Effect of exogenous Ca on the physiology and growth indicators of pakchoi under foliar and root fluorine stress

Fluorine (F) is not an essential element for vegetation and excessive F can be phytotoxic to plant growth, which can cause fluorosis to human beings by ingesting F-contaminated plant. Although there have been some studies focusing on the toxicity of F to plants and the retarding effect of Ca to F-stress plant, atmospheric F contamination to vegetation and the role of the application of foliar Ca are scantly reported. This study investigated several biochemical parameters to evaluate F toxicity under both F-exposure (root and leaf F-exposure) and the remedial effects of foliar Ca. The results showed that F concentration of pakchoi leaves was correlated with exogenous F level positively in both foliar and root F-exposure series, and F concentration of pakchoi roots was only changed under root F-exposure treatments. Ca supplement (0.5 g/L and 1 g/L) significantly decreased plant F concentration. Both F-exposure treatments caused lipid peroxidation in plants and exogenous Ca alleviated the toxicity of F to pakchoi. Meanwhile, chlorophyll-a concentration was decreased by foliar and root F, whereas chlorophyll-b concentration was only affected by foliar F, and chlorophyll-a concentration could be elevated by exogenous Ca but chlorophyll-b could not. It was concluded that both atmospheric and root F can impair pakchoi growth and disturb photosynthesis, and foliar Ca showed an ameliorative effect to F toxicity of pakchoi through alleviating chlorophyll decomposition, increasing protein content and alleviating oxidative damage.

(). It has been suggested that it may be toxic when the intake of fluoride is more than 10 mg day −1 and excessive F may cause skeletal fluorosis and gastrointestinal side effects (Kyzer et al., 2021). For human beings, drinking water and edible plants grown in contaminated environment are the uppermost way to expose to excessive F (Singh et al., 2018). For plant, root absorption of soluble fluoride in soil and foliar absorption of aerial deposition of gaseous fluoride are the most direct approaches to be exposed to F contamination (Gadi et al., 2016a). Several reports about excessive F in edible plants including vegetables were carried out, especially, in the area surrounding F emission factory (He et al., 2021). Although vegetables have always played a vital role in daily diet, studies on F enrichment and resistance in vegetables are scantily reported.
Fluorine ion can be strongly associated with Mg 2+ and Fe 2+/3+ in plant cells, which inhibits biosynthesis of chlorophylls and carotenoids, and then suppresses photosynthesis (Elloumi et al., 2005). Its interactions with Ca 2+ and Mg 2+ could suppress enzyme activities which are strongly related to respiration (Banerjee et al., 2019). Thus, F accumulation could reduce the yield of vegetables and induce phytotoxic effects including lamina tip burning, chlorosis, and even blade necrosis (Fornasiero, 2001). Moreover, F-containing compounds can accumulate exponentially through the food chain into the human body (Han et al., 2021). Such bioaccumulation is not conducive to food security and there is an urgent need to find method to resist fluoride contamination in vegetables.
The uptake of F by plants is affected by other elements in the environment such as Al, B, Cr, and Ca (Ruan et al., 2004;Zhou et al., 2002). In particular, some studies have mentioned Ca as the proven active ingredient to F-contaminated plants (Alvarez-Ayuso et al., 2011a;Ruan et al., 2004;Szostek et al., 2017a). As an essential element to plant cell for its important role in stabilizing membrane and cell wall and participating in information transmission, Ca could also relieve symptoms of fluorosis of plants (Baunthiyal et al., 2015). Previous reports have found the F concentration in plant tissues was inversely correlated with Ca concentration in the surrounding environment, and the inhibitory effect on vegetation growth was relieved with the addition of Ca manifested by a significantly limited influence of F on plant biomass (Alvarez-Ayuso et al., 2011b;Ruan et al., 2004). It has been proposed that the function of alleviating toxicity of F by Ca 2+ was probably because F − can be eliminated by Ca 2+ by forming precipitation, and the channel of F − decreased as the properties of the cell wall or membrane permeability were altered considerably by Ca addition (Banerjee & Roychoudhury, 2019;Boukhris et al., 2018). However, in most studies, Ca was added into the soil or nutrient solution (Fornasiero, 2001;Ruan et al., 2004). Fluorinecontaminated soil, irrigation water and atmosphere constitute the primary routes of plants' exposure to F. However, the uptake of fluoride enters by plant through leaf from atmosphere in dissolved form was overlooked. Moreover, knowledge on the effect of F resistance by the supplementation of foliar Ca is still lacking. Therefore, in the present study, we carried out several experiments to identify the impact of the leaf and root F stress on vegetables and the efficiency of F resistance by exerting different levels of exogenous F and Ca treatments.

Seeds pretreatment
Seeds of pakchoi (Brassica chinensis L.) were purchased from an agricultural station in the central west region of China. Obtained seeds were soaked in deionized water for 4 h to single out plump components. After being disinfected by 0.1% sodium hypochlorite (NaClO) for 10 min and rinsed thoroughly, these seeds were kept in plastic trays filled with deionized water under lucifugal condition for 12 h.

Cultivation conditions
Pre-treated seeds were scattered into seedling trays lined with vermiculite to sprout. Deionized water was rationed daily for the first 7 days, and then, the seeds were cultivated with half-strength Hoagland's nutrition for 15 days until the first leaf was fully unfurled. Afterward, these seedlings were shifted into plastic hydroponic boxes and fixed with high-density purification sponge. Ten liters of half-strength Hoagland's nutrition were added to each hydroponic box for the plant to adapt for 7 days, and then, the culture was continued with the full-strength Hoagland's nutrition.
The pakchoi vegetation in indoor condition was at 22-26 ℃, and each plant was maintained at a level of 60% of the relative air humidity and with photoperiod of 12 h.
Two exposure approaches to F were considered: root F-exposure and foliar F-exposure. Root F-exposure was performed by adding sodium fluoride (NaF) solution to Hoagland's nutrition. Foliar F-exposure was subjected to the increasing contamination of F in a same form of NaF which was sprayed to the pakchoi leaves. The gradient for contamination with F amounted to 0, 5, and 10 mg/L. The nutrient solution was renewed every 7 days.
Calcium nitrate (Ca(NO 3 ) 2 ) was used as the blocking agent by foliar spray, and the level amounted to 0, 0.5, 1, and 1.5 g/L. Each level was applied evenly and quantitatively to the leaves of pakchoi by spraying every 7 days, and the whole experimental cultivation period was kept at about 35 days. The control seedlings were raised identically except without F in growth condition and used deionized water for spraying instead of Ca(NO 3 ) 2 . All treatments were replicated three times, and the schematic is shown in Fig. 1.

Harvest and measurements
After cultivation for 35 days, the plants were harvested and washed by deionized water. Afterward, 8 plants were picked out stochastically in each treatment to measure plant height and root length by vernier caliper, and the fresh weight of the hypogeal and aerial parts of the pakchoi was measured. These plant samples were randomly separated into 2 parts. One part was stored in − 80 ℃ condition after quick-frozen by liquid nitrogen for the determination of concentration of free proline, malondialdehyde and soluble protein, and the other part was stored after being dried to a constant weight and crushed to pass through a 0.15 mm sieve for the F concentration determination.

F and Ca concentration
Powdered samples of the plant roots and leaves were digested with 5 mL concentrated nitric acid (HNO 3 ) in a microwave digestion system (Sineo MDS-6, Shanghai, China), and 10 mL supernatant was taken for F concentration determination by F ion selective electrode method (MP523-04, Sanxin, China). The Ca concentration of plant tissues was analyzed by an inductively coupled plasma atomic emission spectroscopy (PRIDE100, HKTC, China) after nitric-perchloric digestion.

Chlorophyll concentration
Fresh samples of the pakchois were blended with the 1:1 mixture of anhydrous ethanol and acetone under lucifugal condition for 5 h to extract the chlorophyll. The mixture was filtrated and the extractant was used for chlorophyll concentration measurement by spectrophotometric method. An ultraviolet spectrophotometer (UV-1100, MAPADA, Shanghai, China) was used to determine the absorbance of supernatant at 645 nm and 663 nm, respectively. The concentration of chlorophyll-a and chlorophyll-b was calculated by the formula as following (Iram et al., 2016): where A 663 = absorbance at 663 nm, and A 645 = absorbance at 645 nm.

Soluble protein concentration
Soluble protein concentration was determined by Coomassie brilliant blue G-250 method (Goldring, 2019). Briefly, 0.5 g of fresh plant sample was ground into homogenate with 5 mL distilled water. The mixture was centrifuged at 10,000 rpm for 10 min, and the supernatant was left for chromogenic reaction with Coomassie brilliant blue G-250. After fully mixing for 2 min, absorbance was measured by colorimetry at 595 nm. Soluble protein concentration was calculated according to the standard curve.

Proline and malondialdehyde level
Free proline concentration was measured by a rapid determination of sulfosalicylic acid extraction spectrophotometry as reported by Bates et al. (1973). Acid-ninhydrin as the chromogenic agent was prepared by 1.25 g ninhydrin, 30 mL glacial acetic acid and 20 mL 6 M phosphoric acid with agitation until dissolved, and acid-ninhydrin was mixed with the supernatant extracted by 10 mL of 3% aqueous sulfosalicylic acid from 0.5 g fresh sample. Afterward, methylbenzene was used to leach the proline. The absorbance of extraction was determined at 520 nm. Besides, the level of malondialdehyde (MDA) was measured to indicate the level of lipid peroxidation by thiobarbituric acid method according to Dhindsa et al. (1981).

Data analysis
The data of physiology and growth indicators were subjected to one-way analysis of variance (ANOVA) to examine the effect of exogenous F on pakchoi growth and the restorative impact of exogenous Ca on fluorosis symptoms under same F stress. Statistical significance was accepted as p ≤ 0.05. The program packages SPSS and Excel were used to perform statistical analyses.

Plant growth parameters
Total length of the pakchoi stem and root was determined at different concentrations of NaF and Ca(NO 3 ) 2 groups (Fig. 2). More severe inhibitory effect was documented in leaf F-exposure series than in root F-exposure treatments. In the cultivation test of foliar F stress, a significant decrease in stem length compared to that of the control induced by 5 mg/L and 10 mg/L F was recorded (p < 0.05), while no noteworthy difference was observed in root F-exposure treatments. Besides, the effect of F on root length in both F-exposure series was inconspicuous.
Leaf exposure to F significantly decreased the fresh weight of the plants without the neutralizing substance of Ca (Fig. 2). However, root F-exposure series exhibited exactly inverse relationship. The fresh weight was elevated to a large extent under root F stress, and the highest fresh weight of leaves and roots was observed in the root F-exposure treatments with 5 mg/L F, which increased by 149.66% and 185.39%, respectively, compared to control. When treated with 10 mg/L root F-exposure, the fresh weight of leaves and roots of the pakchoi was decreased as the addition of Ca sprayed on leaves, whereas a significantly monotonic augmentation was documented when exogenous Ca was applied in foliar F treatments.
When the pakchois were treated with 5 mg/L and 10 mg/L NaF in foliar series, the addition of Ca(NO 3 ) 2 at 1 g/L and 1.5 g/L evidently increased the plant height compared to that of the control plants that were not treated with Ca. As the foliar F stress was intensified to 10 mg/L, exogenous Ca at a concentration of 1.5 g/L induced a 40.70% increase of Similarly, there was a significant elevation in stem length when 0.5 g/L exogenous Ca was added under 5 mg/L NaF root stress. Besides, no visible effect of Ca(NO 3 ) 2 on root length occurred at any of the F-exposure treatments.
In addition, we discovered that the exposure to F in root and leaf treatments groups did not exhibit apparent fluorosis symptoms in the first 2 weeks of the incubation trials. However, abnormal plant growth was discovered in the beginning of the third week in 5 mg/L and 10 mg/L treatment groups of both F-exposure approaches, which was mostly shown by chlorosis, gradual thinning of old leaves, subsequently shrinking and drying till wilting. Meanwhile, individual leaf curling was observed in the root F-exposure groups, where the leaf surface became uneven and the leaf edges curled inward from both sides. It is worth noting that the number of plants showing chlorosis gradually increased with increasing Ca spraying concentration in the no-F treatment, and with 22.2% and 11.1% of the plants showing chlorosis at a Ca application concentration of 1.5 g/L for pakchoi that absorbed F via roots and leaves, respectively. Exogenous Ca successfully controlled chlorosis in the leaf F-exposure treatments, whereas it had no significant restorative impact on pakchoi in the root F-exposure groups; instead, the application of high concentration of Ca increased the amount of yellow leaves and curled plants.

The uptake of F and Ca by pakchoi
Generally, all exogenous Ca resulted in a significant increase on Ca concentration in the pakchoi. The pakchoi in control groups had leaf Ca concentrations ranging from 5.12 to 7.91%, with a mean value of 6.52%. In root F-exposure treatments, leaf Ca concentration showed a positive correlation with exogenous Ca concentration, whereas a lower Ca concentration was found when leaf was treated with 1.5 g/L Ca in leaf F-exposure treatments than that of 1 g/L Ca addition treatments. Similar results were found in F absent treatments.
The F concentration in roots (RF) and leaves (LF) in F absent treatments was 0 (below the detection limit) and 8.42 mg/kg, respectively. Of both F-exposure groups, environmental F stress caused several folds of accumulation of F in the leaves compared to that of the control (Fig. 3). Moreover, LF positively correlated with exogenous F level, whether it's root or leaf F-exposure. Additionally, LF in leaf F-exposure treatments was 1.43-5.89 times higher than that in root F-exposure groups, and higher F accumulation in roots was found in root F-exposure series, which were approximately one order of magnitude more than the foliar treatments at the same F stress level. Root F-exposure caused the accumulation of F in leaves, which was still far below than that in roots. However, RF in foliar F-exposure treatments was below the detection limit.
When 10 mg/L F was applied in the root F-exposure treatments, exogenous Ca at 0.5 g/L and 1 g/L significantly lowered RF by 16.12% and 25.12%, respectively (p < 0.05), whereas 1.5 g/L exogenous Ca did not exert significant impact as the RF was significantly elevated compared to the series treated with 1 g/L Ca. Foliar Ca did not affect LF in root F-exposure treatment groups, but decreased LF significantly in leaf F-exposure series at a Ca dose of 1 g/L (p < 0.05).
Effects of F and Ca on the concentration of chlorophyll-a, chlorophyll-b, and total chlorophyll Generally, the impact of F stress without neutralizing substance of Ca(NO 3 ) 2 significantly decreased chlorophyll-a, chlorophyll-b, and total chlorophyll concentration in foliar F-exposure series compared to that of the control (Fig. 4). When the given foliar F stress was 5 mg/L, the concentration of chlorophyll-a and chlorophyll-b was significantly decreased by 29.4% and 25.18% (p < 0.05), respectively, manifesting the phytotoxicity of F to photosynthesis. Similar inhibition was recorded when root was exposed to 5 mg/L F in chlorophyll-a and total chlorophyll, whereas the level of chlorophyll-b was unaffected.
On the other hand, when the roots of pakchoi were exposed to 5 mg/L F, the influence of increasing levels of neutralizing substance of Ca on the concentration of chlorophyll-a depended on the dosage of Ca. The concentration of chlorophyll-a significantly increased when 0.5 g/L and 1 g/L Ca were added (p < 0.05), and the highest level was documented at 1 g/L Ca treatment, which was 1.75 times that of Ca absent series. However, the level of chlorophyll-a was evidently decreased as the addition of 1.5 g/L Ca in F-absent series.
With respect to 5 mg/L foliar-F treatments, low concentration of Ca had no apparent effect on chlorophyll-a concentration until 1 g/L and 1.5 g/L of Ca were added. As for chlorophyll-b, however, there was no distinct observation of increase in leaf F-exposure as well as root F-exposure series versus the control, except that a sharp decrease was documented in F absent groups after the addition of 1.5 g/L Ca.
Similarly, the concentration of total chlorophyll in the series with 0.5 g/L Ca manifested an evidently increasing influence compared to the series without Ca addition, whether it was foliar or root F-exposure to 5 mg/L F stress. When exposed to 10 mg/L F, however, this remedial approach lost efficiency.

Soluble protein
In F absent groups, the highest concentration of soluble protein was found when 0.5 g/L Ca was applied. However, exogenous Ca at the level of 1 g/L and 1.5 g/L decreased the soluble protein concentration by 37.30% and 40.39%, respectively (Fig. 5).  In the case of 5 mg/L root F stress, a sharp decrease of the soluble protein level occurred versus the control, after a given F-exposure time for 0.5 g/L and 1 g/L neutralizing Ca for 35 days, and 3.51-and 3.79-folds increase occurred on the soluble protein concentration in contrast to the control, respectively. Likewise, the highest concentration of external Ca limited the elevating effect on internal protein level. Similar results were also obtained under 5 mg/L foliar F stress, and the limit effect caused by 1.5 g/L exogenous Ca was more pronounced.
With respect to application of 10 mg/L F in root F-exposure treatments, exogenous Ca (0.5 g/L and 1 g/L) elevated the soluble protein concentration evidently. As for foliar F stress at 10 mg/L, exogenous Ca at 0.5 g/L and 1.5 g/L elevated the soluble protein concentration by 9.04% and 29.52%, respectively. Oxidative impairment MDA concentration was determined to assess the degree of lipid peroxidation in the cell membrane (Fig. 6). The addition of F significantly elevated the level of MDA in the plant leaves. When 10 mg/L F was applied, MDA concentration increased by 65.85% and 217.56% in root and leaf F-exposure series compared to that of the control, respectively, without neutralizing Ca. When 1.5 g/L Ca added in the foliar F treatments, the MDA level was decreased by 16.39% and 12.20% under 5 mg/L and 10 mg/L F stress, respectively, whereas the MDA concentration was unaffected by exogenous Ca in root F-exposure treatments.
Dissociate proline concentration was also determined under different doses of F stress (Fig. 7). When 5 and 10 mg/L F was added into the nutrient solution, the addition of Ca did not exert an alleviative effect on F toxicity and intensified the proline level. Likewise, similar results were observed in foliar F treatments at a concentration of 5 mg/L. When 10 mg/L F was sprayed to the pakchoi leaves, exogenous Ca at a concentration of 1 g/L significantly decreased the level of proline by 14.38%, whereas 1.5 g/L Ca exerted significantly inverse influence, i.e., increased the proline concentration by 23.26%. In the present study, the concentration of peroxidase showed an evident increase in exogenous Ca treatment when 5 mg/L F was applied in both root and foliage treatments (p < 0.05), whereas no similar phenomenon was observed under 10 mg/L F stress.

Discussion
The contamination of F exerted a serious inhibition on plant growth. In accordance with the present results, previous studies have documented the inhibition of F stress on harvested biomass of plants. Bhargava and Bhardwaj found that 4-20 mg/L NaF caused a significant reduction on plant biomass of both aerial parts and roots (Bhargava et al., 2010). Similar results were also reported in the F-exposure experiments with Abelmoschus esculentus by Iram and Ti who observed that root length, shoot length, root weight and shoot weight reduced by 37.91%, 44.42%, 67%, and 73.37%, respectively, in response to the highest dose of 50 mg/L F stress (Iram & Ti, 2016). Gupta conducted trials on paddy, where 20% and 29% reduction of fresh weight and dry weight of the seedlings were noticed at the highest dose (30 mg/L) of NaF (Gupta, 2009).
Obtained results in the root F-exposure series in the present study which indicated the promoting effect on harvested biomass of the pakchoi may be due to the repellence of the plant. Szostek et al. tested eight plant species and recorded a significant increase in biomass in F-tolerant plants as well as the distinct growth inhibition in F-sensitive plants (Szostek et al.,Fig. 7 The concentration of proline under root and foliar F stress with exogenous Ca concentration gradient 2017b), which indicated pakchoi may have decent resistance to F. Moreover, the increased biomass following Ca application may be due to the precipitation reaction with F which can neutralize phytotoxic effects of F. Szostek et al. (2017b) noted a significant increase in aerial parts of several crops after adding lime, which was proved that Ca was the proven active ingredient to resist F, which limited the negative effect of this xenobiotic by inactivating it. Álvarez-Ayuso et al. reported a raising trend in the biomass of plants with the flue gas desulfurization gypsum addition, which supplied Ca and resulted in the formation CaF (Alvarez-Ayuso, et al., 2011b). It is worth noticing that the application of foliar Ca was easier to make a difference in foliar series where less internal plant pathways to absorb and transport Ca were required. Hence, it was possible that there was excessive Ca as the deficient reaction with F and caused salt effect in root F-exposure groups and thus lowered the biomass in high dose of additive Ca series. Spraying Ca to leaves did not effectively prevent F from entering the plant via the roots, which resulted in fluorosis symptoms-chlorosis of the leaves, while it was discovered that exogenous Ca also caused some damage to the plant leaves to a certain extent in the treatment without F application, which could be the result of Ca over-application. In contrast, foliar Ca administration at 1 g/L and 1.5 g/L under F-exposure treatment considerably reduced the number of chlorosis in the plants and demonstrated good alleviation of fluorosis symptoms in the plants.
Previous literature documented different doses of environmental F stress on F concentration in plant tissues (Abdallah et al., 2006a(Abdallah et al., , 2006bFornasiero, 2001;Saini et al., 2012). Iram et al. reported a significantly positive correlation between inner F level and exogenous F (Iram & Ti, 2016). Gupta demonstrated 26 and 53 times increase of F uptake at 20 and 30 mg/L NaF treatments, respectively, in paddy cultivation experiments (Gupta, 2009). Besides, the positive correlation between exogenous Ca level and the concentration of Ca in plant tissues was also reported by Ruan et al., (2004) who introduced CaO to the soil in which tea plants grew.
Singh et al. noted pathways for F adsorption by plants including apoplastic transport system, anion channel and stomata (Singh et al., 2018). Higher F accumulation of LF in foliar F-exposure than root F-exposure series indicates that atmospheric F contamination may have serious potential hazard. Besides, higher F adsorption in roots than in leaves was also observed by Iram and Ti (2016) and Saini et al. (2012). In foliar F-exposure treatments, RF was unaffected by foliar F stress, while LF increased significantly as the F contamination increased in both foliar and root F-exposure series. These results manifested the important role of atmospheric F in inhibiting plants growth.
Reactions between Ca and F were usually considered being responsible for dynamics of F concentration in plant tissues. Boukhris et al. found the formation of Ca crystals under F stress which were involved in plant detoxification and defense (Boukhris et al., 2018). In the present experiments, the decrease of F concentration occurred in series with exogenous Ca addition verifies previous observations which demonstrated similar results by cultivating plants in contaminated soil and solution. Ruan et al. conducted experiments on tea plants, and they reported a negative correlation between addition of Ca and the LF, while the root and stem F concentrations were unaffected by Ca solution (Ruan et al., 2004). The reason for the discrepancy in the present study could be attributed to the different way of Ca absorption, which can take place through stomatal diffusion through the leaf stomata under atmospheric F stress (Gadi et al., 2016). Moreover, it is possible that Ca in the solution can be precipitated by F in nutrient solution, which was not completely absorbed, and resulted in inadequate utilization of exogenous Ca. Besides, the formation of CaF 2 coatings has been reported on root surface with the addition of soluble Ca under rhizosphere F stress. The large surface area of pakchoi leaves might provide much space for forming CaF 2 coatings to resist F. However, the application of 1.5 g/L Ca in the present experiment restricted the neutralizing effect, and a possible explanation may be the salt effect caused by the highest dose of Ca.
Fluorine in both root and leaf impaired the yield of chlorophyll, which neutralized by exogenous Ca at low concentration (Fig. 4). Gupta (Gupta, 2009) demonstrated a sharp decrease of chlorophyll-a, chlorophyll-b, and total chlorophyll content under 30 mg/L F. Singh et al. noted a typical declining trends of chlorophyll yield under various doses of F (100-500 mg/L) (Singh et al., 2013). A widely accepted interpretation for inhibition of chlorophyll by F was considered as the stimulative breakdown of chlorophyll or inhibition of chlorophyll-biosynthesis. On the one deed, the association of F − with Mg 2+ and Fe 2+/3+ and the decrease of their translocation should be responsible for reduction of chlorophyll-biosynthesis (Elloumi et al., 2005). McNulty et al. documented the quantities of ether-soluble Mg varied in the same directions and with the same magnitudes as did the chlorophylls (McNulty et al., 1961). Another biosynthesis approach which involves γ-aminolevulinic acid can also be suppressed under F stress (Wallis et al., 1974). Moreover, it is suggested that the formation of MgF + can degrade the pigments (Abdallah et al., 2006a(Abdallah et al., , 2006b. Meanwhile, the disintegration of the chloroplasts could also be responsible for chlorophyll degradation as well (McNulty & Newman, 1961).
It is noted that the sequestration of F by forming CaF with Ca 2+ played a crucial role in detoxification (Levy et al., 1973). Nevertheless, in the present study, 1.5 g/L Ca did not have apparently remedial effect on the yield of chlorophyll or even impaired it. That was probably because the salt stress exerted inhibitor influence on chlorophyll-biosynthesis.
The higher level of applied F-contaminated nutrient solution or mist spray induced the lower possibility of resistance to F contamination by Ca. It seems possible that these phenomena are due to that high doses of F usually caused worse damage to plant cell involving the integrity of membrane and activities of enzymes, which could not be remediated by the neutralizer of Ca. It has been reported that higher doses of F had a distinct effect on membrane lipid-protein (Rakowski 1997) and chlorophyll-binding proteins (Banerjee & Roychoudhury, 2019), which are closely related to membrane stability and photosynthesis. Additionally, the elevating effect on protein by Ca could be combined with the physiological parameter of plant weight. For example, 1.5 g/L exogenous Ca caused 3.07 and 1.30 times increase in leaf fresh weight and protein concentration, respectively, when 10 mg/L foliar F was applied, at the same condition compared to the control (Figs. 2, 4).
The stress of F retards the physiological activities of plants may be due to the production of reactive oxygen species (ROS), which caused lipid peroxidation with a production of MDA (Gunes et al., 2007). In the present study, the concentration of MDA which related to severe damage to membrane and protein was significantly elevated under various F stress. Li et al. documented significant accumulation of MDA, which suggested that antioxidants did not sufficiently scavenge excessive reactive oxygen species to protect the tissue from free radical injury under the F stress (Li et al., 2011). Obtained findings in the present study manifested that exogenous Ca could play a part in preventing the lipid peroxidation caused by F contamination; however, 1.5 g/L Ca may aggravate oxidative damage because of the salt effect, and a deeply clear and unambiguous explanation for the remedial mechanism is still lacking.
Proline is also an adaptive response to F stress, which occupies a central place in metabolism by stabilizing cellular structures and acting as a free radical scavenger (Verslues et al., 2010). Datta et al., (2012) reported a monotonic increase of the level of proline under increasing doses of F. Li et al. (2011) reported remarkable increase in proline content in their trial. Besides, in the present study, salt effect did not exert effect on proline concentration even when 1.5 g/L Ca was added.

Conclusion
In the present study, foliar exposure to F disturbed the growth of pakchoi, which was manifested by the significant decrease on plant height and fresh weight of the roots and leaves. Whereas, a sharp increase of fresh weight was reported under various root F stress (5, 10 mg/L). Both F-exposure treatments led to F enrichment in plant tissues except that RF was unaffected by foliar F. Under 5 mg/L F stress, the concentration of chlorophyll-a was adversely affected by both F-exposure pathways, and chlorophyll-b and soluble protein level were adversely affected by foliar and root F-exposure treatments, respectively. The concentrations of MDA, dissociate proline and peroxidase were evidently elevated by increased NaF level in both F-exposure series. Exogenous Ca at 0.5 g/L and 1 g/L efficaciously decreased F concentration in plant tissues. The application of foliar Ca made a difference on the alleviation of lipid peroxidation and promoted morphological features such as plant height and fresh weight under root and foliar F stress. cooperation project between Sichuan University and Yibin municipal government (2019CDYB-19).
Author contribution J-YY contributed to the conception of the study; RL performed the experiment; S-FC contributed significantly to analysis and manuscript preparation; RL performed the data analyses and wrote the manuscript; C-DG helped perform the analysis with constructive discussions.

Funding
The authors have not disclosed any funding.

Conflict of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.