Effects of different nitrogen forms and concentrations on seedling growth traits and physiological characteristics of Populus simonii × P.nigra

Zhongye Liu·Wei Li·Zhiru Xu·Huihui Zhang·Guangyu Sun·Heng Zhang·Chuanping Yang·Guanjun Liu

Abstract Numerous growth and physiological variables of 3-week-old Populus simonii ×P.nigra seedlings were assessed after treatment with either nitrate nitrogen(NO3--N) (0.1，0.5，1，5，or 10 mmol·L-1) or ammonium nitrogen (NH4+-N) (0.1，0.5，1，5，or 10 mmol·L-1) to determine the best nitrogen form and concentration to optimize growth，biomass allocation，pigment content，and photosynthetic capacity.The results of combining membership function and an evaluation index suggested that，5 mmol·L-1 nitrogen，regardless of the form，yielded the highest comprehensive evaluation index and good growth.In addition，a Pearson correlation analysis and network visualization revealed that the total mass，shoot mass，root mass，leaf dry mass，plant height，leaf area，chlorophyll a and total chlorophyll had a physiological index connectivity degree ≥ 15 for both nitrogen forms.Net photosynthetic rate，stomatal conductance，transpiration rate，maximum photochemical efficiency of PSII，total nitrogen content，ground diameter，chlorophyll b，and carotenoid were unique indices for evaluating NH4+-N-based nutrition，which could provide a theoretical basis for evaluating the effects of nitrogen fertilizer on seedlings，cultivation periods，and stress tolerance in P.simonii ×P.nigra.

Keywords Nitrogen form·Nitrogen concentration·Growth traits·Physiological characteristics

Introduction

Plant absorb most of their nitrogen in the form of nitratenitrogen (NO3--N) or ammonium nitrogen (NH4+-N)although some can directly absorb amino acids and organic nitrogen (such as urea) (Azcón et al.1992).Because these different forms of nitrogen have differing effects on plant growth and photosynthesis (Clarkson 1980)，the type of nitrogen amendments must be evaluated to determine optimal forms and concentrations for a plant species and the growing conditions.

When rice (Oryza sativaL.) was supplied with different forms of nitrogen [(NH4)2SO4or Ca(NO3)2]，measures of photosynthetic activity were higher after NH4+-N treatment than after NO3--N treatment at 40 mg·L-1N (Song et al.2007).At the development to eight leaves stage，tobacco(Nicotiana tabacumL.) had higher photosynthetic variables after 24 d of treatment with NH4+-N compared with NO3--N at 40 mg·L-1N (Guo et al.2006).Sugarbeet (Beta vulgarisL.) plants grown for 4 weeks in 3.75 mmol·L-1Ca(NO3)2，then with 3.75 mmol·L-1(NH4)2SO4had lower photosynthetic rates compared with those grown only with 3.75 mmol·L-1Ca(NO3)2(Raab and Terry 1994).Similar findings were found for some photosynthetic variables(photosynthetic rate，intercellular CO2concentration，stomatal conductance，transpiration rate，and photosynthetically active radiation utilization efficiency) for barley plants grown in 0.8 mmol·L-1(NH4)2SO4solution compared with those grown in 0.8 mmol·L-1Ca(NO3)2(Ali et al.2013).When Dai et al.(Dai et al.2003) treated three wheat cultivars with different NH4+/NO3-ratio (0/100，50/50，and 100/0)，NH4+substantially raised leaf chlorophyll content，net photosynthetic rate，and soluble sugar content.Cucumber plants grown with NO3-were superior to those grown with NH4+conditions for growth，photochemical efficiency，Photosystem II O2-independent alternative electron flux，and O2-dependent alternative electron flux (Zhou et al.2011).Sweet pepper plants (Capsicum frutescensL.[syn.C.annuumL.] var.grossumBailey) treated with a low-dose NH4+application had greater growth，root respiration rates，wateruse efficiency，and chlorophyll fluorescence (Pi?ero et al.2018).

The growing interest in exploring various opportunities regarding the fast-growing poplar trees (Populusspp.) (Luo et al.2006;Studer et al.2011)，an important economic and timber tree species globally (Luo et al.2013) has led to more research to optimize growth.Different nitrogen concentrations significantly affect photosynthesis，biomass，and morphology (Luo and Zhou 2019).Low nitrogen can lead to the degradation of photosynthetic pigments and proteins，reduce photosynthesis，and damage the photosynthetic apparatus inPopulus balsamiferasubsp.Trichocarpa(Dejuan et al.2020)，Populus tremuloidesMichx (Luo and Zhou 2019;Coleman et al.1998) andPopulus deltoides(Cooke et al.2010).However，a high nitrogen concentration damaged the photosynthetic system of plants due to a nutrient imbalance inPopulus trichocarpa×P.deltoides(Dong et al.2004).

At present，nitrogen fertilizer is increasingly used in plantation forests to shorten the rotation period and increase yield;thus，understanding the responses of different poplar varieties to NO3--N and NH4+-N is critical for optimizing the nitrogen input to promote the best poplar growth，reduce costs and nitrogen loss，and improve nitrogen-use efficiency.Populus simonii×P.nigrais one of the main poplar cultivars in North China，due to its excellent wood properties，rapid growth，drought tolerance，and high cold resistance (Novaes et al.2009;Luo and Polle 2010).In recent years，some studies have mainly focused on growth traits (Ren et al.2020)，molecular analyses (Liu et al.2020a，b)，and resistance ofP.simonii×P.nigra(Yao et al.2020)，rather than on changes in physiological traits ofP.simonii×P.nigraseedlings in response to different nitrogen forms and concentrations.Here for seedlings ofP.simonii×P.nigra，we thus sought to determine:(1) the optimal level and type of nitrogen(NO3--N or NH4+-N)，and (2) the effects of different nitrogen forms and concentrations on growth and physiological traits ofP.simonii×P.nigra.The results provide a theoretical basis for evaluating nitrogen fertilizers for optimal seedling growth and for stress tolerance ofP.simonii×P.nigra.

Materials and methods

Plant materials and treatments

Young twigs ofP.simonii×P.nigra，which can sprout new roots and leaves，were grown in hydroponic culture at 25°C with 16 h light/8 h dark and40% relative humidity with an irradiance of 1000 μmol·m-2·s-1，and 40% relative humidity.The composition of the LA hydroponic nutrient solution(nitrogen free) is shown in Table 1.Ammonium (NH4Cl) or nitrate (NaNO3) was used as the only nitrogen source at 0.1，0.5，1，5，or 10 mmol·L-1.After 28-d culture，all indexes were determined.

Table 1 Composition of LA nutrient solution (nitrogen free)

Measurement of plant physiological characteristics and biomass

Plant height (Height) and ground diameter (Diameter) were measured with a ruler and vernier caliper，respectively.The leaf area (LA) of the fourth leaf at the top was measured using a leaf area meter (LI-3000，USA).Then，nine seedlings were randomly selected from each group.The aboveground and belowground parts of the seedlings were separated and weighed.Samples were dried in an electrothermal constant temperature drying oven at 120°C for 2 h，then at 65°C until a constant mass was attained and weighed again.

Measurement of total nitrogen and total carbon content

The whole samples were ground in a high-speed pulverizer (Wuxi Jiuping Instrument Co.Ltd，Wuxi China)，then digested in H2S O4-H2O2for 2 h，and nitrogen content was determined using a The Kjeltee 2300 analyser (Foss Tecator AB，Sweden).For carbon，a sample (20 mg) was placed in a Euro EA 3000 Elemental Analyzer (Milan，Italy)，completely burned，then the concentration of CO2released was measured to calculate the total carbon content in the tissue.For each procedure and treatment，nine samples were analyzed.

Measurements of photosynthetic parameters and chlorophyll fluorescence

Chlorophylla(Chla)，chlorophyllb(Chlb)，and carotenoids were extracted from the fourth fully expanded leaf from the apex in 2 mL of 95% ethanol for 1 h，and the optical density of extraction solution was measured at 663，645，and 470 nm using a UV-2550 spectrophotometer (Thermo Fisher，Shimadzu，Japan).Pigment contents (mg·g-1) were calculated as reported by Yaryura et al.(2009).

Net photosynthetic rate (Pn)，intercellular CO2content(Ci)，stomatal conductance (Gs)，and transpiration rate (Tr)were measured under photosynthetically active radiation(PAR)of 1400μmol·m-2·s-1and CO2concentration of 500μmol·mol-1at25°C with using a Portable Photosynthesis System (LI-6400XT，LI-COR，Lincoln，NE，USA).The intensity of chlorophyll fulorescence was measured using the procedure of Xu et al.(2014).The chlorophyll fluorescence parameters of leaf samples were analyzed using a MINIPAM Photosynthesis Yield Analyzer (Imaging PAM，Walz，Germany).The highest PSII photochemistry potential was determined after the leaf samples were fully open following a 30-min dark adaptation.TheFv/Fm，Y(II)，Y(NPQ)，Y(NO)，and ETR were estimated using the formulae below:

where，F(xiàn)mis the maximum fluorescence，F(xiàn)m’ is the maximum fluorescence under light adaptation，F(xiàn)ois the initial fluorescence，F(xiàn)vis the variable fluorescence，F(xiàn)sis the steady-state fluorescence，F(xiàn)v/Fmis the maximum photochemical efficiency of PSII，Y(II) is the actual photosynthetic efficiency of PSII，Y(NPQ) is the quantum yield of controlled energy dissipation in PSII，Y(NO) is the quantum yield of uncontrolled energy dissipation in PSII，and ETR is the electron transfer rate.

Statistical analyses

In this study，physiological traits were in different dimensions，so the data needed to be standardized.The processing method was as Eq.(6)

where，X iis the original physiological data，S iisthe standard deviation，is the mean value of physiological data (i=1，2，3，4，5…mphysiological traits).

A principal component analysis (PCA) was performed using the relative values of physiological traits as several new comprehensive indicators，and the load value in the score matrix was used as the basis for selecting physiological traits to use in the membership function analysis using the following formulas (Han et al.2006):

where，Ris the membership function value of physiological index，X iis the physiological traits measured value，Xmaxis the maximum value of the experimental material index，andXminis the minimum value of the experimental material index.

The new comprehensive indicators were designated asF1，F(xiàn)2…，F(xiàn) n.The score for each principal component and comprehensive evaluation index (D) was estimated using the following equations (Liu et al.2020a，b):

where，Dis the comprehensive evaluation index，X iis the physiological index load value of each principal component under component scoring coefficient matrix，ZX iis the physiological index standardization，P iis the variance contribution rate of principal componentF i，W iis the weight of principal componentF i，andF iis the principal component ofi.

The relationship between the physiological traits with NH4+-N and with NO3--N was deciphered using Pearson’s correlation method.Gephi V0.9.2 software was used to complete the network visualization.The data were obtained from at least nine replications and expressed as means±standard error.SPSS version 19.0 (SPSS，Chicago，IL，USA) was used for all analyses.The impacts of the two forms of nitrogen on the studied variables were assessed using a two-way analysis of variance (ANOVA)，and aP＜0.05 value indicated a significant difference，and the least significant difference (LSD) technique was used to evaluate differences between means.

Results

Effect of nitrogen form and content on plant development and biomass allocation

Both nitrogen form and concentration had different effects on the plant samples.As shown in Fig.1，under NO3--N-based nutrition，plant height，ground diameter，and leaf area ofP.simonii×P.nigraseedling were significantly higher at 5mM NO3--Nthan at 1mMNO3--N，but were not significantly affected by 10 mMNO3--N.Asimilar trendwas found for the NH4+-N treatments.The two-way ANOVA test revealed that only leaf area was significantly affected by nitrogen form and plant height and ground diameter significantly increased with increasing nitrogen concentration(P＜0.05).Nitrogen form and concentration had a signif ciant interaction (P＜0.05) on plant height and leaf area.

Fig.1 Effect of nitrogen form and content on A plant height，B ground diameter，and C leaf area.Values are mean±standard errors(n=9).Nitrogen content bars with the same letters were not significantly different (p ＞ 0.05).Non-significance is denoted by NS，Significance levels at p＜0.05，p＜0.01，and p＜0.001 were represented by *，**and ***，respectively.a，b，c，d，e and fwere the results of multiple comparisons，and the same letter indicated there was insignificance between each other.The same as below

As shown in Fig.2，under NO3--N-based nutrition，total mass，shoot mass，root mass，and leaf dry mass ofP.simonii×P.nigraseedling were higher at concentrations below 5 mM NO3--N，while lower at 10 mM NO3--N.The trends for total mass，shoot mass，root mass，and leaf dry mass under NH4+-N-based nutrition was consistent with those under NO3--N-based nutrition.Conversely，at5 mM N O3--N，plants had higher total mass and root mass but lower leaf dry mass than plants receiving 5 mM NH4+-N.In addition，shoot mass did notdiffer between the NO3--N-and the NH4+-N-based nutrition.The two-way ANOVA revealed that nitrogen form had a significant effect on total mass，root mass，dry leaf mass，nitrogen concentration，and the interaction between nitrogen form and nitrogen concentration significantly affected plant mass.

Fig.2 Effect of nitrogen form and content on the A total mass，B shoot mass，C root mass，and D leaf dry mass

Effects of nitrogen form and content on nitrogen and carbon content

As shown in Fig.3，under NO3--N-based nutrition，P.simonii×P.nigraseedling had higher nitrogen content，which was highestat 5 mMNO3--N.The nitrogen con tent of plant seedlings under NH4+-N-based nutrition was generally higher than that under NO3--N-based nutrition，except at the 0.5 mM nitrogen content.However，the total carbon content in plant seedlings did not differ significantly under the different nitrogen content，except at 0.1 mM NO3--N.The two-way ANOVA revealed that nitrogen form，nitrogen content，and nitrogen form×nitrogen content significantly affected nitrogen content.The interaction of nitrogen form×nitrogen concentration had a significant effect on carbon content.

Fig.3 Effect of nitrogen form and concentration on A nitrogen content and B carbon content

Effect of nitrogen form and concentration on pigment content，photosynthetic variables，and chlorophyll fluorescence

Chla，Chlb，carotenoids，and total Chl in leaves are shown in Fig.4.Under NO3--N-based nutrition and NH4+-N-based nutrition，P.simonii×P.nigraseedling chlaand total Chl contents increased significantly at 5 mM NO3--N concentration compared to those at 1 mM NO3--N concentration，while they were not significantly affected by 10 mM NO3--N concentration.At 5 mM nitrogen concentration，Chlbreached the highest levels (4.76 mg·g-1with，3.75 mg·g-1NH4+-N).Most of the photosynthetic pigment contents under NH4+-N-based nutrition were higher than under NO3--N-based nutrition at the same nitrogen concentration.The two-way ANOVA revealed that nitrogen form，nitrogen content，and nitrogen form×nitrogen content significantly affected the content of all the measured pigments.

Fig.4 Effect of nitrogen form and concentration on pigment contents，including A chlorophyll a，B chlorophyll b，C carotenoids，and D total chlorophyll

As shown in Fig.5，net photosynthetic rate was highest at 5 mM nitrogen content (NO3--N:0.161 mol·m-2·s-1，NH4+-N:3.58 mmol·m-2·s-1)，whereas the intercellular CO2content reached the highest levels (NO3--N:256.11 μmol·mol-1，NH4+-N:292.14 μmol·mol-1)at 1 mM nitrogen.Under NO3--N-based nutrition，stomatal conductance and transpiration rate reached the highest levels (NO3--N:0.161 mol·m-2·s-1，NH4+-N:3.58 mmol·m-2·s-1) at 1 mM NO3--N concentration.Under NH4+-N-based nutrition，5 mM NH4+-N yielded the highest stomatal conductance (0.232 mol·m-2·s-1)and transpiration (6.39 mmol·m-2·s-1).Photosynthetic variables under NH4+-N-based nutrition were higher than those under NO3--N-based nutrition at the same nitrogen concentration，except for 1 mM nitrogen.The two-way ANOVA revealed significant effects of nitrogen form，nitrogen concentration，and nitrogen form×nitrogen concentration on all photosynthetic variables (P＜0.05).

Fig.5 Effect of nitrogen form and content on photosynthetic parameters，including the net photosynthetic rate (Pn，A)，intercellular CO2 content(Ci，B)，stomatal conductance (Gs，C)，and transpiration rate (Tr，D)

In Fig.6，F(xiàn)v/Fmwas highest at 10 mmol·L-1NO3--N(0.806) and 5 mmol·L-1NH4+-N (0.804).Under NO3--N-based nutrition and NH4+-N-based nutrition，Y(II) initially increased，then decreasedY(II);however，Y(NPQ) had the opposite trend;Y(II) was highest at 1 mmol·L-1(NO3--N:0.036，NH4+-N:0.034).Y(NO)was highest at 0.01 mmol·L-1(NO3--N:0.278，NH4+-N:0.299).In addition，the results of two-way ANOVA test revealed that nitrogen concentration had significant effects onFv/FmandY(NO)，and nitrogen form and nitrogen concentration had significant effects onY(II)，Y(NPQ)，and ETR;nitrogen form and concentration had no significant interaction on any of the variables.

Fig.6 Effect of nitrogen form and content on chlorophyll fluorescence including A Fv/Fm，B Y(II)，C Y(NPQ)，D Y(NO)，and E ETR

PCA of different physiological traits and nitrogen concentrations

When the physiological traits were converted into several new comprehensive indicators to eliminate less-significant factors and improve data reliability，PCA was conducted based on NO3--N-or NH4+-N-based treatment.Under NO3-N-based nutrition，the first and second principal component (PC) contributed，respectively，to 62.86% and 23.94%of the variation (Table 2).Thus，PC1 and PC2 represent 86.8% of the variation for all measured traits，which were selected as the comprehensive indicators.PC1 included net photosynthetic rate，chlorophyll a，plant height，andFv/Fm;for PC2，the essential factor was intercellular CO2concentration (Table S1).Under NH4+-N-based nutrition，PC1 contributed 73.14% and PC2 19.33% of the variation(total=92.47%) and were adopted as the comprehensive indicators.For PC1，the main contributors were total mass，shoot mass，leaf dry mass，and net photosynthetic rate;for PC2，Y(NPQ) was the main factor (Table S1).

Comprehensive evaluation of concentrations

Based on PCA results，the load values in the matrix were obtained and then used in membership function analysis(Table S1).Under NO3--N-based nutrition，net photosynthetic rate，intercellular CO2concentration，F(xiàn)v/Fm，Y(II)，Y(NO)，Chla，total chlorophyll，total carbon content，plant height，ground diameter，leaf area，total mass，shoot mass，root mass，and leaf dry mass were selected as indicators to use in the membership function analysis (Table S2).Under NH4+-N-based nutrition，net photosynthetic rate，stomatal conductance，transpiration rate，carotenoid，F(xiàn)v/Fm，total mass，shoot mass，root mass，plant height，ground diameter，leaf area and leaf dry mass were selected as indicators to use in the membership function analysis (Table S3).The maximum membership function values were 0.530 (under NO3--N-based nutrition) and 0.559 (NH4+-N-based nutrition) for 5 mmol·L-1treatment.

Based on the score value in the score coefficient matrix and the standardized value of the different indexes，composite scores estimated and sorted were calculated and arranged(Tables S4 and S5).Meanwhile，it showed the highest composite score level (0.742) under 5 mmol·L-1NO3--N treatment (Table 3).The highest composite score (0.931) was found at 5 mmol·L-1NH4+-N.These two results suggested that，under 5 mmol L-1nitrogen，the comprehensive evaluation index was the highest and plants grow well.

Table 2 PCA results of P.simonii ×P.nigra seedling under NO3--N-and NH4+-N-based nutrition

Table 3 Composite scores of different traits of P.simonii ×P.nigra seedling under NO3--N-and NH4+-N-based nutrition

Pearson correlation analysis of different indicators

Pearson correlation analysis was carried out to understand better the characteristics of different indicators (Fig.7).Under NO3--N-based nutrition，total nitrogen content was positively correlated withFv/Fm，leaf dry mass，plant height，ground diameter，leaf area，chlorophyll a，and chlorophyll b，while chlorophyll was negatively correlated withY(NO).Under NH4+-N-based nutrition，total nitrogen content was highly positively correlated with net photosynthetic rate，transpiration rate，F(xiàn)v/Fm，total mass，shoot mass，root mass，leaf dry mass，plant height，ground diameter，leaf area，Chla，Chlb，total Chl，and carotenoid.The correlation parameters among different traits under NH4+-N-based nutrition were higher than those under NO3--N-based nutrition，which indicated that the positive effect of NH4+-N-based nutrition on physiological traits was more than that of NO3--N-based nutrition.

Fig.7 Depiction of Pearson correlation coefficients of all of P.simonii × P.nigra seedling traits under A NH4+-N and B NO3--N based nutrition，respectively.These were determined using the Pearson correlation.*and **represents significant difference at the 0.05 and 0.01 levels，respectively.Height，plant height;Diameter，ground diameter;LA，leaf area;TM，total mass;SM，shoot mass;RM，root mass;LDM，leaf dry mass;Chl a，chlorophyll a;Chl b，chlorophyll b;carotenoid，carotenoids;Total Chl，total chlorophyll; Pn，net photosynthetic rate; Ci，intercellular CO2 concentration; Gs，stomatal conductance; Tr，transpiration rate; Fv/Fm，maximum photochemical efficiency of PSII;Y(II)，actual photochemical efficiency;ETR，electron transfer rate; Y(NPQ)，quantum yield of regulatory energy dissipation in photosystem II; Y(NO)，quantum yield of nonregulatory energy dissipation in photosystem II;N，total nitrogen content，C，total carbon content

Following partial Pearson correlation under NO3--N and NH4+-N based nutrition，the Gephi software was used to construct images to depict the complex inter-relationships (Fig.8;Tables S6 and S7).Under NO3--N-and NH4+-N-based nutrition，the high degree of connectivity was composed of net photosynthetic rate，F(xiàn)v/Fm，actual photosynthetic efficiency of PSII，quantum yield of non-regulatory energy dissipation in photosystem II，total carbon content，total mass，shoot mass，root mass，leaf dry mass，plant height，leaf area，Chla，Chlband total Chl Combined with the high Pearson correlation，the results suggested that total mass，shoot mass，root mass，leaf dry mass，plant height，leaf area，Chla，and total Chl were the core physiological indicators under NO3--N based nutrition (Table S8).Under NH4+-N-based nutrition，the high degree of connectivity was composed of net photosynthetic rate，transpiration rate，total mass，shoot mass，root mass，leaf dry mass，plant height and leaf area.Combined with the high Pearson correlation，the results suggested that net photosynthetic rate，transpiration rate，F(xiàn)v/Fm，total mass，shoot mass，leaf dry mass，plant height and leaf area are the core physiological indicators under NH4+-N-based nutrition (Table S8).Therefore，total mass，shoot mass，leaf dry mass，plant height，and leaf area are the preferred indices for evaluating different nitrogen treatments and thus，vastly decreasing future workloads.

Fig.8 Configuration images of A NO3--N and B NH4+-N based nutrition using partial Pearson correlation.The size of the circle represents the degree of connectivity of physiological indicators.The line width determines a correlation coefficient.The color of each line depicts the type of relationship:red and turquoise denote positive and negative，respectively

Discussion

In this study，plant height and ground diameter ofP.simonii×P.nigraseedlings under NO3--N-based nutrition were slightly higher than those under NH4+-N-based nutrition，consistent with the results for blueberry (Claussen and Lenz 1999)，rice (Qian et al.2004)，sweet pepper (Xu et al.2001) and corn (Lewis et al.1989).Some studies showed that plants had smaller leaf areas when given NH4+-N compared with NO3--N (Chaillou et al.1986;Leidi et al.1992;Khan et al.1994;Raab and Terry 1994;Walch-Liu et al.2000).This effect was attributed to reduced osmotic regulation and expansion of leaf cells(Raab and Terry 1994;Chaillou et al.1986;Heuer and Bruria 2008).The leaf area ofP.simonii×P.nigraunder NO3--N-based nutrition was smaller than that under NH4+-N-based nutrition，which is consistent with the results of tomato (Kalaji et al.2014;Horchani et al.2010).The main reason for this phenomenon might be due to different plant species differing in their nitrogen uptake and utilization.

The concentration of nitrogen also had a significant effect on the morphology of the seedlings.The appropriate nitrogen concentration can increase cell number and volume，thus promoting plant growth in height，ground diameter，and leaf area (Reddy et al.1996;Lawlor 2002).Plant growth increases as the nitrogen concentration increases within an acceptable range (Jin et al.2015);low or high nitrogen levels can reduce the rate of cell division and cell expansion(Reddy et al.1996).Here，the changes in height，ground diameter，and leaf area ofP.simonii×P.nigraseedlings were consistent with this trend.

In terms of biomass，total mass，shoot mass，root mass，and leaf dry mass of the seedlings receiving NO3--N were higher than for those with NH4+-N-based nutrition.Similar results were found in cucumber，rice，cedarwood，and slash pine plants (Zhou et al.2011;Ye et al.2018;Kou et al.2015).These results might be due to differences in tissue assimilation;nitrate nitrogen absorbed by plant roots is mainly reduced and assimilated in leaves，while ammonium nitrogen is primarily assimilated in the roots.During root growth，NH4+-N assimilation in roots competes with leaf photosynthates，leading to shorter roots，reducing lateral roots，and decreasing root activity，thus affecting root morphology and physiology (Drew et al.1973).The total mass，shoot mass，root mass，and leaf dry mass ofP.simonii×P.nigraseedlings increased with the increase of nitrogen concentration below 5 mmol·L-1as previously reported (Jin et al.2015;Biemond and Vos 1992;Vos et al.2005).However，these variables decreased when the nitrogen concentration was 10 mmol·L-1，indicating that higher nitrogen concentrations inhibit plant growth.As reported，When NH4+-N is used as the only nitrogen source，excessive NH4+-N can lead to ammonium toxicity and damage plants，and even cause plants to wilt (Zebarth et al.2012).When NO3--N is the only nitrogen source，excess NO3--N is stored in vacuoles，but can also inhibit plant growth due to an over-accumulation of nitrogen (Zebarth et al.2012;Zhu et al.2020).

The nitrogen content inP.simonii×P.nigraseedlings under NH4+-N-based nutrition was generally higher than under NO3--N-based nutrition，except at 0.5 mM nitrogen;root growth and water absorption is apparently less inhibited than with NH4+-N，resulting in the higher nitrogen content(Ti-Da et al.2008).However，the total carbon content ofP.simonii×P.nigraseedlings was not significantly affected by different nitrogen concentrations，except at 0.1 mM NO3--N.The chlorophyll content in leaves is one of the critical indicators reflecting the photosynthetic capacity of plants (Gou et al.2020)，and it increased significantly inP.simonii×P.nigraseedlings at 5 mM NO3--N compared to 1 mM NO3--N was not further affected by 10 mM NO3--N.Chloroplast structure is degraded by high nitrogen levels，resulting in lower chlorophyll content (Wu et al.2011).Total Chl content under NH4+-N-based nutrition was higher than that under NO3--N-based nutrition at the same nitrogen concentration.This trend is similar to that found for nitrogen content because nitrogen is a primary constituent in compounds such as amino acids and chlorophyll.

Interestingly，at least 70% of leaf nitrogen is involved in photosynthesis (Xing and Ma 2015).In addition，nitrogen forms also affect the chloroplast construction，photosynthetic reaction processes，and the variables related to photosynthetic rate (Tao et al.2017).Nitrogen forms significantly affect photosynthesis in barley (Ali et al.2013)，wheat (Dai et al.2003)，rice (Song et al.2007)，and flue-cured tobacco(Guo et al.2007).Factors such as transpiration rate，gasexchange，and intercellular CO2concentration can also influence photosynthetic capacity (Dong et al.2006).Stomatal conductance mainly reflects the degree of stomatal opening，which directly affects photosynthesis.The greater the stomatal conductance，the higher the concentration of CO2in mesophyll cells，and the greater the photosynthetic rate(Guo et al.2007).In the present study，photosynthetic variables under NH4+-N-based nutrition were higher than under NO3--N-based nutrition at the same nitrogen concentration，except at 1 mM nitrogen.A similar result was reported for advanced plants andTrifolium repens(Hoegh Jensen and Schjoerring 1997;Guo et al.2002).Under NO3--N-based nutrition or NH4+-N-based nutrition at 10 mM here，net photosynthetic rate，intercellular CO 2 concentration，stomatal conductance，and transpiration rate did not reach the highest levels.

Nitrogen concentration can also inhibit the photosynthetic rate to some extent (Wu et al.2011)，because it can damage the plant biofilm，weaken the oxidative phosphorylation reaction，and reduce photophosphorylation and non-photosynthetic phosphoric acid coupling，resulting in a decrease in ATP generation，thereby inhibiting carbon fixation by CO2and reducing the photosynthetic rate of plants (Negi et al.2015).Similarly，intercellular CO2concentration，stomatal conductance，and transpiration rate of plants were inhibited to some extent under a high concentration of pure NO3--N or NH4+-N (Kim et al.2002).

Chlorophyll fluorescence variables of leaves are mainly used to describe functioning of the photosynthetic light system such as absorption，transmission，dissipation，and distribution of light energy (Li et al.2006).Chlorophyll fluorescence is a widely used indicator of environmental impacts on plants and the responses of specific photosynthetic processes under different stresses (Wang et al.2015).Plant photoinhibition levels can be determined using indicators such asFv/Fmchanges.Photosystem II (PSII) pigments absorb three forms of light energy-Y(II)，Y(NPQ)，andY(NO)-that are given offas heat and can serve as stress indicators and predict damage levels to photosynthesis (Queiroz et al.2016).In addition，ETR can predict photosynthetic homeostasis under different situations (Long et al.2013).In a specific range of nitrogen concentrations，nitrogen had a particularly positive effect on the integrity of the PSII structure of seedling leaves in the present study.As a result，F(xiàn)v/Fm，Y(II)，Y(NPQ)，and ETR of PSII of seedling leaves increased with the increase of nitrogen concentration.However，PSII system integrity and the capacity for efficient photosynthesis can decline under extremely high or low nitrogen concentrations high.

The membership function is used to screen the stress resistance index for different varieties of the same species after the same stress treatment to find varieties with higher stress tolerance，tolerance to low nitrogen in broomcorn millet (Liu et al.2020a，b)，salt tolerance in soybean (Suo et al.2020)，drought tolerance in poplar (Hong et al.2020) and superior agronomic traits in hybrid maize (Li et al.2019).In this study，we used the membership function to screen stress tolerance indices for the same plant variety under different stress levels and then evaluated plant growth.However，selection and evaluation only using membership function analysis is often one-sided，so principal component analysis(PCA) is used to analyze large data sets through the dimension-reduction method.Its advantage is to change multiple physiological indicators into several comprehensive indicators that cover comprehensive information to make up for the knowledge gap in the membership function analysis caused by only having data on major stress tolerance indicators.Our results from these two methods combined suggested that at 5 mmol·L-1nitrogen，the comprehensive evaluation index was the highest，and plant growth was the best.

In addition in our study，the Pearson correlation coefficient and network visualization revealed relationships among different physiological indicators.Interestingly，the connective degree of photosynthetic pigment，photosynthetic parameters，and chlorophyll fluorescence parameters under NH4+-N based nutrition was higher than those of under NO3--N-based nutrition，indicating that only NH4+-N based nutrition was the nitrogen source，the photosynthetic pigment，photosynthetic parameters，and chlorophyll fluorescence had a more significant influence，which was not available for NO3--N-based nutrition.

In our study，there was a high positive correlation coefficient between net photosynthetic rate and plant biomass under NO3--N-and NH4+-N-based nutrition，which agrees with the results of (Poorter et al.2013) since photosynthesis determines the growth status of plants and thus biomass accumulation (Peng et al.1991).The lower the net photosynthetic rate，the less biomass accumulates (Ghosh et al.2017).Total Chl content in leaf can also reflect changes in physiological activity and is closely related to net photosynthetic rate (Qian et al.2021).In this study，there was a positive correlation between net photosynthetic rate and total Chl content in the two nitrogen groups，in line with the findings of (Huang et al.2019).In conclusion，there was a positive correlation between net photosynthetic rate，plant biomass and total Chl content.In addition，the correlation coefficient between net photosynthetic rate and total Chl content under NH4+-N-based nutrition was higher than that under NO3--N-based nutrition，likely from the generally higher net photosynthetic rate and total Chl content under NH4+-N-based nutrition.

Conclusion

Nitrogen form and concentration affected plant development，biomass allocation，pigment content，and photosynthetic capacity.The combined membership function and comprehensive evaluation index suggested that the plant growth was the best when the nitrogen concentration was 5 mmol·L-1.In addition，Pearson correlation analysis and network visualization revealed that the connective degree of photosynthetic pigments，photosynthetic variables，and chlorophyll fluorescence variables under NH4+-N-based nutrition was higher than under NO3--N-based nutrition，indicating that photosynthesis had a remarkable effect on other physiological traits under NH4+-N-based nutrition.Meanwhile，several key evaluation indices based on Pearson correlation analysis and network visualization were selected to improve evaluation efficiency in the future.