the plant was defined as the height of the plant regrowth per hour from the time 0 of the experiment after defoliation. The leaf width and tiller number were measured 1 day before and 17 days after defoliation. The aboveground and belowground biomass of individual plants was harvested on the 17th day. Then we brought them to the laboratory for oven drying at 70 • C to a constant weight, with three biological replicates of 6 independent plants in each treatment. The rate of nitrate uptake by roots was determined by 15 N labeling. At 2 days after defoliation, the plant was gently sucked onto a paper towel and then immediately washed with 0.1 mM CaSO 4 for 1 min to remove any compounds adsorbed on the root surface and then exposed to a basic nitrogen-free Hoagland medium added with 0.05 mM or 5 Mm 15 N-labeled KNO 3 (atomic% 15 N: 10%) for 1 h [6]. At the end of the incubation period, roots were immediately washed with 0.1 mM CaSO 4 for 1 min, frozen in liquid nitrogen, and stored at −80 • C. The 15 N and total nitrogen contents in the samples were determined using an isotope ratio mass spectrometry.

Photosynthetic Pigments and Anthocyanin Content Measurement
Chlorophyll and carotenoid contents were extracted with 100% acetone from leaf powder ground with liquid nitrogen, according to Lichtenthaler [28]. All extractions and measurements were performed under low light conditions, and the absorbance was measured with a spectrophotometer at 661.6 nm, 644.8 nm and 470 nm, respectively. All spectrophotometric analyses were conducted on a SHIMADZU UV-2600 spectrophotometer. Anthocyanin contents were extracted using a method from Rabino and Mancinelli [29].

Gas Exchange Measurement
Photosynthetic rate, stomatal conductance, intercellular carbon dioxide concentration, and transpiration rate of treated plants were measured on fully expanded leaves by using the LI-6400 photosynthesis system (Li-Cor, Inc., Lincoln, NE, USA). During the measurements, leaf temperature was maintained at 23 ± 0.5 • C, the vapor pressure deficit was 2.8 ± 0.05 kPa, steady photosynthetic photon flux density was set as 1000 µmol m −2 s −1, and the reference CO 2 in the cuvette was 400 ± 5 µmol mol −1 and the airflow was constant at 500 µmol s −1 . Measurements were performed on at least five independent plants per treatment.

Chlorophyll Fluorescence Measurement Analysis
After dark-adapting plants for a minimum of 30 min, a Dual-PAM-100 fluorometer (Walz, Effeltrich, Germany) was used to measure the maximum photochemical efficiency of photosystem II (PSII; Fv/Fm), the quantum yield of PSII (ΦPSII), the electron transfer rate (ETR), photochemical quenching coefficient (qP) and non-photochemical quenching coefficient (NPQ). Photosynthesis measurements were performed on fully expanded leaves on at least five independent plants per treatment.

Assays of Antioxidant Enzyme Activity and Soluble Protein
For extraction of antioxidant enzymes and soluble protein, leaf powder samples were homogenized with 50 mM NaH 2 PO 4-Na 2 HPO 4 buffer (pH = 7.00) containing 0.2 mM EDTANa 2 and 1% polyvinylpyrrolidone (PVP). The resultant supernatant was used for the determination of Ascorbate peroxidase (APX), Superoxide dismutase (SOD), Catalase (CAT) and Peroxidase (POD) activity, as well as the soluble protein content.
APX activity (EC 1.11.1.11) was estimated using the method described by Nakano and Asada [30]. The decrease in absorbance was recorded at 290 nm. One-unit APX activity was defined as the absorbance change of 0.01 units per minute [31]. The activity of SOD (EC 1.15.1.1) was determined as the ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT). Absorbance was recorded at 560 nm, and 1 unit of SOD activity was defined as the amount of enzyme required to inhibit 50% of the photoreduction of NBT [32]. CAT (EC 1.11.1.6) activity was obtained based on the decline in absorbance at 240 nm for 3 min due to the decomposition of H 2 O 2 according to the method of Maehly and Chance [33], and the absorbance change of 0.01 units per minute was defined as one unit CAT activity. For POD (EC 1.11.1.7) activity, the increase in absorbance at 470 nm was recorded due to the oxidation of guaiacol [32]. One unit POD activity was defined as the absorbance change of 0.01 units per minute. The soluble protein concentrations were determined via the Coomassie brilliant blue G-250 staining method [34]. Absorbance was recorded at 595 nm with bovine serum albumin as a protein standard.

Determination of Soluble Sugar Content, Proline Content and Lipid Peroxidation
The soluble sugar was extracted from boiling leaf tissue samples in a water bath for 1 h and determined by the anthrone-sulfuric acid method [35]. The absorbance was measured at 630 nm, and the amount of soluble sugar was calculated based on a standard curve with sucrose as a standard. Proline content was determined by the sulfonylsalicylic acid method following Du et al. [36]. The degree of membrane lipid peroxidation was measured in terms of malondialdehyde (MDA) content by the method of Buege and Aust [37].

RNA Isolation and Quantitative RT-PCR
Total RNA was extracted from 100 mg of frozen sample powder using the RNase Plant Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. The extracted RNA was then converted into cDNA using a quantitative reverse transcription kit (Qiagen). Reverse transcription-quantitative PCR (RT-qPCR) was used to detect the relative expression of the nitrate transporter (NRT) gene in the treatment groups. The Nitrate transporters were identified by our previous study [6], and the primers for LpNRT2.1 (Forward: GCTGGTGGTAACGTGGGTGCAG, Reverse: AGCGGGAGTTCTCKGCRAACTTTTG) was designed by Primer Premier 6.20. Three biological replicates were performed, and 3 independent plants were pooled as 1 biological replicate. Primer The reaction system was a 15 µL volume system containing 1 µL of 10-fold diluted cDNA, relevant primers and homemade SYBR Green master mix. The relative expression (fold change) of the target gene was corrected using the geometric means of the 2 reference genes, LpELONGATION FACTOR (eEF − 1α) and LpGAPDH, and calculated using the 2 −∆∆Ct method as described in a previous study [6].

Statistical Analysis
SPSS 26.0 software was used for the statistical analysis of the data, and Graphpad prism 9.3 was used for the production of the figures. The N content was analyzed statistically using the t-test. In addition, in the case of normally distributed data and homogeneous variance, one-way analysis of variance (ANOVA) with least significant difference (LSD) multiple comparison test at the 0.05 level of probability was used to analyze the remaining data.

Comparison of Plant Regrowth under LN and MN Treatments
The effect of nitrogen addition on ryegrass regrowth after defoliation was assessed. The growth rate of plants after defoliation was much higher than that of intact plants (

Effect of Defoliation and Nitrogen Treatments on Photosynthetic Pigments and Gas Exchange Parameters
Before defoliation, the nitrogen content of MN plants was significantly higher than that of LN plants, both in the stubble and in the roots (Figure 3a). After defoliation, the content of photosynthetic pigments in plants changed significantly with the supply of different nitrate concentrations. The 5 mM N addition following defoliation resulted in more chlorophyll content in the regrowth leaf tissue of plants, which was more apparent in LN plants than MN plants (Figure 3b

Effect of Defoliation and Nitrogen Treatments on Photosynthetic Pigments and Gas Exchange Parameters
Before defoliation, the nitrogen content of MN plants was significantly higher than that of LN plants, both in the stubble and in the roots (Figure 3a). After defoliation, the content of photosynthetic pigments in plants changed significantly with the supply of different nitrate concentrations. The 5 mM N addition following defoliation resulted in more chlorophyll content in the regrowth leaf tissue of plants, which was more apparent in LN plants than MN plants (Figure 3b Compared with intact plants, the net photosynthetic rate of regrowth leaves was increased following defoliation, especially in plants grown in low nitrate conditions ( Table  1). The addition of 5 mM nitrate following defoliation significantly induced the net photosynthetic rate of plants grown in LN condition. The net photosynthetic rate and leaf chlorophyll content of plants showed a similar trend among different treatments ( Figure  3b and Table 1). Compared with plants grown in MN conditions, the LN plants were more sensitive to the defoliation treatment. Similarly, stomatal conductance and transpiration rate also showed similar dynamic changes with the net photosynthetic rate under different nitrogen addition following defoliation (Table 1).     Compared with intact plants, the net photosynthetic rate of regrowth leaves was increased following defoliation, especially in plants grown in low nitrate conditions ( Table 1). The addition of 5 mM nitrate following defoliation significantly induced the net photosynthetic rate of plants grown in LN condition. The net photosynthetic rate and leaf chlorophyll content of plants showed a similar trend among different treatments (Figure 3b and Table 1). Compared with plants grown in MN conditions, the LN plants were more sensitive to the defoliation treatment. Similarly, stomatal conductance and transpiration rate also showed similar dynamic changes with the net photosynthetic rate under different nitrogen addition following defoliation (Table 1).

Chlorophyll Fluorescence Response under Defoliation and Nitrogen Treatments
To further characterize the photosynthetic apparatus, the photoresponse curves of PSII quantum yield (ΦPSII), electron transport rate (ETR), and redox state of the QA electron acceptor of PSII (1 − qP) and Fv/Fm were analyzed following 16 days after defoliation. (Figure 4 and Figure S2). The ΦPSII and ETR were much higher in the LN-defoliation-MN treatment group (Figure 4a,b). In contrast, 1 − qP was significantly reduced in the LN-Defoliation-MN plants, while the continued 0.05 mM N addition of LN plants after defoliation resulted in the highest 1 − qP among all treatments (Figure 4c). We also monitored non-photochemical quenching (NPQ) induction during light and dark transition periods. After dark adaptation, plants were exposed to 450 µmol of photons m −2 s −1 for 5 min to analyze the induction and relaxation of NPQ (Figure 4d). The results showed that the NPQ induction value of LN plants under the condition of adding 5 mM N after defoliation was significantly higher, and the induction rate was slower than that of plants with 0.05 mM N addition after defoliation.

Plant Antioxidant Responses under Defoliation and Nitrogen Treatments
In order to explore the role of nitrogen addition following defoliation in the antioxidant defense system, the activities of several antioxidant enzymes ten days after defoliation were measured ( Figure 5 and Figure S3). The activity levels of APX, CAT, SOD, and POD reflect the ability of plants to clear reactive oxygen species. Ten days after defoliation, the APX, CAT and POD activities of the plant stubble were significantly enhanced with the addition of 5 mM nitrate compared with the addition of 0.05 mM N, even exceeding the activities of intact plants, especially in LN plants (Figure 5d-f). In the leaves of MN plants, the patterns of APX, CAT, and POD activity were consistent with that of the stubble (Figure 5a-c). In LN plants, the activities of CAT and POD increased obviously with the addition of 5 mM N,  Figure S3a). resulted in the highest 1−qp among all treatments (Figure 4c). We also monitored nonphotochemical quenching (NPQ) induction during light and dark transition periods. After dark adaptation, plants were exposed to 450 µmol of photons m −2 s −1 for 5 min to analyze the induction and relaxation of NPQ (Figure 4d). The results showed that the NPQ induction value of LN plants under the condition of adding 5 mM N after defoliation was significantly higher, and the induction rate was slower than that of plants with 0.05 mM N addition after defoliation. Different letters indicate significant differences (p < 0.05); the same letter indicates no significant differences between the treatments.

Plant Antioxidant Responses under Defoliation and Nitrogen Treatments
In order to explore the role of nitrogen addition following defoliation in the antioxidant defense system, the activities of several antioxidant enzymes ten days after defoliation were measured (Figures 5 and S3). The activity levels of APX, CAT, SOD, and POD reflect the ability of plants to clear reactive oxygen species. Ten days after defoliation, the APX, CAT and POD activities of the plant stubble were significantly enhanced with the addition of 5 mM nitrate compared with the addition of 0.05 mM N, even exceeding the activities of intact plants, especially in LN plants (Figure 5d-f). In the leaves of MN plants,

Changes in Anthocyanin Content under Defoliation and Nitrogen Treatments
The anthocyanin concentration of LN plants was higher than that of MN plants under the influence of different amounts of nitrogen supply, and the anthocyanin content of regrown leaves of plants exposed to the defoliation was significantly lower than that of old leaves (Table 2). Meanwhile, the accumulation of anthocyanins in the regrown leaves of LN plants was enhanced significantly under the 0.05 mM N addition compared with the 5 mM N addition after defoliation, showing a similar pattern to that of MN plants ( Table 2). After defoliation, the anthocyanin content in the stubble of LN plants was strongly increased under the 0.05 mM N addition, while it significantly decreased under the 5 mM N addition ( Table 2). After the fifth day of regrowth, the anthocyanin content in the stubble of MN plants had little difference (Table 2). In contrast, anthocyanin content in the stubble of MN plants was apparently increased in the treatment with 0.05 mM N addition after defoliation on the 10th day, while this enhancement was alleviated with 5 mM N addition (Table 2). the patterns of APX, CAT, and POD activity were consistent with that of the stubble (Figure 5a-c). In LN plants, the activities of CAT and POD increased obviously with the addition of 5 mM N, while the activities of APX and SOD were inhibited, showing opposite trends (Figures 5a-c and S3a).

Changes in Anthocyanin Content under Defoliation and Nitrogen Treatments
The anthocyanin concentration of LN plants was higher than that of MN plants under the influence of different amounts of nitrogen supply, and the anthocyanin content of regrown leaves of plants exposed to the defoliation was significantly lower than that of old leaves (Table 2). Meanwhile, the accumulation of anthocyanins in the regrown leaves of LN plants was enhanced significantly under the 0.05 mM N addition compared with the 5 mM N addition after defoliation, showing a similar pattern to that of MN plants ( Table  2). After defoliation, the anthocyanin content in the stubble of LN plants was strongly increased under the 0.05 mM N addition, while it significantly decreased under the 5 mM

Response of Lipid Peroxidation and Proline Concentration to Defoliation and Differential N Supply
The malondialdehyde and proline content in the stubble of LN plants and MN plants was investigated after ten days of regrowth to show the degree of plant recovery after defoliation ( Table 3). The MDA content in the stubble of LN plants was decreased under the addition of 5 mM N, whereas there was no significant difference in MN plants (Table 3). Meanwhile, the free proline content in LN plants was greatly increased in the addition of 5 mM N after defoliation, suggesting improved tolerance to defoliation treatment.  Values are means ± SE (n = 3 pools of six plants each). Different letters indicate significant differences (p < 0.05); the same letter indicates no significant differences between the treatments.

Effect of Defoliation and Nitrogen Treatments on the Content of Soluble Sugar and Protein in Ryegrass
In comparison with intact plants, a significant reduction in soluble sugar content was observed in LN and MN plants after defoliation ( Table 2). The addition of 5 mM N in LN plants after defoliation resulted in a significant reduction in the soluble sugar concentration of the stubbles and leaves compared with the 0.05 mM N addition (Table 2). However, there was little difference between different nitrogen treatments after defoliation in MN plants ( Table 2). In addition, the soluble protein content of leaves and stubbles was significantly enhanced under the 5 mM N addition after the defoliation of LN plants and the leaves of MN plants (Table 3).

Nitrate Uptake Rate and NRT Transcript Levels
To better understand the changes in nitrate uptake in response to defoliation and nitrogen addition, nitrate uptake rates and NRT gene expression two days after defoliation was measured ( Figure 6). The results showed that the rate of nitrate uptake in the first two days of plant regrowth was significantly reduced by defoliation stress, and the decrease was more pronounced in MN plants (Figure 6a). As a candidate gene of ryegrass high-affinity transport system (HATS), LpNRT2.1 is more susceptible to inducible expression in a low nitrogen environment. In our findings, LpNRT2.1 was strikingly higher in LN plants than in MN plants, and the expression of LpNRT2.1 was significantly down-regulated by the addition of 5 mM N after defoliation (Figure 6b). two days of plant regrowth was significantly reduced by defoliation stress, and the decrease was more pronounced in MN plants (Figure 6a). As a candidate gene of ryegrass high-affinity transport system (HATS), LpNRT2.1 is more susceptible to inducible expression in a low nitrogen environment. In our findings, LpNRT2.1 was strikingly higher in LN plants than in MN plants, and the expression of LpNRT2.1 was significantly downregulated by the addition of 5 mM N after defoliation (Figure 6b).  −1α) and LpGAPDH, and calculated using the 2 -∆∆Ct method. Values are means ± SD (n = 3 pools of six plants each). Different letters indicate significant differences (p < 0.05); the same letter indicates no significant differences between the treatments.

Discussion
Growth, development, and regulation directly depend on micro-and macronutrients to maintain proper photosynthesis and physiology in plants [19,38]. Nitrogen limitation is a key factor leading to plant growth dysfunction [19]. At the same time, nitrogen is an indispensable component for plants to resist adversity [20,22,24]. In this study, we found that the regrowth rate, leaf width, tiller number, and biomass of LN plants after defoliation were significantly enhanced under the addition of 5 mM N ( Figure 2). Therefore, to understand the specific physiological responses of perennial ryegrass that alleviated defoliation stress when plants were supplied with 5 mM N, our study investigated the plant photosynthetic response and antioxidant defense system under both moderate and low nitrogen supply. Each data point is corrected using the geometric means of the two reference genes, LpELONGATION FACTOR (eEF − 1α) and LpGAPDH, and calculated using the 2 −∆∆Ct method. Values are means ± SD (n = 3 pools of six plants each). Different letters indicate significant differences (p < 0.05); the same letter indicates no significant differences between the treatments.

Discussion
Growth, development, and regulation directly depend on micro-and macronutrients to maintain proper photosynthesis and physiology in plants [19,38]. Nitrogen limitation is a key factor leading to plant growth dysfunction [19]. At the same time, nitrogen is an indispensable component for plants to resist adversity [20,22,24]. In this study, we found that the regrowth rate, leaf width, tiller number, and biomass of LN plants after defoliation were significantly enhanced under the addition of 5 mM N ( Figure 2). Therefore, to understand the specific physiological responses of perennial ryegrass that alleviated defoliation stress when plants were supplied with 5 mM N, our study investigated the plant photosynthetic response and antioxidant defense system under both moderate and low nitrogen supply.

Nitrate Supply Significantly Affects the Establishment of Ryegrass Leaf Photosynthesis after Defoliation
As important photosynthetic pigments, chlorophyll and carotenoids participate in the absorption, transmission, and distribution of light energy in photosynthesis and play a key role in plant growth and development [39]. Increasing nitrogen application can enhance the content of chlorophyll and carotenoids, thereby increasing the yield of photoassimilates and the growth rate [40][41][42]. This is consistent with our study that MN addition significantly promoted chlorophyll and carotenoid contents in plant leaf parts after defoliation (Figure 3b-e). The chlorophyll content of regrowth leaves was significantly higher than that of older leaves of intact plants because the decline of chlorophyll content could present the beginning of senescence [40]. Photosynthetic efficiency depends on photosynthetic pigment content [43]. As chlorophyll and carotenoid content of plants grown under LN conditions supplied by moderate nitrogen following defoliation increased, the net photosynthetic rate also improved significantly (Figure 3b-e and Table 1). Previous studies have also shown a lower photosynthetic rate of maize, rice, and rapeseed (Brassica campestris) when plants were grown under nitrogen starvation conditions that did not meet their respective recommended nitrogen application levels [19,44,45]. In the present study, we also found that the stomatal conductance and transpiration rate of perennial ryegrass leaves were significantly increased under a supply of moderate nitrogen after defoliation (Table 1), which was consistent with the studies of Shen et al. [46] and Yue et al. [47]. These results further demonstrated that the change of photosynthetic rate under nitrogen deficiency could be due to stomatal limitation, but non-stomatal limitation, such as the membrane structural integrity of the photosynthetic apparatus, the energy supply of the photosynthetic reaction center and the related enzyme activities, also affected the net rate of plant photosynthesis [48]. Therefore, under the condition of defoliation stress, adding an appropriate amount of nitrate to increase the chlorophyll content might be an important way to improve plant photosynthesis and further alleviate the defoliation stress of perennial ryegrass.
Chlorophyll fluorescence assay is a powerful tool for analyzing the photosynthetic response of plants under abiotic stress, reflecting the basic functions of the photosynthetic apparatus and the performance of photosynthesis [19,49]. Appropriate nitrogen application rate can ensure higher photosynthetic pigment content, enhance light energy capture ability, improve the photochemical efficiency and the ratio of the open reaction center of PSII, and improve the quantum efficiency and self-protection ability of PSII [11]. Our results emphasized that the actual quantum yield of PSII photochemistry (ΦPSII) and electron transport rate of PSII photochemistry (ETRII) were efficiently enhanced under moderate nitrogen addition following defoliation (Figure 4a,b). This mechanism may be related to the catalytic effect of nitrogen fertilizer on the activity of photoactivated enzymes in leaves, thereby enhancing the energy capture efficiency of the PSII reaction center [50]. At the same time, the QA electron acceptor of PSII (1 − qP) under LN plants with 0.05 mM N addition following defoliation was much higher than that of LN plants with 5 mM N addition (Figure 4c), reflecting a more highly oxidized plastoquinone pool in LN-Defoliation-LN plants. The induction kinetics of NPQ showed that the NPQ under LN-Defoliation-MN plants was induced faster, and the values were significantly higher compared with the LN plants with 0.05 mM N addition after defoliation. After switching to darkness, the NPQ relaxation of LN-Defoliation-MN plants was also faster than that of LN plants with 0.05 mM N addition, indicating that nitrogen application after defoliation alleviated the damage to the photosynthetic system caused by defoliation stress (Figure 4d). The above findings were consistent with the study by Mu et al. [51] that both ΦPSII, qP and ETR were evidently decreased in maize under low nitrogen supply.

Moderate Nitrate Supply Alleviates Defoliation Stress by Enhancing Antioxidant Defense System
Plant redox homeostasis is disrupted under stress conditions [52]. As a result of defoliation, plant leaves will be mechanically damaged, the photosynthetic leaf area will be reduced, and the cytosol will extravasate, which will eventually lead to the imbalance of leaf energy and water. At this time, the photosynthetic rate of the regrowth leaves of the plant will increase rapidly. The photosynthetic process can trigger the accumulation of oxygen free radicals, thereby activating the antioxidant enzyme protection system [53]. Meanwhile, low concentrations of nitrogen induce senescence and reactive oxygen species (ROS) production in plant cells [54]. ROS can cause lipid peroxidation and damage cell membranes [55]. Nitrogen deficiency directly affects the production of antioxidant enzymes, and once the ability of plants to scavenge ROS is lower than the production of ROS, it will lead to oxidative stress [56]. Our study showed that the CAT and POD activities in leaves and stubbles of LN plants under moderate nitrate supply after defoliation were significantly increased compared with LN-Defoliation-LN plants ( Figure 5). Moreover, the content of MDA, the membrane lipid peroxidation product, was significantly decreased in stubbles of LN-Defoliation-MN plants after defoliation (Table 3). Additionally, the SOD activity of ryegrass was decreased under the addition of moderate nitrate after defoliation (Supplementary Figure S3). This was consistent with the study by Garrido et al. [57] that the defoliation of plants resulted in a decrease in SOD activity. Anthocyanins can be induced and accumulated under abiotic stress conditions [58,59]. In this study, we found that the anthocyanin content of LN plants was significantly reduced under the addition of moderate nitrate after defoliation ( Table 2), indicating that nitrogen addition alleviated the damage to plants caused by defoliation stress. The accumulation of osmotic regulatory substances maintains the normal metabolic environment of cells and reduces damage when plants are under stress. The addition of moderate nitrate compared with low nitrate resulted in an increase in soluble protein and proline in LN plants after defoliation (Tables 2 and 3). The above results showed that the supply of moderate nitrate after perennial ryegrass defoliation might occur mainly through the enhancement of the activities of two antioxidant enzymes, CAT and POD, and the two osmotic regulators, soluble protein and proline, to scavenge newly generated ROS, maintain cellular water balance, prevent membrane damage, and mitigate oxidative damage.
In addition, we found that on the second day of regrowth, the nitrate uptake rate of MN plants decreased significantly (Figure 6a), which was consistent with the study of Guo et al. [6]. In the early stage of regrowth, the nitrate absorption and assimilation ability of perennial ryegrass were limited [6]. Studies have shown that after plant defoliation, when photosynthesis fails to meet the needs of plants for carbon sources, stored carbohydrates will migrate, and carbon migration will occur through fructan hydrolysis [7]. Fructans stored in the growing area of leaves are hydrolyzed and recirculated in this area to maintain leaf growth immediately after defoliation [8]. However, it is the depletion of carbohydrate reserves that limits the uptake of nitrate in ryegrass in the early stages of regeneration [6]. This was also shown in our results. The soluble sugar content of ryegrass decreased significantly after defoliation compared with that of intact plants, and the decrease was greater in plants with moderate nitrogen addition after defoliation. Guo et al. [6] showed that with the degradation and depletion of fructans after defoliation treatment for 48 h, the expression of the nitrate transporter gene LpNRT2.1, which is responsible for high affinity, was significantly inhibited by the regulation of carbon and nitrogen balance, which was consistent with our research results. After the addition of moderate nitrogen, the expression of LPNRT2.1 in LN-defoliation-MN plants was significantly down-regulated ( Figure 6b). This may be due to the mobilization of stored carbohydrates for regrowth after plant defoliation [6,7], thus reducing the energy supply for nitrate uptake. However, the addition of exogenous glucose remedied the inhibition of nitrate absorption efficiency caused by defoliation [6], which further proved the dependence of the nitrate absorption system on carbon sources in plants.

Conclusions
Defoliation, as the main utilization method of perennial grassland, will cause certain mechanical stress to grassland plants. In our study, the addition of 5 mM nitrate after defoliation promoted the increase of chlorophyll content, which enhanced photosynthesis and photosynthetic capacity, promoted plant regrowth, and also enhanced plant resistance by improving antioxidant enzyme activities and osmotic regulatory substances, thereby mediating the damage of defoliation stress. This suggests that nitrate addition after defoliation in perennial ryegrass alleviated defoliation stress by modulating photosynthesis and antioxidant defense systems. Therefore, nitrogen is beneficial to the growth and development of perennial ryegrass, and proper management of nitrogen after plant defoliation is important, especially for plants grown in nitrogen-deficient areas. As our understanding of nitrogen and defoliation responses continues to deepen, we can apply this knowledge to pasture production for grazing to improve pasture yield. In addition, it is necessary to further explore the appropriate amount and time of nitrate addition to ryegrass after defoliation, as well as its molecular regulation mechanism to optimize the nitrogen use efficiency of ryegrass.