Effects of Soil Exogenous Nitrogen on Bamboo Shoots, Photosynthetic Characteristics, and Nitrogen Metabolism in Dendrocalamus Latiflorus Munro


 BackgroundNitrogen (N) is an essential nutrient element that is involved in almost every aspect of a plant's physiological mechanism. Therefore, the current research aims to determine the optimal amount of N fertilizer to bamboo seedlings for better nutrient management practices to minimize N pollution in bamboo forests. We evaluated the physiological response of Dendrocalamus latiflorus Munro grown under five varying levels of N fertilizer; such as N0, N1, N2, N3, N4, and N5 (0, 1.5, 3.0, 4.5, 6.0, and 7.5 g·pot-1, respectively). ResultsN4 treatment had a significant effect on the number of shoots, which was greatly correlated with net photosynthetic rate (Pn) and photosynthetic pigment (Car, Chls, and Chl a/b) as well as N-related indices [leaf N, leaf ammonium N (NH4+-N), and nitrate reductase (NR)]. N supply significantly increased soil carbon and N contents, which could be conducive to the accumulation of leaf chlorophyll content, improving leaf photosynthesis mechanism, and accelerating N metabolism and conversion through an enzymatic reaction. ConclusionsOverall, the N application of 6 g·pot-1 was advantageous to improve physiological characteristics and shoot production of seedlings. As a consequence, we suggest that optimal nitrogen supply can be effective to improve soil fertility to attain high bamboo production.

Background Nitrogen (N) is an essential macronutrient element that is required by plants in higher amounts relative to other essential nutrients (Kirova et al. 2005). N is involved in almost every aspect of a plant's physiological metabolism being a most essential nutritional component (Kishorekumar et al. 2020a). At the physiological level, N may trigger both the NO 3 assimilatory mechanism and the regulation of carbon (C) metabolism that can provide C skeletons and reductants for this process (Stitt 1999). At the developmental stage, N regulates activities such as leaf expansion (Walch-Liu et al. 2000), root branching (Forde & Lorenzo 2002), and resource allocation between shoot and root growth (Scheible et al. 1997). N and other elements in the plant are responsible for the synthesis of amino acids, proteins, nucleic acids, chlorophyll, and other compounds (Talukder et al. 2016, Wen et al. 2020. Furthermore, N can also regulate the adaptation mechanism of plants to the environment and play an important role in the adaptation of plants to adversity (Krouk et al. 2010). Simultaneously, exogenous N can signi cantly affect the N metabolism in plants as well as a certain degree of impact on its related enzymatic activities, thereby affecting plant growth (Xing et al. 2018). N, being a necessary component, has the potential to change plant proteins as well as plant photosynthesis (Kishorekumar et al. 2020b, Liu et al. 2020. In general, when the N concentration increases, the leaf's photosynthetic rate (P n ) and transpiration rate (T r ) increases while the intercellular CO 2 concentration (C i ) decreases , Mu & Chen 2021. Furthermore, increasing the quantity of N fertilizer used may enhance the chlorophyll content of leaves and. In addition, increasing the amount of N fertilizer can also increase the leaf's chlorophyll contents and alleviate the reduction in photosynthetic e ciency (Zhou et al. 2017a). Previous studies have shown that N fertilizer application can effectively increase the chlorophyll contents and net photosynthetic rate of agricultural or horticultural crops, such as rice (Kurai et al. 2011, Zhou et al. 2017b), wheat (Kataria & Guruprasad 2015), peanut (Liu et al. 2019b), etc. As a result, in the current research, we attempt to gain insight into the photosynthetic capacity of Dendrocalamus lati orus Munro grown under varying N levels.
Soil is a part of the ecosystem, where the contents of essential nutrients can not only reveal the utilization of soil nutrients but also explore the regulatory and metabolic balance mechanisms of elements such as C and N in plants and soil (Kleinhenz et al. 2003). Since, both the C and N are essential nutrient elements in the soil and are required for plant growth and development (Krapp & Castaings 2012). Plants uptake the nutrients from the soil via roots, which are greatly in uenced by the external environment, especially by fertilizers (Bargaz et al. 2018). Therefore, the nutritional changes (C and N) in plants can explore the nutrient distribution ratio and status of plants, as well as the internal connection between soil and plants.
N metabolism is one of the important physiological metabolic processes in plants (O'Brien et al. 2016).
Nitrate reductase (NR) is the main N metabolizing enzyme of higher plants. The metabolic enzyme activity of plant leaves and roots is related to the accumulation of N in plants (Fu et al. 2020b).
Glutamine synthetase (GS) is the rst enzyme to be isolated, puri ed, and identi ed from plants, and it is also the rst enzyme to be found to be related to the storage form of plant N (Seabra & Carvalho 2015).
GS converts the inorganic ammonia absorbed by plants into an organic form in glutamine (Gln) and glutamic acid (Glu) as N donors in the biosynthesis of N-containing organics in higher plants (Yang et al. 2016). NR and GS are all inducible enzymes that affect the metabolism and transformation of N in plants, which play an important role in the absorption and transformation of nutrient elements to affect plant growth.
Fertilization is an important measure to improve bamboo cultivation. As one of the main factors affecting bamboo growth, N has a great in uence on the growth and development of bamboo. Previous research showed that optimum N fertilization can effectively improve photosynthetic capacity, growth, and productivity of shoots and timber in bamboo forests (Xu et al. 2014, Gao et al. 2016). At present, N fertilization in bamboo forests is relatively systematic in China, but the phenomenon of soil deterioration and environmental degradation caused by fertilization still exists (Zhu & Chen 2002). Increased use of N fertilizers will also exacerbate the increase in nitrous oxide (N2O) emissions from the soil (Xu et al. 2014).
In order to increase the yield of bamboo forests and protect the ecological environment, it is necessary to further improve the utilization e ciency of N fertilizer in bamboo forests. D. lati orus Munro has become the main cultivated bamboo species for shoots in China by the features of its strong adaptability, long shoot period, and high yield of bamboo shoots. D. lati orus Munro has a large leaf area and continuous rainfall in the early stage of bamboo shoots in southern China, which would exacerbate the lack of sunlight in the forest understory. Nutrient management can also alleviate the stress caused by low light, improve the photosynthetic capacity of bamboo, and increase its productivity. Therefore, in the current study, D. lati orus Munro seedlings were established under various levels of N-based fertilizer, to assess its e cacy on shoot growth. The study will provide valuable insights into optimum N requirements for the growth of D. lati orus Munro. The main goals of our experiment were (i) to distinguish the effect of different levels of N on physiological and biochemical attributes of D. lati orus Munro; (ii) to identify the effect of N application on soil properties and the growth pattern in D. lati orus Munro; (iii) to determine the reasonable amount of N applied to bamboo shoots.

Study sites
The greenhouse experiment was carried out in College of Forestry, Fujian Agriculture and Forestry. The study site is located (119°13′51.18″ E, 26°05′4.35″ N) in Fuzhou, Fujian province, China ( Figure. 1). The site has a subtropical monsoon climate, with an annual average temperature of 19.9 ℃, an extreme maximum temperature of 42 ℃, and a minimum temperature of 0 ℃. The annual average sunshine is 1 755.4 h, and the frost-free period is more than 360 days.

Plant material and soil
Two-year-old seedlings of D. lati orus were grown in PVC pots with a diameter of 30 cm and a height of 33 cm. The potting substrate material was yellow soil and peat (volume ratio 3:1) with a weight of 15 kg per pot. The basic properties of soil were as follows: pH value, 5.77; organic C, 13.67 g·kg -1 ; total N, 0.35 g·kg -1 ; total phosphorus (P), 0.50 g·kg -1 ; total potassium (K), 50.01 g·kg -1 . The basic growth attributes of D. lati orus Munro seedlings cultured from October 2018 were as follows: mean height, 105.51 cm; mean DBH, 4.67 mm; north-south crown, 71.11 cm; east-west crown, 68.95 cm.

Experiment design
In the current research, we used ve N treatment combinations; N0, N1, N2, N3, and N4, like 0, 1.5, 3, 4.5, 6, 7.5 g of N in each pot, respectively. N was applied three times such as April (30%), May (40%), and June (30%) 2019, respectively. The amount of exogenous N applied to the seedlings of D. lati orus Munro seedlings has been presented in Table 1. All treatments were replicated 15 times. Leaf gas exchange parameters were measured after six months, after that, the leaf tissues were collected for the estimation of chlorophyll content, N-related indices, and C content. Soil samples were also collected to measure Nrelated indices and C content. Urea (mass fraction 46%) was applied as N fertilizer, calcium superphosphate (P 2 O 5 , mass fraction 12%), and potassium chloride (K 2 O, mass fraction 60%) were used as phosphate and potassium fertilizer, respectively. Among them, phosphate and potassium fertilizer were applied only once, and the dosages were 6 and 1.5 g·pot -1 , respectively. Weeding and other cultural practices were carried out regularly during the entire experiment.

Investigation of the bamboo shoots
After every two days, the number of shoots from each replicate was recorded from beginning to end. The bamboo shoots were harvest after they attained a height of approximately 50 cm.

Determination of leaf gas exchange parameters and chlorophyll content
Li-6400 photosynthesis system (Li-Cor Inc., Lincoln, USA) was used to determine the gas exchange parameters [leaf net photosynthetic rate (P n ), stomatal conductance (Cond), intercellular CO 2 concentration (C i ), transpiration rate (T r ), and water use e ciency (WUE)] from 2-3 mature functional leaves during consecutive sunny days. The LED (red and blue) light source was used with 1600 μmol·m -2 ·s -1 photosynthetically active radiation (PAR). The readings were collected from all replicates, however, before the measurement, the sampling leaves were induced under a light intensity of 1600 μmol·m -2 ·s -1 PAR for 20-30 min.
Following the assessment of gas exchange parameters, fresh leaves were cut to determine the concentrations of the photosynthetic pigments. After the separation of the midrib, the fresh leaves were cut into pieces and weighed 0.2 g. A mixed solution (pure acetone: absolute ethanol: distilled water = 4.5: 4.5:1) of 25 ml (Gao 2006) was used to extract the photosynthetic pigments directly in the dark for 48 h. Chlorophyll a (Chl a), b (Chl b), total content (Chls), and carotenoid (Car) contents were calculated by optical density (OD) values according to the equations of Lichtenthaler (Lichtenthaler 1987), and the ratios of Chl a/b and Car/Chls were further calculated.

Determination of leaf N Indices and C content
After the extraction of leaf chlorophyll, part of fresh leaves was chopped, mixed, weighed, stored in liquid N, and then stored at -80℃ refrigerator for measuring. Fresh leaves were used to estimate the glutamine synthetase (Gs), nitrate reductase (NR), leaf ammonium N (NH 4 + -N), and nitrate N (NO 3 --N) using the kit manufactured by Suzhou Keming Biotechnology Co., Ltd. After chopping and mixing, another part of fresh leaves was oven-dried at 105°C for 15 minutes and later at 85 °C to dry to constant weight. Furthermore, the dried samples were grinded with an ultra-high-speed pulverizer (HUANGCHENG HC-300Y, China) and sieved through a 0.149 mm sieve. The total C and N contents were determined using an element analyzer (VARIO MAX, ELEMENTAR, Germany), and their ratio was further calculated.

Determination of soil C and N indices
Similarly, the fresh soil samples were taken from a distance of 10 cm and a depth of 10 cm around the bamboo stump. After removing stones and roots, each soil sample was separated into two portions: One portion was used to determine soil NH 4 + -N and NO 3 --N using the chemical kits. The other was air-dried, grinded, and sieved to 0.149 mm to determine total C and N contents using an element analyzer (VARIO MAX, ELEMENTAR, Germany), and their ratios were also calculated.

Data Analysis
All the data were expressed as means and standard errors. Analysis of variance (one-way ANOVA) was performed using SPSS 20.0 to determine the effect of N treatments, and Tukey HSD test was used to identify signi cant differences (α=0.05) between mean values. Origin 9.5 and Prism 8.0 were used for graphical illustrations.

Impact of N application on bamboo shoots
We noticed that N application signi cantly in uenced the number of bamboo shoots (Figure 2). D. lati orus Munro established under N4 treatments increased their shoots up to 68.75% (P 0.05) compared to N0.

Leaf photosynthetic pigments concentrations under various N application rates
Under various N application rates, the leaves photosynthetic pigments (Car, Chls, and Chl a/b) concentrations increased as depicted in Figure 3. Compared to N0, under N4 and N5, the increases of 100% and 109.60%, 99.08% and 110.52%, 14.01% and 13.13% (P 0.05) were observed for Chls, Car, and Chl a/b, respectively ( Figure 3A-C). Besides, for Car/Chls ratio, compared to N0, seedlings established under the N application did not show any difference ( Figure 3D).

Leaf gas exchange parameters under various N application rates
Page 7/22 The N application had a signi cant (P 0.05) effect on leaf P n ( Figure 4A). Speci cally, N4 and N5 treatments resulted in a signi cant rise in leaf P n relative to N0, whereas N4 was found the most effective over all other treatments combinations. In addition, the rise in Cond was parallel to the N application rate ( Figure 4B) and seedlings established under N4 greatly (P 0.05) enhanced their plant Cond (106.57%) as compared to N0. In contrast, compared to N0, none of N based treatments in uenced the C i and T r signi cantly ( Figure 4C, D). Besides, seedlings under N4 exhibited a relatively greater WUE compared to all other treatments ( Figure 4E).
The N and C indices of leaf and soil under various N application rates Compared to N0, N treated seedlings accumulated signi cantly (P 0.05) greater soil N and C contents ( Figure 5). Overall, N4 treated seedlings responded with maximum concentrations for leaf N and C contents (31.97 and 433.23 g·kg -1 , respectively) ( Figure 5A, B). In contrast, the N5 treatment for the contents of soil N and C were higher relative to other treatments ( Figure 5D, E). Except for N5, the leaf C/N ratio decreased under N application as the level of concentration increased ( Figure 5C). Additionally, seedlings amended with N4 treatment responded with maximum soil C/N ratio ( Figure 5F).

Leaf GS and NR activities under various N application rates
The activities of leaf GS and NR were highly in uenced by N application (Figure 6). Compared to N0, GS activities greatly (P 0.05) increased by 140.55% and 121.13% under N3 and N4 treatments ( Figure 6A). N application showed no signi cant impact on NR activities, while N3 and N4 enhanced NR activities by 82.73% and 70.43%, respectively, when compared to N0 ( Figure 6B).
Leaf and soil NH 4 + -N and NO 3 --N under various N application rates N application determined higher leaf NH 4 + -N in seedlings compared to N0 ( Figure 7A). N1, N2, and N3 treatments showed a signi cant (P 0.05) impact on soil NH 4 + -N, while N4 decreased soil NH 4 + -N by 18.88% compared to N0 ( Figure 7C). In contrast, there was a decline under N application amended seedlings for leaf and soil NO 3 --N ( Figure 7B, D).

Evaluation of all indices under different N treatments by principal component analysis (PCA)
Principle component analysis (PCA) revealed that the cumulative variance contribution rate of the rst two principal components reached 74.47%, suggesting an overall variation of the data (Fig. 8). PC1 showed that the number of shoots had a highly positive correlation with photosynthetic pigments (Car, Chls, and Chl a/b), gas exchange parameters (P n , Cond, T r , and WUE), and C and N metabolism related

Discussion
In the current study, we noticed that different N levels impacted the number of shoots, leaf photosynthetic characteristics, soil and leaf N-related indices in D. lati orus Munro seedlings. The optimal N application can substantially increase leaf N and photosynthetic pigments, as well as improve plant photosynthetic e ciency and leaf physiological activities (Peng et al. 2021). The increase in soil C and N contents is conducive to the accumulation of leaf chlorophyll content, improving leaf photosynthetic capacity, and accelerate C and N metabolism and conversion (Manna et al. 2005, Zhang et al. 2019) ultimately increasing the number of shoots, which was also con rmed by our research.
The formation of bamboo shoots is inseparable from the intensity of photosynthesis, N utilization, and transportation (Dordas & Sioulas 2008). Increasing the application of N fertilizer up to some extent may increase the chlorophyll contents, prolong the function period of the leaves, and improve the photosynthetic e ciency, thereby increasing the production capacity of the plant (Liu et al. 2019a, Fu et al. 2020a. The current study concluded that N application enhanced the chlorophyll accumulation and improved the photosynthetic e ciency in leaves (Figures 3, 4). For instance, N4 treatment had a positive effect on the accumulation of leaf chlorophylls and P n in leaves (Figures 3, 4), indicating that optimal N supply can effectively increase leaf chlorophyll contents, which was conducive to improving the photosynthesis mechanism of D. lati orus Munro seedlings.
In plants, nutrient elements play a vital role in maintaining leaf C balance and sustaining photosynthetic e ciency (Matthews et al. 2017). In the current research, N application signi cantly increased P n to x relatively large amounts of C for photosynthesis ( Figure 4A), which is closely related to N assimilation and metabolic processes (Huang et al. 2013. We reported that N4 was advantageous compared to other N treatments in terms of increasing P n to enhance the C sequestration ability of D. lati orus Munro ( Figure 4A). Higher Cond leads to enhancing leaf photosynthetic biochemical pathways and the accumulation of photosynthetic products (Lichtenthaler et al. 2007a, Lichtenthaler et al. 2007b. Compared with N0, the values of Cond, C i , T r, and WUE increased in a certain degree of N treatments ( Figure 4B-D), which can improve photosynthetic performance in the growth of D. lati orus Munro.
N application can effectively increase the contents and reserves of soil organic C and total N content (Huang et al. 2013). The C:N ratio in the soil can re ect the utilization e ciency of the soil, and the application of fertilizer can affect the nutrient element in the soil (Vitousek 1982). In our research, N treatments signi cantly improved N availability and increased soil N contents ( Figure 5D). Similarly, N provided a carbon source and improved environment in the soil, which promoted the conversion of organic C and increased the organic C content (Cheng et al. 2020), where all N treatments signi cantly increased soil C content compared to N0 ( Figure 5E). Additionally, previous studies have shown that there is a positive correlation between soil C and N content and crop yield (Wang et al. 2019, Zhang et al. 2019). Our research found that shoot, SC, and SN all had positive correlations (Figure 8).
In addition, some studies have shown that the application of N fertilizer signi cantly increases the N accumulation in leaves, thereby reducing the C to N ratio of the leaves (Liu et al. 2014, Wu et al. 2019, which are consistent with our research (Figure 5A-C). These changes would be conducive to increasing productivity, as well as changing the degradation and mineralization of leaves, which in turn would be conducive to the return of C and N to the soil (Stubbs et al. 2009, Pierik et al. 2011, to ensure the supply essential nutrient to meet the needs of bamboo shoot production. NR and GS are the key enzymes for N assimilation and ammonia assimilation in plant physiological processes (Xie et al. 2014, Lin et al. 2017. Zhu et al. (2016) found that fertilization can effectively promote the N metabolism of Phyllostachys edulis, and enhance the activities of both NR and GS, which is related to the direct promotion of fertilizers and the absorption of N by plants. Our research came to the same conclusion that the activities of NR and GS were enhanced with the application of N fertilization (Figure 6), which can improve transformation of N metabolism directly. Furthermore, N application would encourage plants to absorb more N to synthesize more NR and GS indirectly.
Compared to N0, the soil and leaf NH 4 + -N was at a signi cant level under N supply (Figure 7). Soil NH 4 + -N was greatly reduced under N4 treatment with comparison to NO 3 --N ( Figure 7C, D), indicating that roots of seedlings had higher absorption of NH 4 + -N. The enhanced NR activity under N application could promote the reduction of NO 3 to more NH 4 + (Maeda et al. 2014), which possibly promoted to accumulation of NH 4 + -N of leaves in our research ( Figures 6B, 7A). Additionally, compared with other N treatments, the enhanced activity of GS under N4 treatment could be conducive to catalyzing inorganic NH 4 + -N to producing more organic N ( Figure 6A), which could provide su cient N not only for bamboo shoot production but also to synthesize more chlorophyll to ensure the photosynthesis of seedlings (Cruz et al. 1993). However, NO 3 --N of soil and leaves had an insigni cant negative correlation with bamboo shoots ( Figure 8), which had an adverse effect on shoots. Our research was consistent with the previous ndings that NH 4 + -N can promote leaf chlorophyll synthesis and increase plant production (Sanchez-Zabala et al. 2015, Heuermann et al. 2021. The PCA analyzed with strong correlations may be screened out and can be used to assess D. lati orus Munro adaptation to varying N levels. According to the PCA results, N4 treatment seems to be advantageous for shoots growth (Figures 2, 8), owing to increased leaf photosynthetic characteristics and soil nutrients availability. In addition, the increased number of bamboo shoots under N4 treatment exhibited strong correlations with chlorophylls pigments and leaf N-related indices (Figure 8), which may regulate the photosynthesis mechanism and promote N accumulation in D. lati orus Munro to ful ll the requirements of a greater number of bamboo shoots.

Conclusions
Exogenous N has varying degrees of in uence on the photosynthetic characteristics, nutritional element contents, and N metabolism-related indices of D. lati orus Munro seedlings. We conclude that applying N fertilizer (6 g·pot -1 ) to maximize the growth and shoot production of D. lati orus Munro seedlings is advantageous. We elucidated the N absorption mechanism of the bamboo seedlings as well as the related physiological change mechanism after the application of N fertilizer. These ndings not only provide a theoretical basis for improving the N utilization e ciency of D. lati orus Munro but also the production of high-yield and high-quality bamboo shoots in the future.

Declarations
Availability of data and materials The data and materials presented in this study have been available in the article.

Competing interests
The authors declare that they have no competing interests Funding This work was supported by the "National Key R&D Program of China" (2018YFD0600100); program for scienti c and technological innovation teams for universities of Fujian province (No.2018[49]).

Authors' contributions
LLF performed all the experiments, analyzed the data, and wrote the manuscript. MWKT revised the manuscript. YZH and WFH helped in performing the experiments. JDR and TYH helped in designing the study. YSZ designed and directed the study. All authors have read and approved the nal manuscript.
Different letters indicate signi cant differences (P < 0.05) of mean between different N treatments.