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EFFECTS OF EXOGENOUS CaCl 2 ON THE PHOTOSYNTHETIC FUNCTION AND ACTIVE OXYGEN METABOLISM OF SALIX VIMINALIS LEAVES UNDER PB STRESS

. In order to provide some basic data for revealing the mechanism of exogenous CaCl 2 on improving drought resistance of Salix viminalis seedlings. The experimental results showed that: Spraying exogenous of different CaCl 2 concentrations significantly alleviated the damage degree of S. viminalis seedlings leaves caused by heavy metal stress, and the effect of 30 μmol·L -1 CaCl 2 was the most significant. Spraying exogenous CaCl 2 could regulate stomatal limitation of S. viminalis seedlings leaves under heavy metal stress, which was beneficial to water holding capacity of S. viminalis seedlings leaves under heavy metal stress, and enhanced photosynthetic carbon assimilation capacity of leaves under heavy metal stress; Spraying exogenous CaCl 2 can reduce the energy pressure of PSII reaction center by increasing the non-photochemical quenching (NPQ) of leaves of S. viminalis seedlings, alleviate the photoinhibition of PSII, and promote the electron transfer process, especially on the receptor side of PSII; Spraying exogenous CaCl 2 effectively reduced the production of reactive oxygen species in leaves of S. viminalis seedlings, as well as the degree of membrane peroxidation, which was also one of the important reasons for alleviating the inhibition of photosynthetic capacity.


Introduction
Heavy metal stress is an important limiting factor in agricultural production, and the yield of crops caused by heavy metal stress alone exceeds the sum of all pathogens. Lead (Pb) is one of the most harmful heavy metals in the environment. In recent years, with the rapid development of economy, mining, agriculture and recycling of waste metals have significantly increased the concentration of Pb in the soil, and the damage dealt by Pb pollution to the environment has gradually intensified, and the impact on human health has become a hot topic (Soffianian et al., 2014;Sauliutė and Svecevičius, 2015). The majority of Pb enters the human body through the soil-plant-human pathway and causes harm. To a certain extent, Pb in the soil will have a series of adverse effects on plant physiological metabolism, such as the increase of cell membrane permeability, the accumulation of reactive oxygen species, and the aggravation of membrane lipid peroxidation, and even lead to plant death in severe cases (Shu et al., 2012;Ogbomida et al., 2018).
Plant photosynthesis is one of the most sensitive processes to heavy metals. Persistent heavy metal stress will cause irreversible damage to plant photosynthetic apparatus (Zhang et al., 2018a), such as inhibition of photosynthetic pigment degradation (Albert et al., 2011;Guadagno et al., 2017;Chen et al., 2018;Zhang et al., 2018), photosynthetic phosphorylation and electron transport. Oxidative stress caused by oxidative stress in plant cell membrane caused by oxidative stress (Gill et al., 2010;Wang et al., 2017). Plant photosystem II (PSII) is one of the sensitive parts to heavy metal stress. A series of photosynthetic physiological processes such as light energy absorption, water photolysis and electron transfer are closely related to PSII (Allahverdiyeva et al., 2013;Chen et al., 2017). To ensure the stability of photosynthetic function, especially PSII function, of plant leaves under heavy metal stress plays an important role in maintaining the normal growth of plants and improving the stress resistance of plants.
Calcium is not only an essential mineral nutrient element for plants, but also a second messenger for intracellular physiological and biochemical reactions. Therefore, the stress resistance of plants can be improved by stabilizing cell wall and cell membrane structures and inducing the expression of specific genes (Ryan and Kochian, 1993;Larkindale and Huang, 2004). At present, there are a lot of reports about CaCl2 can protect the normal physiology of PSII and increase the content of antioxidant enzymes in plant leaves under stress. S. viminalis is a Salix plant of Salix family. S. viminalis has the advantages of rapid growth, high biomass and strong ability to accumulate heavy metals is widely used in phytoremediation and biomass energy development in heavy metal contaminated soil areas, and has certain ecological and economic value (Zhai et al., 2016). Therefore, improving the resistance of S. viminalis seedlings to heavy metal stress is the key to ensure the survival and growth of seedlings after transplanting. However, the regulation of exogenous CaCl2 on physiological function of S. viminalis seedlings under heavy metal stress, especially the regulation mechanism of PSII function, was less studied. In this experiment, the effects of spraying different concentrations of exogenous CaCl2 on PSII function of S. viminalis leaves under heavy metal stress were studied, in order to provide some basic data for improving the drought resistance ability of S. viminalis.

Test materials and treatment
The experiment was conducted in the laboratory of soil science of Jilin Agricultural University (Changchun, Jilin Province, China) from March to June 2019. The annual seedlings were raised by cutting. The culture substrate was fully mixed with peat soil and quartz sand; the ratio was 2:1 (V / V). It was cultured in an artificial climate box with temperature of 25/23 ℃ (light / dark), light intensity of 400 μmol·m -2 ·s -1 , photoperiod of 12/12 h (light/dark), and relative humidity of about 75%. After the seedlings had long functional leaves, 1/2 of Hoagland nutrient solution was irrigated once a week. When the seedlings grow to six leaves and one heart, they are transplanted. Before transplanting, the seedlings are irrigated once to make the relative water content of the soil basically reach saturation. The seedlings are transplanted into a cultivation bowl with a diameter of 12 cm and a height of 15 cm, and one plant is planted in each pot.
After transplanting, the seedlings with the same growth were selected as the experimental seedlings. The treatment group was sprayed with CaCl2 solution with concentration of 15 and 30 μmol·L -1 (PH=7), and the control group was sprayed with water (CK). The foliar spray was carried out at 4:00 p.m., and the front and back sides were evenly sprayed until the solution on the leaves formed fine mist like uniform droplets ready to drop, after the water on the leaf surface evaporates naturally and the CaCl2 solution is completely absorbed, repeat spraying 1 time with the same amount each time. 10 plants in each treatment were repeated. After spraying CaCl2, the water on the surface of leaves is naturally evaporated. In order to fully absorb the spraying CaCl2 on the leaf surface, after the CaCl2 solution on the leaf surface evaporates, spray once more, and the usage and dosage are the same as the first time. The content of Pb in the test Ning et

Determination parameters and methods
The growth parameters were determined: Vernier caliper was used to measure plant height and leaf area was measured by leaf area meter.
Photosynthetic gas exchange parameters were measured: A CIRAS-2 portable photosynthesis system (PPsystem Company, UK) was used to determine the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO2 concentration (Ci) of the second fully expanded functional leaf of the penultimate leaf of S. viminalis seedlings was selected. The CO2 concentration was fixed at 400 μl·L -1 in a CO2 cylinder. The light intensity PFD was set to 1000 μmol·m -2 ·s -1 with the light source built in the instrument. The net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and intercellular CO2 concentration (Ci) of S. viminalis leaves under different treatments were measured. Repeat 5 times.
Chlorophyll fluorescence parameters were measured: The electron transfer rate (ETR) and non-photochemical quenching (NPQ), the maximum photochemical efficiency (Fv/Fm) and the actual photochemical efficiency (ФPSⅡ) of PSII reaction center under light acclimation were measured by FMS-2 (Hansatch company, UK) for 5 times.
The chlorophyll fluorescence kinetic curve and its parameters were determined: After 30 min dark adaptation, the OJIP curve of leaves after dark adaptation was measured by handy pea (Hansatech compan, UK). According to Strasser et al.'s method (1995), the OJIP curves were standardized by VO-P=(Ft-Fo)/(Fm-Fo) and VO-J=(Ft-Fo)/(FJ-Fo) respectively. The relative variable fluorescence (VJ and VK) of J point at 2 ms and K point at 0.3 ms were obtained, respectively. The differences between the standardized VO-P and VO-J curves of different treatments and the control were calculated, expressed as VO-P and VO-J, respectively. The measured OJIP curve was analyzed by JIP test. The maximum photochemical efficiency (Fv/Fm) of PSII and the photosynthetic performance index (PIABS) based on absorbed light energy were measured.

Determination of ROS metabolism and other physiological indices:
The rate of production of O2 • was measured using the method of Zhang et al. (2007), The malondialdehyde (MDA) content was determined using the thiobarbituric acid method. The conductivity was measured by DDS-11C. Relative conductivity was used to express the electrolyte leakage rate. The content of superoxide dismutase activity (SOD) was determined using the NBT method. The activity unit (U) was 50% of the enzyme that inhibited the photochemical reduction of NBT in 1 ml of reaction solution in 1 h. The activity of ascorbic acid peroxidase (APX) was determined as described by Shen et al. (1996). An activity unit (U) is defined as the amount of enzyme that catalyzes 1 μmol ascorbic acid oxidation in one minute. Each index was measured five times.

Data and analysis
Excel and SPSS software (Version. 22) were used to conduct statistical analyses on the measured data. The data in the figure was denoted as mean ± standard deviation (SE). One-way ANOVA and least significant difference (LSD) were used to compare the differences among different data groups.

Effects of exogenous CaCl2 on leaf growth of S. viminalis seedlings under Pb Stress
Under Pb stress, the leaf growth of S. viminalis seedlings changed significantly, and the increase of leaf area of S. viminalis seedlings without spraying exogenous CaCl2 was significantly delayed (Fig. 1). Compared with CK, the plant height of S. viminalis seedlings was decreased by 20.36% (P＜0.01), reaching a very significant difference level. However, spraying different concentrations of exogenous CaCl2 significantly alleviated the reduction of leaf area of S. viminalis seedlings under heavy metal stress, of which 15 and 30 μmol·L -1 were sprayed under heavy metal stress. The leaf area of S. viminalis seedlings treated with 30 μmol·L -1 exogenous CaCl2 was 26.51% (P˂0.05) and 37.21% (P˂0.05), respectively.

Effects of exogenous CaCl2 on Photosynthetic gas exchange parameters of S. viminalis seedlings under Pb Stress
Compared with CK, Pn, Gs and Tr of S. viminalis seedlings leaves under Pb stress were significantly decreased, but spraying different concentrations of exogenous CaCl2 significantly alleviated the reduction range of Pn, Gs and Tr. Except that there was no significant difference between spraying different concentrations of exogenous CaCl2 and spraying CaCl2 under heavy metal stress, spraying 15 and 30 μmol·L -1 in leaves of S. viminalis seedlings leaves Under CaCl2 Treatment, Pn and Gs in leaves of S. viminalis seedlings were significantly increased compared with those without CaCl2 treatment. It can be seen from Fig. 2D that spraying different concentrations of exogenous CaCl2 also significantly alleviated the Ci reduction of S. viminalis seedlings under heavy metal stress, but there was no significant difference between the Ci of S. viminalis seedling leaves treated with 30 μmol·L -1 exogenous CaCl2 and that without spraying exogenous CaCl2.

Effects of exogenous CaCl2 on Fv/Fm,ФPSⅡ, ETR and NPQ of S. viminalis seedlings under Pb stress
Under Pb stress, Fv/Fm, ФPSⅡ,and ETR of S. viminalis seedlings decreased significantly, while NPQ increased significantly (Fig. 3)

Effects of exogenous CaCl2 on OJIP curve of S. viminalis seedlings under Pb stress
Compared with CK, the relative fluorescence intensity of O point and J point had no significant change under Pb stress, while the relative fluorescence intensity of I and P decreased significantly, especially that of P point (Fig. 4). However, spraying different concentrations of exogenous CaCl2 could significantly reduce the change range of OJIP curve of S. viminalis seedlings under heavy metal stress.

Effects of exogenous CaCl2 on standardized O-P curve, VJ and VI of S. viminalis seedlings under heavy metal stress
Compared with CK, the relative variable fluorescence (VJ) of each point on the VO-P curve of S. viminalis seedlings leaves under Pb stress increased significantly with the relative variable fluorescence VJ of J point at 2 ms (Fig. 5). the increase range of VJ in leaves of S. viminalis seedlings treated with different concentrations of exogenous CaCl2 was significantly less than that of no CaCl2 treatment. At 0.3 ms, the change range of K point relative to variable fluorescence VK was small, and it was not significantly affected by spraying CaCl2.

Effects of exogenous CaCl2 on reactive oxygen species and membrane peroxidation of S. viminalis seedlings under Pb Stress
Compared with CK, the results showed that the treatment of 30 μmol·L -1 exogenous CaCl2 had the most obvious effect, and the O2 •production rate and H2O2 content decreased by 25.83% (P˂0.05) and 32.22% (P˂0.05), respectively, which resulted in the decrease of MDA content and electrolyte leakage rate of membrane lipid peroxidation by 21.43% (P˂0.05) and 25.14% (P˂0.05), respectively (Fig. 6). Under Pb stress, the photosynthetic carbon assimilation capacity of S. viminalis seedlings leaves was limited, which was mainly manifested in the decrease of Pn, along with the decrease of Gs, Tr and Ci. According to Farquhar's photosynthetic stomatal factor analysis theory (Farquhar et al., 2003), the reason for the decrease of photosynthetic carbon assimilation capacity of S. viminalis seedlings under heavy metal stress was directly related to the decrease of stomatal conductance Although the decrease of water loss can effectively prevent the loss of water, it also directly reduces the supply of carbon assimilation material (CO2), which limits its carbon assimilation capacity. In this experiment, spraying different concentrations of exogenous CaCl2 increased Pn in different degrees, which was similar to the change of Gs, and accompanied by the increase of Ci, indicating that spraying exogenous CaCl2 could improve the photosynthetic capacity of S. viminalis seedlings by improving stomatal limitation.
Under Pb stress, the dark response of plant leaves was inhibited, and the accumulation of assimilative capacity (ATP and NADPH) would feedback inhibit the light response process, resulting in the excess of electrons in the photosynthetic electron transport chain (Li et al., 2000;Liu et al., 2006). In addition, the decrease of PSII activity also inhibited the process of light energy absorption and electron transfer. In this experiment, although Fv/Fm, ETR and, ФPSⅡ of S. viminalis seedlings were significantly decreased under Pb stress. Fv/Fm and, ФPSⅡ were important indexes reflecting the photochemical activity of PSII, and the sensitivity of ФPSⅡ was significantly higher than that of Fv/Fm (Kalaji et al., 2016). Therefore, the results showed that the photochemical activity of PSII in leaves of S. viminalis seedlings was inhibited under heavy metal stress, and the process of PSII electron transport was hindered. However, the decrease of PSII photochemical activity was alleviated by spraying different concentrations of exogenous CaCl2, especially 30 μmol·L -1 . The results showed that exogenous CaCl2 could alleviate the photoinhibition of S. viminalis seedlings under heavy metal stress and promote electron transfer. NPQ was positively correlated with heat dissipation dependent on xanthophyll cycle. Under Pb stress, NPQ in leaves of S. viminalis seedlings increased significantly, which means that the excess excitation energy in PSII could be dissipated by increasing NPQ under Pb stress, so as to reduce the pressure of PSII reaction center. The NPQ of S. viminalis seedlings leaves treated with different concentrations of exogenous CaCl2 increased to varying degrees. Ivanov et al. (1995) found that CaCl2 treatment enhanced the xanthophyll cycle in barley seedlings, which enhanced the opening degree of photosystem II reaction center and promoted the utilization rate of light energy. Therefore, the reason why spraying exogenous CaCl2 to alleviate the decrease of PSII in leaves of S. viminalis seedlings under heavy metal stress may be related to the mechanism of energy dissipation dependent on xanthophyll cycle induced by CaCl2. Under stress conditions, the blocking sites of photosynthetic electron transfer often occur on the electron donor side and receptor side of PSII reaction center, especially the transfer of QA to QB is the main inhibition site (Zhang et al., 2018a). The increase of J point relative to variable fluorescence VJ at 2 ms on the normalized O-P curve indicated that the electron transfer from QA to QB was blocked in the photosynthetic electron transport chain Zhang, 2018b). The increase of relative variable fluorescence VK at 0.3 ms on the normalized O-J curve was considered as a specific marker of damage to OEC activity of PSII electron donor side oxygen complex (Zhang et al., , 2016. In this experiment, the VJ of S. viminalis seedlings leaves increased significantly under Pb stress, but the VK did not change significantly, which indicated that the reason for the decrease of PSII photosynthetic electron transfer rate under Pb stress mainly occurred in PSII receptor side. However, the change of VK was not only affected by the donor side injury of PSII, but also by the receptor side injury of PSII. When the injury degree of the recipient side was greater than that of the donor side, the VK did not increase significantly Zhang et al., 2018c). Therefore, in this study, the effect of heavy metal stress on the donor side of PSII may be due to the insensitivity of OEC to Pb stress, or it may be due to the excessive damage on the receptor side. However, spraying different concentrations of exogenous CaCl2 significantly alleviated the increase of VJ, but the effect on VK was not significant, which indicated that exogenous CaCl2 could promote the electron transfer from QA to QB in PSII receptor side of S. viminalis seedlings under Pb stress.
Pb usually leads to excessive reduction of photosynthetic electron transport chain in plants, and produces a large number of ROS in chloroplasts and mitochondria (Ramachandra et al., 2004;Asada, 2009;Ahmed et al., 2009). Excessive ROS breaks the redox balance in plants, causes membrane peroxidation, damages membrane system, and causes oxidative damage to cell components and structures (Jiang and Zhang, 2004;Gill et al., 2010). Under normal conditions, the production and elimination of intracellular free radicals are in a dynamic equilibrium state. When the concentration of exogenous Pb 2+ reaches a certain level, this dynamic balance will be broken, resulting in membrane lipid peroxidation and osmotic stress (Amhed et al., 2009; Li and Li, 2011). The results also showed that under Pb stress, the relative permeability of cell membrane increased, the production rate of O2 •and the content of MDA increased, which caused lipid peroxidation in the inner membrane of S. viminalis. Under Pb stress, O2 •production rate and H2O2 content were significantly increased, MDA content and electrolyte leakage rate were also significantly increased, which indicated that excessive ROS caused membrane system peroxidation and increased membrane permeability. However, spraying different concentrations of exogenous CaCl2 significantly alleviated ROS production and membrane lipid peroxidation of S. viminalis seedlings, especially the treatment of 30 μmol·L -1 CaCl2 was the most effective, which was consistent with the change of photosynthetic parameters of S. viminalis seedlings. Spraying exogenous CaCl2 could alleviate ROS production by enhancing photosynthetic capacity and reducing PSII damage degree of S. viminalis seedlings, and the decrease of ROS production could also alleviate the photoinhibition of S. viminalis seedlings leaves under heavy metal stress.

Conclusion
The photosynthetic capacity of S. viminalis seedlings was decreased under Pb stress, mainly due to stomatal factors, and also related to the decrease of PSII photochemical