This experiment was carried out in the greenhouse of Shenyang Agricultural University from April 2019 to July 2019. The experimental materials were seedlings of the annual poplar P. simonii × P. euphratica, which is a fast-growing, salt-tolerant species called “Liao Hu NO.1” poplar in China. Six poplar seedlings were planted into one pot with dimensions of 32 cm length × 18 cm width × 14 cm height. After the poplar seedlings were planted, 7 L of DI water was added to each pot. We ensured that the conditions were uniform in all the pots. After 20 days of recovery, the lateral bud of each poplar seedling was treated, so that only one lateral bud was selected for each seedling at the same position and treated with calcium.

The nutrient solution was formulated with deionized water according to Xie (2014). The pH of the solution was maintained at 5–6 by adding NaOH. In this culture solution, five different concentrations of Ca²⁺ (0, 2.5, 5, 10, and 20 mmol·L⁻¹) were added and labeled as Ca0, Ca1, Ca2, Ca3, and Ca4, respectively (Paiva et al., 1998). Calcium was provided by anhydrous CaCl₂, and other compounds that provided a large number of elements were KNO₃, MgSO₄·7H₂O, KH₂PO₄, NaNO₃, EATA-Na₂, and FeSO₄·7H₂O. Trace elements were provided by H₃BO₃, MnCl₂·4H₂O, CuSO₄·5H₂O, ZnSO₄·7H₂O, and H₂MoO₄·H₂O. After the seedlings were stable, the nutrient solution was poured every 5 days before the growth boom and once every 3 days during the growth period. During the growth of seedlings, each treatment was equipped with an air pump, which was continuously ventilated from 7:00 to 19:00 h and ventilated 1 h for every 2 h after 19:00 h. Other management measures were carried out in accordance with the routine procedures.

The total carbon and nitrogen contents were measured using a stable isotope mass spectrometer (DELTA V Advantage Isotope Ratio Mass Spectrometer). To measure total P content, samples were first digested with nitric acid and hydrogen peroxide, and then the extracted solutions were analyzed using an ultraviolet photometer (Shang et al., 2018; Zhang et al., 2018). The concentrations of potassium and calcium were measured using a flame atomic absorption spectrophotometer (Jing et al., 2018).

Excel and SPSS 22.0 software programs were used for sorting and drawing, statistical analysis, differential analysis, correlation analysis, and principal component analysis. All experiments were conducted with three replicates, and the results are expressed as the mean ± standard error (SE) values. The different letters in the chart indicate that the differences in each index between different calcium treatments reached the significance level of 5%.

In general, the indicators of growth and biomass showed an increasing trend initially and then decreased with increasing calcium concentrations (Figures 1, 2; Table 1). With the increasing gradient of calcium, the best value for all indicators was observed in the treatment with 5 mmol·L⁻¹ of calcium, and there were significant differences with other treatments except for root biomass (P < 0.05). The maximum plant height and diameter increased by 17.94 and 25.91%, respectively, compared to plants with no calcium treatment (Figure 2). When Ca²⁺ concentration was 5 mmol·L⁻¹, the root biomass, stem biomass, leaf biomass, and total biomass increased by 15.41, 24.21, 13.05, and 17.31%, respectively, compared to the plants with no calcium treatment (Table 1). However, when excess calcium was applied, there was no significant increase in the biomass of poplar seedlings compared to no calcium treatment.

In general, the indicators of photosynthetic characteristics first showed an increasing trend and then a decreasing trend with increasing calcium concentration (Figure 3). With the increasing gradient of calcium addition, the best value for all photosynthetic indicators occurred in the treatments with 5 mmol·L⁻¹ calcium, and there were significant differences with other treatments (P < 0.05). The Pn, Gs, Ci, and Tr levels increased by a maximum of 149.94, 191.67, 23.14, and 86.14%, respectively, compared to those in the absence of calcium treatment. A similar increasing trend was also observed for the indicators of chlorophyll-a, chlorophyll-b, leaf soluble sugar, and leaf starch levels, which increased by 45.54, 45.80, 29.00, and 33.31%, respectively. However, when excess calcium was applied, the photosynthetic capacity of poplar seedlings was weakened.

In general, the chlorophyll fluorescence parameters showed an increasing trend in the beginning and then decreased with increasing calcium concentration (Figure 4). With the increasing gradient of calcium addition, the best values for Fv/Fm and Fv/F0 ratios occurred in the treatments with 5 mmol·L⁻¹ calcium, and there were significant differences with other treatments (P < 0.05). The ratios increased by 2.57 and 16.01%, respectively, compared to no calcium treatment. However, when the calcium concentration was 20 mmol·L⁻¹, the values of all the chlorophyll fluorescence parameters were lower than those obtained without calcium treatment (P < 0.05).

In general, the indicators of antioxidant enzyme activities and iWUE showed an increasing trend in the beginning and then decreased with increasing calcium concentration (Figure 5). With the increasing gradient of calcium addition, the best values for SOD, CAT, POD, and iWUE occurred in the treatments with 5 mmol·L⁻¹ calcium, and there were significant differences with other treatments (P < 0.05). The parameters increased by 15.57, 84.19, 124.30, and 14.28%, respectively, when compared to no calcium treatment. However, when the calcium concentration was 20 mmol·L⁻¹, the antioxidant enzyme activities and iWUE were lower than those obtained without calcium treatment.

The results showed that endogenous concentrations of C, N, P, and K in the leaves, stems, roots, and the whole plant are influenced by different Ca supply levels and showed an increasing trend initially and then decreased (Figures 6A,B,D,E,G,H,J,K). The 5 mmol·L⁻¹ calcium treatment resulted in the highest endogenous concentrations of C, N, P, and K, followed by 2.5 mmol·L⁻¹ and 10 mmol·L⁻¹ calcium treatments (except for the best P content of leaves and stems was observed with the 2.5 mmol·L⁻¹ and 10 mmol·L⁻¹ concentrations). There were significant differences in the concentrations of all other nutrient elements in all other organs with different calcium treatments, except for the N content of roots (P < 0.05). A different trend was observed for the Ca concentration in plant organs; the Ca accumulation rate in each tissue increased gradually with an increase in the doses of Ca ions (Figures 6M,N). Without Ca supply, the Ca concentration was significantly lower. The 10 mmol·L⁻¹ and 20 mmol·L⁻¹ Ca treatments led to a higher accumulation of Ca in the leaves, stems, and whole plant, and there are significant differences with other treatments except for roots (P < 0.05).

The percentage of nutrient elements in each organ in all the tissues reflects the distribution of nutrient elements in the trees and the regularity of the migration of nutrient elements in these tissues (Xing et al., 2021). The results showed that C, N, K, and Ca in the 5 mmol·L⁻¹ Ca treatment group were mainly distributed in the leaves (Figures 6C,F,L,O), but P was mainly distributed in the roots (Figure 6I). With the increase in the concentration of Ca, the P, K, and Ca distribution rates of leaves first showed a decreasing trend and then an increasing trend, and roots showed an adverse trend. The N distribution rate of leaves first showed an increasing trend and then a decreasing trend, and roots showed a decreasing trend first and then an increasing trend, while there was no significant change in the distribution rate of Ca in leaves, stems, or roots. It is evident that an appropriate Ca supply could promote the distribution of N to the leaf system and P and K distribution to the root system, but since Ca is known to have a low transference capacity in plants, the Ca distribution rate was not significantly different between the different treatments, except for the 0 mmol·L⁻¹ treatment.

The overall analysis results showed that (Figure 7) there was no correlation between the concentration of exogenous calcium and C/N, C/P, and N/P ratios of poplar roots or between the concentration of exogenous calcium and the C/P ratio of poplar leaves (P > 0.05). However, exogenous calcium concentration had a significant negative correlation with C/N (P < 0.05) and a significant positive correlation with N/P (P < 0.05) ratio in the leaves of poplar seedlings, and there was a significant negative correlation between exogenous calcium concentration and the C/N, C/P, and N/P ratios in stems (P < 0.05). With increasing exogenous calcium concentration, C/P in leaves gradually increased, while C/P in leaves and C/N, C/P, and N/P ratios in stems gradually decreased.

Calcium is an essential nutrient for plant growth. Exogenous calcium can regulate the expression of plant growth genes, maintain cell function, promote plant growth and development, and improve plant tolerance to the environment (Naeem et al., 2017; Pathak et al., 2020). Previous studies showed that adding calcium fertilizer could significantly increase the biomass of Chinese cabbage, and when the calcium concentration was 600 mg·kg⁻¹, the biomass of Chinese cabbage was the highest (Liu et al., 2009). For Zoysia japonica, an increase in the above- and below-ground fresh biomass was observed for all CaCl₂ pretreatment groups, and the greatest increase was observed for the 10 mmol·L⁻¹ CaCl₂ pretreatment group (Xu et al., 2013). Foliar Ca²⁺ markedly improved maize growth, photosynthesis, stomatal conductance, transpiration rate, and accumulation of total soluble sugars (Naeem et al., 2018) and also improved the quality of sweet cherries (Correia et al., 2019). The foliar application of Ca²⁺ to sugar beet enhanced the plant biomass (Hosseini et al., 2019), and calcium fertilizer promoted the growth of poplar roots (Petrochenko et al., 2019). When plants are deficient in Ca, the cell wall cannot be formed, because the cell division and formation are affected. Lysine degradation is affected, leading to the accumulation of pipecolic acid at the onset of this metabolic pathway and resulting in a decrease in the accumulation of plant biomass and the possible development of physiological diseases (Cinčerová, 1976; De Freitas et al., 2016). However, excessive Ca not only results in cellular toxicity and destroys normal biochemical metabolism, nutrient element metabolism, and other processes in plants, but also has an impact on plant morphology and alterations in the internal structure, such as overly rigid cell walls (Tyler and Olsson, 2009; Conn et al., 2011; Cybulska et al., 2011). For example, Ca deficiency or Ca overload in tomato plants reduces leaf size and plant biomass (Hao and Papadopoulos, 2004). The present study also showed similar results. When exogenous calcium was applied to poplar seedlings, the biomass of each organ and the total biomass of poplar seedlings first increased and then decreased, reaching a maximum when the calcium gradient was 5 mmol·L⁻¹. Therefore, the amount of calcium can influence plant growth and biomass accumulation. The application of exogenous calcium led to distinct changes in the levels of nutrients in leaves, such as Mg, N, P, etc., which further led to an increase in the photosynthetic pigment and photosynthetic product levels and eventually enhanced plant growth and biomass.

Photosynthesis is the process by which green plants absorb light energy, assimilate carbon dioxide and water, make raw and processed materials, and release oxygen (Okafor and Okeke, 2017). It is the basis for dry matter accumulation in plants. Photosynthesis is affected by interactions with various environmental factors, such as light, temperature, water, and minerals (Ke, 2001; Feller and Vaseva, 2014; Rubenovna and Ramazanovich, 2018). Chlorophyll is an important photosynthetic pigment that absorbs and transforms light energy (Kumara et al., 2021). Its content is closely related to thephotosynthetic capacity of plants (Mohsenpour and Willoughby, 2013). Chlorophyll fluorescence kinetics can be used to monitor multiple photosynthetic reactions and reflect the success of plants. Changes in chlorophyll fluorescence are closely related to the absorption, transfer, dissipation, and distribution of light energy. By measuring changes in chlorophyll fluorescence parameters, the adaptability of plants to stressful environments can be well identified (Peng et al., 2021; Zheng et al., 2021). When Fv/Fm ratio is less than 0.8, it indicates that plants are under stress and their growth and development are severely inhibited. Calcium is involved in the process of electron transport and phosphorylation in plant photosynthesis, which directly affects the plant photosynthetic mechanism (Ferguson and Drobak, 1988). Several studies have established an essential role of Ca in maintaining photosynthesis by modulating gas exchange in leaves, PSII processes, carbohydrate metabolism, and expression of chlorophyll synthesis-related genes (Zhang et al., 2019). For example, the stress related to calcium deficiency reduces the chlorophyll content of lettuce (Fan and Yin, 2002), and the net photosynthetic rate, stomatal conductance, and other parameters of potato plants showed a downward trend (Xin, 2008). An appropriate amount of exogenous calcium enhanced the net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration, maximum quantum efficiency of photosystem II photochemistry, actual photochemical efficiency of PSII, photochemical quenching coefficient, and non-photochemical quenching coefficient (He et al., 2018). When the calcium concentration was more, the plant height, biomass, net photosynthetic rate, and chlorophyll content of tomato plants decreased significantly (Zhou et al., 2012). Additionally, a transient increase in Ca causes short-term stomatal closure; therefore, a high Ca concentration does not seem to be directly related to transpiration (Xing et al., 2021). In addition, other studies have found that calcium can affect leaf bud differentiation. When the calcium concentration was 0, the number of differentiated buds decreased, and the greatest number was achieved at 2 MS, but growth was inhibited at 4 MS, at which concentration the number of green leaves was moderate (Li and Liu, 2007). In our study, we found that the photosynthetic characteristics of poplar seedlings were significantly affected by the addition of calcium, and the chlorophyll content, photosynthetic parameters, and chlorophyll fluorescence parameters increased first and then decreased with increasing calcium concentration. This is similar to previous research results. Therefore, calcium at an optimal level is critical for plant growth. It can affect the gas exchange process related to photosynthesis by regulating stomatal movement (Wang et al., 2019). Moreover, a proper amount of calcium ions may improve the integrity and stability of chloroplast structure, and enhance the activities of Rubisco and PEP carboxylase enzymes, thus improving the carboxylation efficiency of carbon dioxide and the activity of ATPase on the membrane and thereby the photosynthetic level of plants.

The parameters like POD, CAT, SOD, and iWUE were measured to identify the resistance capacity of the plant to stress conditions. iWUE reflects the energy conversion efficiency in plant production, is an index used to measure the relationship between crop yield and water consumption, and is also one of the comprehensive indices used to evaluate plant growth suitability under water deficit. In current forest ecology, the water use efficiency (iWUE) of trees is very important in the study of water resources and for the restoration of forest vegetation (Xi et al., 2018). The antioxidant enzyme system can scavenge free radicals in the cell and protect the cell membrane system from damage. Some of the antioxidant enzymes often found in plants are POD, SOD, and CAT (Guo et al., 2018). Previous studies on Allium sativum and Pinus massoniana seedlings showed the effects of elevated calcium concentration on the expression levels of POD, SOD, and CAT. In Allium sativum, these antioxidant enzymes first increased and then decreased. In contrast, in Pinus massoniana needles, these antioxidant enzymes first decreased and then increased at different growth stages (Li et al., 2013; Li and Zhou, 2017). In addition, under some stress factors, such as cadmium stress, suitable exogenous calcium can improve the activities of SOD, POD, and CAT in plant seedlings (Siddiqui et al., 2012), and optimal Ca supplementation upregulated the activities of the assayed antioxidant enzymes and the contents of non-enzymatic antioxidants (ascorbate, glutathione, and tocopherol), thereby reflecting the amelioration of NaCl-induced oxidative damage (Elkelish et al., 2019). Calcium is closely related to plant water use efficiency; for example, a suitable concentration of calcium fertilizer can increase iWUE, fresh yield, and fruit quality of tomato (Hong-Bo et al., 2008; Yang et al., 2012). The results showed that the iWUE and antioxidant enzyme activity of poplar seedlings first showed an increasing trend and then a decreasing trend, and reached maximum values when the calcium concentration was approximately 5 mmol·L⁻¹. At this concentration, the stress resistance of poplar seedlings was the best.

In conclusion, for poplar seedlings, when the concentration of exogenous calcium was 5 mmol·L⁻¹, the photosynthetic capacity, stress resistance, and growth effect were maximum.

Nutrient elements play an important role in plant growth and development by affecting plant physiological processes, such as stomatal conductance, photosynthetic rate, and water use efficiency, which can directly or indirectly affect plant growth (Ma, 2017). In higher plants, interactions between nutrients occur when the supply of one nutrient affects the absorption, distribution, or function of another nutrient. Interactions between nutrients can be assessed by examining both the relationship between nutrient supply and growth and the relationship between nutrient concentrations in plants and growth (Robson and Pitman, 1983; Sevanto et al., 2020). Currently, a large number of studies on the C, N, and P ecological stoichiometry changes in plants focus on global changes, such as elevated ozone levels (Shang et al., 2018), plant management strategies (Heyburn et al., 2017; Chen and Chen, 2021), N deposition (Huang et al., 2018), elevated CO₂ concentrations (Du et al., 2018), and their interactions. However, few studies have been conducted that determined the effect of calcium on C, N, and P ecological stoichiometry, and most studies that evaluated the effect of calcium on plant growth are mainly focused on cash crops, such as pepper (Capsicum annuum) (Akladious and Mohamed, 2018), rice (Liang et al., 2021a), and tomato (Guo et al., 2018). Therefore, it is of great significance to explore the relationship between nutrient stoichiometry characteristics and the growth of poplar seedlings to reveal suitable vigor theory and select growth evaluation indices.

This study showed that N, P, K, and C levels in each organ of poplar seedlings first increased and then decreased with increasing application of exogenous calcium content, and their maximum levels were reached when the concentration of exogenous calcium was 5 mmol·L⁻¹. In contrast, calcium levels in each organ of poplar seedlings increased continuously with increasing exogenous calcium content. These results are similar to those of previous studies. For example, the addition of calcium sulfate to nutrient solution significantly increased leaf K⁺, Ca²⁺, and N levels in tomatoes (Tuna et al., 2007). Also, exogenous Ca²⁺ was shown to promote the absorption of K⁺ in poplar roots after prolonged exposure to salinity and to retain K⁺/ Na⁺ homeostasis in roots (Sun et al., 2009). Another study showed that foliar application of Ca²⁺ improved the photosynthetic capacity of leaves, thus increasing the accumulation of C in plants and enhancing dry matter production (Song et al., 2020). After calcium treatment, the content of calcium and phosphorus increased significantly (Niu et al., 2018). The reasons for these findings may be as follows: An appropriate amount of Ca²⁺ can maintain the integrity of the plant cell membrane and ensure the selective permeability of the cell membrane, and when the concentration of Ca²⁺ is appropriate, it can exhibit a synergistic effect with other ions (such as N) to promote the absorption of other nutrient elements. In addition, with the increase in exogenous calcium content, the proportion of C in roots, stems, and leaves did not change, while the proportion of N in leaves first increased and then decreased, and the proportion of other elements, such as P, K, and Ca, in leaves first decreased and then increased.

In this study, the calcium content of poplar was found to be positively correlated with the N/P ratio in leaves and negatively correlated with the N/P ratio in stems. However, N/P <14 indicates that plant growth in this study was mainly restricted by N content. At the same time, the C/N ratio in the leaves and stems of poplar and the C/P ratio in stems are significantly negatively correlated with exogenous calcium content, indicating that leaves need more N and stems need more N and P to fix C under the conditions of calcium deficiency and that the NUE and PUE were low. No correlation was found between exogenous calcium or C/P in leaves and stoichiometry in roots.