Calcium acetate enhances both drought tolerance and arsenic accumulation in Pteris vittata

ABSTRACT Pteris vittata, the first discovered arsenic (As) hyperaccumulator, has great potential in As-contaminated soil remediation. However, as a fern, it is sensitive to water deficiency. In this study, acetic acid and calcium acetate were added to As-contaminated soils in pots to investigate their effects on the drought tolerance, growth and As remediation efficiency of P. vittata. Comparing with the plants pretreated with municipal drinking water, plants pretreated with 20 mM acetic acid or calcium acetate solutions exhibited strikingly increased drought tolerance. In addition, 20 mM calcium acetate significantly increased shoot As concentration by 165% and the total shoot As amount by 55.1%, probably due to the elevated soil pH and a subsequent 14.2% increase of soil available As. Taken together, this study demonstrates that the appropriate application of calcium acetate can enhance both drought tolerance and As accumulation in P. vittata, which is of significance for the phytoremediation.


Introduction
Arsenic (As) is a toxic and carcinogenic metalloid that is widely distributed in nature [1,2].Due to anthropogenic activities such as mining and industrial production, As and its compounds have been increasingly discharged into the environment, causing soil and water As pollution [2].Currently, As pollution has become one of the most severe forms of heavy metal pollution worldwide.Eating plants grown in As-contaminated soils is one of the main routes of human As exposure, threatening human health [3][4][5].Thus, the remediation of Ascontaminated soil is of great significance.
Many physical engineering technologies have been used to remove As from contaminated soil, but these technologies may be environmentally destructive, difficult to carry out in practice, unstable, or costly [6].In this context, phytoremediation is recognized as an economical and effective remediation technology [5].Phytoremediation relies on As hyperaccumulators, which can be highly tolerant to As and accumulate a large amount of As in their aboveground tissues [3].Pteris vittata, the Chinese brake fern, is the first discovered As hyperaccumulator, and can survive in highly As-contaminated soil and accumulate > 23 g kg −1 As in the fronds [7].With its strong ability to extract As from soil and translocate As to its aboveground tissues, this fern is regarded as an ideal plant to remediate Ascontaminated soils.
However, as a fern, P. vittata has a high demand for soil water [8].Soil moisture is one of the most critical factors affecting plant growth, the available As in soil, and phytoremediation efficiency [8].A pot experiment showed that the remediation efficiency of As was the highest when the soil moisture was between 35%-45% [9].In addition, a field study found that both the yield and As uptake of P. vittata were increased by laying water-retaining materials to prevent the evaporation of water [8].
At present, drought caused by drastic climate change severely threatens the survival of plants worldwide [10].Under field remediation conditions, P. vittata may also suffer drought stress [11], which can increase the permeability of the plant cell membrane, decrease the chlorophyll content, lead to stomatal closure, and inhibit plant growth [12,13].As a result, drought can easily affect the efficiency of phytoremediation and even cause the death of phytoremediation plants.In this context, it is of vital importance and great necessity to develop strategies to enhance the drought tolerance of P. vittata and its As extraction efficiency under drought stress.
Recently, the external application of low doses of acetic acid (AA), a simple low-molecular-weight organic acid, has been reported to effectively enhance the drought tolerance of plants [14,15].AA has been demonstrated to be effective in Arabidopsis, rice, wheat, maize, rapeseed, and cassava [14,16,17].However, whether AA or acetate are capable of enhancing the drought tolerance of ferns such as P. vittata is unclear.In addition, low-molecular-weight organic acids such as oxalic, malic, and citric acid may solubilize minerals in soil, promoting plant growth and affecting heavy metal uptake.However, little is known regarding the effects of AA or acetate on the uptake of As by P. vittata.Therefore, this study aimed to determine the impact of the external application of low doses of AA and acetate on the drought tolerance and As uptake of P. vittata.Calcium acetate was used as an acetate, as P. vittata is a calciphilous plant [18].The specific objectives of this study were to (1) study the effects of AA and calcium acetate on the drought tolerance of P. vittata; (2) investigate the effects of AA and calcium acetate on the uptake of As by P. vittata; and (3) examine the influence of added AA and calcium acetate on the phytoremediation efficiency of P. vittata.

Soil samples and plant growth
The As-contaminated soil was collected from Qixia Mountain, Nanjing, Jiangsu Province (118.96°E,32.16° N) [19].The samples were air-dried, crushed, and sieved to < 8 mm.Spores of P. vittata were obtained from Nanjing, Jiangsu.Spores (0.05 g) were added to 500 mL water, thoroughly mixed, and sprayed onto wet potting soil [20].The pots were placed in a tray and covered with a transparent plastic cover to retain the moisture and sprayed with water every 3 d.The spores developed into small gametophytes in about 20-30 d.Sporophytes appeared on the gametophytes after ~2 months of cultivation and reached the two-to-three -frond stage ~3 months after the spores were sown.The sporophyte seedlings were then transplanted to new pots and cultivated for another 2 months before the experiments began.
Uniform sporophyte seedlings with four to five fronds were chosen to transplant into 10-cm diameter pots with 2.5 kg As-contaminated soil (one plant per pot) and cultivated for 2 weeks to allow the plants to take root into soil.The pot soils were then watered thoroughly (0 d) and subjected to AA and calcium acetate treatments at 10 d, 20 d, 30 d, and 40 d.Each pot was watered with 200 mL (about half water holding capacity) AA or calcium acetate solution at 10 d, 20 d, and 30 d, with municipal drinking water as a control (Figure 1).The pots were watered with 400 mL of different solutions or drinking water at 40 d to the maximum water holding capacity of the soil.The plants were then subjected to a drought stress test.The growth of the plants was observed and photos were taken for record.Plants were rewatered when severe drought symptoms appeared.All the plants were collected for As determination after the experiment.
During the experiments, all pots were randomly placed in a greenhouse with a 15 h light/9 h dark photoperiod, a temperature of 22°C to 26°C, and 40% humidity.

As determination
Soil samples were air-dried and sieved to < 0.325 mm.For plant samples, P. vittata plants were separated into the roots and fronds, washed with deionized water, and then dried at 70°C for 24 h.All the soil and plant samples used for total As analysis were digested with 10 mL 50% HNO 3 and 2 mL 30% H 2 O 2 in a graphite digestion furnace (GDANA, DS-360-36X, China) using USEPA Method 3050B [21].For the determination of available As in soil, 2 g soil was extracted with 20 mL 0.5 M NaH 2 PO 4 solution for 2 h (20°C ± 5°C, 250 rpm), and then centrifuged at 3000 rpm for 10 min, after which the As content in the supernatant was analyzed [22].
The total As and soil available As were determined using inductively coupled plasma mass spectrometry (ICP-MS; PerkinElmer NexIOX 300X, USA).The quality assurance and quality control [QA/QC) method of 23, was followed.Briefly, indium was used as an internal standard and added to the samples, calibration standards, and blanks.The check recovery of indium was within 90-110%.During measurement, a standard solution at 5 ppb As was measured every 20 samples to monitor the stability of ICP-MS.In addition, blanks and certified reference material for plant samples (GSB 21, Chinese geological reference materials] were included for quality assurance to confirm that analyzed values fit within expected values.

Soil water and pH analysis
Soil water was measured using an electrical soil moisture meter (TOP Instrument, TZS I, China).For each pot, the soil water contents at a depth of ~10 cm were measured.
For soil pH measurement, 4 g of dried soil was mixed and shaken with 20 mL 0.01 M CaCl 2 solution for 1 h (20°C ± 5°C, 200 rpm).After standing for 30 min, the supernatant of the mixture was used for pH measurement with a pH electrode (INESA Instrument, PHS-3C, China) [20].

Statistical analysis
Data are presented as the mean of four replicates with standard errors.Significant differences were determined using a two-way analysis of variance with Tukey's multiple comparisons test at P < 0.05.

Acetic acid and calcium acetate enhanced the drought tolerance of P. vittata
The properties of selected As-contaminated soils from Qixia Mountain were analyzed, with a pH of 6.04, and a total As of ~110 mg kg −1 .To investigate the effects of AA and calcium acetate, P. vittata seedlings were transplanted to As-contaminated soils and subjected to municipal drinking water (control), AA or calcium acetate solution treatments and subsequent drought treatments (Figure 1).
After a 28-d drought treatment in contaminated soil, control P. vittata pretreated with drinking water showed significant drought stress symptoms, with the pinnae on mature fronds withered (Figure 2).For the plants pretreated with 10 mM AA or 10 mM calcium acetate, no watering for 28 d also led to severe water loss in mature fronds, similar to the control.In contrast, plants pretreated with 20 mM AA or 20 mM calcium acetate exhibited strikingly increased drought tolerance, with uncurling fronds (Figure 2).These results showed that 20 mM AA and 20 mM calcium acetate were effective in enhancing the drought tolerance of P. vittata.After water deprivation, the drought symptoms appeared after 16 d for the control group, while drought symptoms appeared after 23 d for the 10 mM AA and calcium acetate groups and after 28 d for the 20 mM AA and calcium acetate groups.These results demonstrated that both AA and calcium acetate enhanced the drought tolerance of P. vittata, and the proper dose was critical for coping with drought stress.
The plants were rewatered at 28 d after the drought treatment began, and were harvested at day 32.The fresh and dry weights of both the shoots and roots of P. vittata were measured, and the relative water contents of plant shoots were calculated.The fresh weights of control plants were 2.74 ± 0.24 g, while the fresh weights of plants pretreated with 20 mM AA were 4.78 ± 0.77 g, 74% higher than the control.However, the dry weights of the 20 mM AA group (1.68 ± 0.11 g) showed no significant difference compared to the control group (1.53 ± 0.25 g) (Figure 3A).The dry weights of P. vittata pretreated with 20 mM calcium acetate were significantly lower than compared to the control, indicating that 20 mM calcium acetate may negatively affect the growth of the fern to some degree.
The relative water contents in P. vittata roots were 78.2-83.6% and exhibited no significant differences among different treatments (Figure 3B).In contrast, the relative water content in the plant shoots of the control group was merely 34.4% (Figure 3B), showing severe water loss under drought conditions.The plants treated with either AA or calcium acetate showed obviously higher relative water contents in their shoots than the control (Figure 3B).For plants pretreated with 20 mM AA and calcium acetate, the shoots contained 67.9% water and 66.1% water, respectively, or 97.4% and 92.2% higher than control, respectively (Figure 3B).For plants pretreated with 10 mM AA and calcium acetate, the water contents in the shoots were also 37.4% and 40.0%higher than that of control, respectively (Figure 3B).These results are consistent with the growth statuses of the plants, shown in Figure 2, and further indicate that 20 mM AA and calcium acetate can effectively decrease the water loss of P. vittata and enhance its drought tolerance.
14,have demonstrated that exogenous AA can induce the production of jasmonic acid and the acetylation of histone, which in turn endows Arabidopsis with drought tolerance.In addition to Arabidopsis, 14,also found that the external application of AA successfully enhanced drought tolerance in rapeseed, maize, rice, and wheat.Other studies showed that AA could enhance the drought tolerance of cassava (Manihot esculenta Crantz) [17], soybean (Glycine max) [24], and begonia (Begonia × hybrida) [25].Although AA has been shown to protect diverse plants from drought, it has not previously been evaluated for application on ferns, which have strict demands for water.This study first demonstrated that AA and acetate (calcium acetate) were also capable of enhancing the drought tolerance of a fern plant.
The molecular mechanism of how AA functions in enhancing plant drought tolerance is well understood.In plants, the activation of the acetate biosynthesis and the resulting acetate increase are crucial for drought tolerance [14].Exogenous AA increases the acetate levels in plant cells, which promotes jasmonate synthesis and histone acetylation, and thus stimulates the jasmonate signaling pathway and confers drought tolerance in plant [14].This acetate function mechanism is evolutionarily conserved in plants as a critical survival strategy against drought stress, which indicates that acetate has effects not only in monocots and dicots [14,24] but also in ferns.
Recently, reports also showed compelling evidence as to how AA application promotes drought acclimation responses in plants according to physiological attributes [24].It was reported that AA reduced stomatal conductance and transpiration rate, maintained a higher leaf relative water content, and reduced wilting, indicating that AA can be used as a low-cost antitranspirant [17,[24][25][26][27].This was consistent with the present results, in which reduced water loss and wilting were observed in the fronds of P. vittata pretreated with AA and calcium acetate (Figure 3B).In addition, it was also found that the water contents in soils varied in different pots (Figure 3C).After withholding water for 16 d, the pots previously watered with municipal drinking water reserved little water in soil (1.6%), while the pots watered with AA and calcium acetate solutions maintained significantly higher water contents in soils.For 20 mM AA and calcium acetate treatments, the water contents reached 11.5% and 10.1%, respectively, which was 7.4-and 6.7-fold higher than that of the control.This was probably because AA and calcium acetate reduced plant transpiration rate and water absorption from soil.Moreover, the decreased biomass caused by the inhibited transpiration may further decrease the plant water utilization and increase the soil water content.This was also consistent with the higher water contents and the enhanced drought tolerance in the plants.
In the present study, AA and calcium acetate showed limited effects on fern biomass.Although 20 mM AA significantly increased the fresh weight of the shoot biomass, it had little impact on shoot dry weight.In contrast, 20 mM calcium acetate significantly decreased the shoot dry weight by 36%, which may be a negative effect that accompanies the reduced stomatal conductance and transpiration rate under drought stress.

Calcium acetate significantly increased As concentrations in the shoots of P. vittata
After drought stress, As concentrations in P. vittata were also analyzed.The results showed that P. vittata pretreated with AA contained 41.5-50.9mg kg −1 As in its shoots, 35.5-45.5% lower than the control (Figure 4).In contrast, pretreatments with calcium acetate increased the As concentration in P. vittata shoots.For 20 mM calcium acetate pretreatment, the As levels in P. vittata shoots significantly increased by 165% and reached 201 mg kg −1 , which was supposed to benefit the phytoremediation of As-contaminated soils using P. vittata (Figure 4).
To understand how AA and calcium acetate affected As uptake and accumulation in P. vittata, total and available As in the soil were analyzed after P. vittata harvest.The total As in soil showed no significant differences among different treatments (data not shown), as the As extracted by P. vittata only made up a small proportion of the total As in soil.Under AA treatments, the soil available As was 8.14-8.26mg kg −1 slightly decreased compared to the control (Figure 5), which was consistent with the decrease of As accumulation in P. vittata pretreated with AA (Figure 4).In contrast, soil available As pretreated with 10 mM and 20 mM calcium acetate increased by 6.5% and 14.2%, respectively (Figure 5).The higher availability of As was maintained after calcium acetate application, which explained the higher As contents in P. vittata shoots than that under AA treatment and control (Figure 4).Many factors affect As availability and subsequent As uptake by P. vittata, and soil pH is among the most important [28,29].Generally, lower soil pH leads to lower As availability, while higher pH increases As availability [30][31][32][33][34]. Dissolved As increased significantly when the soil pH was above 6.2-6.3 [35], and the solubility of As (V) increased upon the increase of pH in a range from 3 to 8 [36].Increasing pH by adding lime to soil could increase the availability of As, which would consequently enhance As uptake by P. vittata [28].It has been reported that the application of phosphate (5 g kg −1 ) to soil can increase soil pH, and increase NaHCO 3 -extractable As by 45% [37].In this study, the soil pH in control group was 5.95, which is close to initial pH of 6.04.Being an organic acid, AA may decrease soil pH, as the pH values of 10 mM and 20 mM AA solutions were 4.22 and 3.77.The pretreatments with 10 mM and 20 mM AA lowered the soil pH from 5.95 to 5.90 and 5.85, respectively (Figure 5), which was consistent with the reduced soil available As (Figure 5) and the lower As in P. vittata shoots (Figure 4).In contrast, as a strong base-weak acid salt, calcium acetate is weakly alkaline and the pH values of the 10 mM and 20 mM calcium acetate solutions were 7.17 and 7.30.Thus, 10 mM and 20 mM calcium acetate application elevated the soil pH from 5.95 to 6.15 and 6.35, respectively (Figure 5).This was in agreement with the higher available As in soils (Figure 5) and higher As accumulation in plants (Figure 4).In addition, as an essential element for plant growth, calcium may also play a role in promoting As uptake by P. vittata [38; 18].

Calcium acetate improved as extraction efficiency by P. vittata
For phytoremediation, two crucial factors determine the remediation efficiency: plant biomass and shoot heavy metal concentration [3].In this study, although AA enhanced the drought tolerance of P. vittata, it reduced As accumulation in this fern, which impaired  the phytoremediation of As.Unlike the AA treatment, calcium acetate increased both the drought tolerance and shoot As levels of P. vittata.Although 20 mM calcium acetate decreased the shoot biomass (dry weight) of P. vittata by 36% during cultivation with drought stress (Figure 3A), it may still improve phytoremediation efficiency, considering that the shoot As concentrations in P. vittata soared to 2.5-fold of that of control (Figure 4).According to the calculated total amount of As in the shoots of P. vittata, AA pretreatments significantly reduced As amounts by 41.1-50.1% (Figure 6).In contrast, calcium acetate increased the total amount of As in shoots, and 20 mM calcium acetate significantly increased shoot As by 55.1% (Figure 6).These results further demonstrate that calcium acetate has a great potential to improve the As extraction efficiency by P. vittata.

Conclusions and environmental implications
As a fern, P. vittata has strict requirements for its growth environment, especially for soil moisture and air humidity.Its drought tolerance and adaptability to arid environments are relatively weak, which limit its application in the phytoremediation of Ascontaminated soil under complex environmental conditions.Thus, it is vital to enhance P. vittata drought tolerance under the occasional period of dry weather, as well as improve As remediation efficiency under these adverse environmental conditions.
In this study, both AA and calcium acetate enhanced the drought tolerance of P. vittata.The pretreatments with AA lowered the soil pH and reduced soil available As, and thus decreased As level in P. vittata shoots.In contrast, calcium acetate application elevated the soil pH and increased soil available As, resulting in enhanced As concentrations in P. vittata shoots.Though 20 mM calcium decreased plant biomass, it still significantly increased the total amount of As accumulated in the plant shoots by 55.1%, which greatly benefited phytoremediation.Overall, this work provided a simple calcium acetate irrigation method by which P. vittata could be adopted to arid environments and enhanced its remediation efficiency.These findings shed light on the improvement of phytoremediation under practical, complex, and occasional dry conditions.

Figure 1 .
Figure 1.Schematic diagram of the experimental process.

Figure 2 .
Figure 2. Effects of acetic acid and calcium acetate pretreatments on Pteris vittata drought tolerance.(A-E) Representative photographs of P. vittata plants exposed to drought for a period of 28 d after pretreatment with municipal drinking water (A), 10 (B) or 20 (C) mM acetic acid, and 10 (D) or 20 (E) mM calcium acetate.(F-J) Magnified view (5×) of representative mature fronds of treatments A-E, respectively.The experiments were performed in triplicate.Scale bars, 5 cm.

Figure 5 .
Figure 5. Available As and pH of soil at different treatments.Error bars, mean ± SD.Means marked with different letters indicate significant differences (P < 0.05).