Flue gas desulfurization gypsum by-products alters cytosolic Ca^{2 +} distribution and Ca²⁺-ATPase activity in leaf cells of oil sunflower in alkaline soil

Abstract

Soil salinization in arid zones is a major factor that resulted in the reduction in the yield and quality of many important crops in Northwestern China. In this study, the potential mechanism of flue gas desulfurization gypsum by-product (FGDB) mediated amendment of alkaline soils was investigated in an oil sunflower model by accessing the Ca²⁺ distribution and Ca²⁺-ATPase activity in leaf cells. Our results demonstrated an increased calcium concentration, as well as intact chloroplast structure with increasing calcium precipitates in the cell wall, intercellular space, and vacuole of leaf cells in the plants grown in alkaline soils supplied with FGDB or CaSO₄. Additionally, a dose-dependent Ca²⁺-ATPase activity was detected in the plasma membrane and tonoplast of leaf cells from the plants grown in FGDB or CaSO₄ supplemented soils. These results implied that the Ca²⁺-ATPase activity cause cytosolic Ca²⁺ efflux. The Ca²⁺ influx is through the Ca²⁺-channels, and increasing cytosolic Ca²⁺ concentration might benefit the stability and integrity of cell membrane and cell wall, sequentially alleviated the injury of oil sunflower against alkali stress.

Keywords:

• flue gas desulfurization gypsum by-products
• CaSO₄
• alkaline soils
• Ca²⁺ distribution
• oil sunflower

Introduction

Salinity in arid zones is one of the major abiotic factors limiting agricultural and forestry productivity, rendering an estimated one-third of the world's irrigated land unsuitable for crops (Frommer et al. 1999). The amount of salt-affected soil is increasing due to the increase in the evaporation rate and excessive cultivation, which has become a serious issue in Northwestern China. Alkaline soil accumulates salt on its surface, and it is difficult for plants to grow. Salt tolerance of a plant is a complex trait involving responses to cellular osmotic and ionic stresses, as well as their consequent secondary stresses and whole plant coordination. The complexity and polygenic nature of salt stress tolerance are important factors contributing to the difficulties associated with breeding salt-tolerant crop varieties. Hence, the amendment of alkaline soils, as well as a better understanding of the how the plant mechanisms adapt to salinity stress and maintain their growth will ultimately help human with the selection of stress-tolerant plants for exploiting saline soil (Sudhakar et al. 2001; Rahnama & Ebrahimzadeh 2005; Lecourieux et al. 2006).

Calcium is required as a secondary messenger for certain processes in plant defense mechanisms (Messiaen et al. 1993; Levine et al. 1996; Sanders et al. 1999; Ma et al. 2009). Increasing number of studies have suggested that Ca²⁺ widely participated in the processes of signal transduction that is induced by various stimuli and environmental stresses, such as salt, oxidation drought, cold, diseases, and metallic ions, in many plants (Li et al. 1997; Kiegle et al. 2001; Kadota et al. 2004; Kawano et al. 2004). Particularly, Ca²⁺ also participated in many cytosolic signal transduction processes induced by various stimuli outside of cells (Kiegle et al. 2001; Kawano et al. 2004; Liu et al. 2010), which were mainly based on the alteration of the concentration, channel type, spatial distribution, and open mechanism of Ca²⁺. These studies suggested the important roles of Ca²⁺ concentrations, Ca²⁺ distribution, and activity of Ca²⁺-ATPase in the regulation of plant physiological responses to stress-resistance.

The reclamation of sodic soil involves the replacement of exchangeable Na⁺ with Ca²⁺ that could be supplied by the presence or addition of gypsum (Oster & Frenkel 1980; Keren & Miyamoto 1990). Flue gas desulfurization gypsum by-product (FGDB), 80% of its component is calcium sulfate (CaSO₄·2H₂O), has shown a capacity for amending alkaline soils. The FGDB is the most common alkaline soil amendment since it has the advantages of being nontoxic to plants, easy to handle, and moderately soluble. Thus the FGDB has been utilized as an efficient and a low cost-effective soil amendment for reclaiming alkaline soil (Keren & Miyamoto 1990; Ishak et al. 2002). Importantly, recent studies have demonstrated that FGDB was capable of decreasing the soil pH and exchangeable sodium percentage (ESP), improving the physical shape of soil moisture, as well as increasing soil aggregate structure in Northwestern China (Feldhake & Ritchey 1996; Sakai et al. 2004; Xiao et al. 2010). However, limited information about the mechanism of FDGE-mediated soil amendment, as well as the detail effects, and its main component CaSO₄ on plants is currently available. Therefore, we hypothesized that the function of FDGE on amendment of alkaline soil might be mediated by a mechanism of alteration of cytosolic calcium distribution and Ca²⁺-ATPase activity in the plant cells. To test this, hypothesis against alkali stress was investigated in an oil sunflower model. In this study, the effects of the supplementation of FGDB or CaSO₄ on the Ca²⁺ distribution and Ca²⁺-ATPase activity in leaf cells were evaluated in an oil sunflower model grown in an alkaline soil.

Materials and methods

Experimental soil

Soil used in this study was collected from Pingluo County (38°50′N, 106°32′ E) of Ningxia Province in the Northwest of China, The region is a typical arid zone with a monsoon climate. Mean annual temperature is 8.5°C. The soil texture at this geographic site is classified as mainly clay, with clay 250–450 g kg⁻¹, silt 0–450 g kg⁻¹, and sand 100–550 g kg⁻¹. The total salt content ranges from 2.5 to 6.5 g kg⁻¹. The range of pH is 8.0–10.4. Electrical conductivity (Lecourieux et al. 2006) ranges from 0.34 to 2.96 dsm⁻¹.

Plant material and treatment

The experiment was conducted in pots which were filled with 5 kg of soils collected 0–20 depth from Pingluo County in Ningxia Province in the Northwest of China, The components of soil were: total salt 10.80 g kg⁻¹; pH 8.66; total alkalinity 0.60; organic matter 13.00 g kg⁻¹; total phosphorus 0.57 g kg⁻¹; total potassium 21.30 g kg⁻¹; anion 0.60 g kg⁻¹; 11.29 g kg⁻¹; Cl⁻ 6.73 g kg⁻¹; and cation Na⁺ 14.70 g kg⁻¹; Ca²⁺ 2.34 g kg⁻¹; Mg²⁺ g kg⁻¹. FGDB was obtained from Ningxia Maliantai power plant (containing 800.5g Kg⁻¹ of CaSO₄·2H₂O). A CaSO₄ control was included to validate that it was a main and effective compound in the FGDB in this study. The soil and FGDB were mixed at ratios (w/w) of 5 kg:26.66 g (T₁) and 5 kg:53.33 g (T₂). The soil and CaSO₄ were mixed at ratios (w/w) of 5 kg:21.33 g (T₃) and 5 kg:42.66 (T₄); a control soil (CK) without FGDB or CaSO₄ was also included. The plants were grown in a greenhouse at Ningxia University in China, started on 10 April 2009. ‘KWS203’ cultivar oil sunflower was chosen as the experimental plant for this study. Twenty seeds were sown in each pot, and the plants were thinned leaving three of them to grow in the pot after the crops were established. A total of 30 plants grown in 10 pots for 15–20 d were analyzed for each condition in this report.

Preparation of protoplasts

The leaves of oil sunflower were treated with hydrolysate solution (0.2% BSA, 0.5% PVP, 1.0% cellulase R-10, 0.1% pectinase Y-23, and 0.4 M D-sorbitol pH 5.7) in a small beaker by shaking at 55 rpm for 3 h at 27°C in darkness, followed by filtering the solution with a 50 µm nylon mesh. The filtrate was then centrifuged for 5 min at 100× g, and the protoplast pellet was washed twice with the buffer solution containing 5 mM 2-(N-morpholino)ethanesulfonic acid buffer (MES), 1 mM CaCl₂, 2 mM MgCl₂, 0.4 M D-sorbitol, pH 5.7. Then the protoplasts were suspended in buffer solution and stored at 4°C before use.

Protoplast Fluo-3AM loading

Fluo-3AM stock solution (Sigma, St. Louis, MO, USA) prepared with anhydrous dimethyl sulfoxide (DMSO) was added to protoplast suspension at a final concentration of 5 µM, followed by gentle mixing and incubating at 4°C in darkness for 1 h before it was washed with buffer solution (5 mM MES, 1 mM CaCl₂, 2 mM MgCl₂, 0.4 M D-sorbitol, and pH 5.7) for three times by centrifugation at 100× g. The protoplasts were then used for experimental analysis.

Confocal scanning laser microscopy

Protoplasts loaded with Fluo-3AM were mounted in a chamber with a clean cover slip attached to the bottom. Fluorescence from the protoplasts loaded with Fluo-3AM was visualized using a LSM 510 confocal microscope. Excitation was at 488 nm and detection was under 510–525 nm.

Fixation and transmission electron microscopy of leaves

The distribution of Ca²⁺ in the leaf was accessed by a transmission electron microscopy, since Ca²⁺ was capable of binding potassium pyroantimonate to form dark particles, an indication of the distribution of cellular Ca²⁺. Briefly, the oil sunflower leaves were cut into approximately 1 mm² pieces and fixed in a fixative containing 3% glutaraldehyde, 2% potassium pyroantimonate, and 50 mM potassium phosphate (pH 7.6) at 4°C for 4h, followed by washing three times in 50 mM potassium phosphate and 2% potassium pyroantimonate (pH 7.6) for 30 min each. Samples were subsequently fixed in 1% OsO₄ containing 2% potassium pyroantimonate and 50 mM potassium phosphate (pH 7.8) for 12–16 h at 4°C, rinsed in 50 mM potassium phosphate buffer for three times (30 min each), dehydrated in a graded acetone series, and embedded in Epon 812 resin (Qin et al. 2005). Ultra-thin sections were obtained using a Leica UC6i ultramicrotome and observed using a JEM-1230 transmission electron microscopy.

Ca²⁺-ATPase activity of plasma membrane and tonoplast extraction

Plasma membrane and tonoplast vesicles of leaves were isolated according to a previously described method with modifications (Wang et al. 2001). Briefly, a 30 g of leaves was washed with cold deionized water and homogenized in 60 ml of extraction solution (50 mM Tricine-Tris, 5 mM EGTA, 3 mM MgSO₄, 0.5% (w/v) polyvinylpyrrolidone, 5 mM dithiothreitol, 1 mM Phenylmethylsulfonyl fluoride (PMSF), 5% (v/v) glycerol and 10 mM sucrose, and pH 7.8). The homogenate was filtered through four layers of cheesecloth and centrifuged at 10,000× g for 20 min. The pellet was resuspended in suspension solution (250 mM sucrose, 5 mM Tris–MES, 1 mM DTT, PMSF 0.5 mM, and pH 7.0) and ultracentrifuged with 22%, 35%, and 46% (w/w) sucrose of discontinuous gradient density centrifuge at 60,000× g for 2 h. The tonoplast and plasma membrane vesicles were carefully collected at interfaces of 22–36% and 36–45% of sucrose, respectively. The vesicles were frozen in liquid nitrogen and stored at –70°C before further use. All steps of the procedure were performed at 2–4°C. The Ca²⁺-ATPase hydrolytic activity of plasma membrane or tonoplast was ascertained by determined the amount of inorganic phosphate released in the absence and presence of 3.0 mM Ca(NO₃)₂, according to a previous method (Chifflet et al. 1988). The enzymatic reaction was performed at 37°C for 30 min by mixing 20 µg of membrane vesicle protein in 800 µl of assay buffer (30 mM Tris–MES, pH 7.0, 0.1 mM (NH₄)₂ MoO₄, 1 mM NaN₃, 50 mM NaNO₃, 0.01% (v/v) Triton X-100, and 3 mM Na₂-ATP), followed by adding 50 µl of 55% trichloroacetic acid to terminate the reaction. Spectrophotometer readings were taken at 660 nm.

Total calcium contents assay

The leaves were milled in a pestle after they were dried at 80°C, and the total calcium contents was ascertained essentially according to a previously described method using an atomic absorption spectrophotometry (TSA-900, Beijing, China) (Manual 1976).

Statistical analysis

Statistical analyses were conducted using the SPSS for windows (versions 17.0). Duncan test was used to establish differences between the groups. A value of p<0.05 was considered a significant difference.

Results

Effects of FGDB and CaSO₄ on cytosolic Ca²⁺ fluorescence intensity

To test whether FGDB or CaSO₄ affect the extracellular Ca²⁺ influx in plant leaves, Fluo-3AM, a Ca²⁺-specific fluorescence indicator was preloaded into protoplasts, and the changes of cytosolic Ca²⁺ concentrations were monitored using a laser scanning confocal microscopy (LSCM). Figure 1 showed the effects of FGDB or CaSO₄ on the abundance of free cytosolic Ca²⁺ in protoplasts isolated from sunflower leaves with different treatments. The Ca²⁺ fluorescence intensities were increased by 1.77-fold and 4.07-fold in those plants grown in T₁ and T₂ soils in comparison with those CK soil, respectively. Similarly, 1.75-fold and 5.23-fold of increased Ca²⁺ fluorescence intensities were also observed in the plants grown in T₃ and T₄ soils that supplied with exogenous CaSO₄, as compared to CK soil, respectively. A more enhanced intensity of Ca²⁺ fluorescence was observed in the groups grown in soils with higher ratio of FGDB or CaSO₄ (T₂ or T₄) than that in lower ratio of FGDB or CaSO₄ (T₁or T₃), and CaSO₄ showed to be more effective than that of FGDB (Figure 1). These data implied a dose-dependent effect of FGDB or CaSO₄ on extracellular Ca²⁺ influx.

Figure 1. A dose-dependent cytosolic Ca²⁺ fluorescence intensity in the leaf protoplasts of oil sunflowers grown in soils supplied with FGDB or CaSO₄. (A–E) Examples of fluorescence intensity of protoplasts preloaded with Fluo-3AM and (F) the changes of cytosolic calcium concentrations induced by FGDB or CaSO₄. A: T₁ soil; B: T₂ soil; C: T₃ soil; D: T₄ soil; and E: CK control soil. Bar=5 µm. Compared to the CK control soils, *p<0.05, **p<0.01. Data represented as mean±SD of an N of 30 for each condition.

Effects of FGDB and CaSO₄ on Ca²⁺ distribution in leaf cells

As shown in Figure 2, the damaged structure of deformed chloroplasts (Figure 2H), as well as few calcium precipitates, was observed in the chloroplast and cytoplasm of plants grown in CK soil with alkali stress (Figure 2G–I and data not shown). While intact, chloroplasts, as well as increasing abundance of calcium precipitates with increasing ratio of FGDB or CaSO₄, were found in the cell wall, intercellular space and vacuole of the plants grown in soils mixed with FGDB or CaSO₄ (Figure 2A–F and data not shown). Notably, more calcium precipitates in CW were found in plants treated with lower doses of FGDB or CaSO₄, while more calcium precipitates in intercellular space and vacuole were observed when a higher dose was added. This result indicated that the alkali stress-induced deformity of chloroplast could alleviate the addition of Ca²⁺.

Figure 2. Locations of calcium precipitates in leaf cells of oil sunflowers. Examples showed calcium precipitates in the leaf cells of oil sunflowers grown in the soils with addition of FGDB (A–C, left panel), CaSO₄ (D–F, central panel), or CK control soil (G–I, right panel). A–C: calcium precipitates were observed in cell wall, intercellular space, and vacuolar of leaf from plants grown in T1 (A) and T₂ (B, C) soils; D–F: calcium precipitates were found in intercellular space, cell wall, and chloroplast of leaf cells in plants grown in T₃ (D) and T₄ (E, F) soils; G–I: Only infrequent calcium precipitates were observed in leaf cells in the plants grown in CK control soils. V: Vacuolar; CW: Cell wall; Chl: Chloroplast; IS: Intercellular space; CP: Cytoplasm. Arrows showed the calcium precipitates. Bar=10 µm.

Effects of FGDB and CaSO₄ on the activities of plasma membrane and tonoplast Ca²⁺-ATPase

A dose-dependent increase of Ca²⁺-ATPase activity was observed in the plasma membrane (Figure 3A) and tonoplast (Figure 3B) of plants grown in soils supplied with desulfurization gypsum or CaSO₄, and the Ca²⁺-ATPase activity in tonoplast was higher than that in plasma. The Ca²⁺-ATPase activities were increased by 1.78-fold and 2.67-fold in plasma membrane and by 1.36-fold and 1.60-fold in tonoplast in T₁ and T₂ soil groups over the CK, respectively. Of note, the Ca²⁺-ATPase activities were increased by 2.04-fold and 2.79-fold in plasma membrane and by 1.41-fold and 1.48-fold in tonoplast of plants grown in soils of T₃ and T₄, in comparison with that of CK, respectively. Similar to the cytosolic Ca²⁺ fluorescence intensity assay, CaSO₄ was more effective on Ca²⁺-ATPase activity in plasma membrane (specially comparing T₁ and T₃); however, in tonoplast, the Ca²⁺-ATPase activities were similar to CaSO₄ or to FGDB (Figure 3A–B).

Figure 3. Activities of Ca²⁺-ATPase in plasma membrane (A) and tonoplast (B) of leaves from the oil sunflowers grown in soils mixed with different dosage FGDB or CaSO₄. Compared to the CK control soils, *p<0.05, **p<0.01. Data represented as mean±SD of an N of 30 for each condition.

Figure 4. Total calcium content of leaves from the oil sunflower grown in soils mixed with different dosage FGDB or CaSO₄. Compared to the CK control soils, *p<0.05. Data represented as mean±SD of an N of 30 for each condition.

Effects of FGDB and CaSO4 on total calcium contents

Increasing total calcium contents were also found in the plants after applying FGDB or CaSO₄ (Figure 4). The concentrations of total calcium in the plants treated with T₁ and T₂ soils were significantly increased by 5.16% and 12.9% over that of CK (p<0.05), respectively. And the total contents of calcium in plants treated with T₃ and T₄ were 0.64% and 24.5% (p<0.05) higher than the control, respectively.

Discussion

Sodic soil contains exchangeable sodium ions on soil colloids and soluble carbonates in the forms of Na₂CO₃ and NaHCO₃. Sodium is responsible for high pH (>8.4), clay dispersion, swelling, and overall physical properties of the soil (Suarez et al. 1984; Gupta & Abrol 1990). The principle of using FGDB to improve sodic soil involved the replacement of exchangeable Na⁺ with Ca²⁺ and discharging Na⁺ through the leaching of soil. However, there was no direct evidence of whether the FGDB had the capacity to enhance plant sodium resistance through the regulation of cytosolic Ca²⁺ levels as has been reported. The mechanism of how the Ca²⁺ in soil was absorbed by the plant under alkali stress has not been established yet either. In this study, cytosolic Ca²⁺ fluorescence intensity analysis demonstrated that the FGDB or CaSO₄ was able to induce Ca²⁺ elevation, which might be due to the extracellular Ca²⁺ influx and the participation of Ca²⁺ in the signal transduction process of alkali stress. However, further study is necessary to test the effect of calcium channel inhibitors on the cytosolic Ca²⁺ fluorescence intensity.

Calcium is an important nutrient for plants. The role of calcium in the plant life cycle is well established and has been reviewed extensively (Bush 1995; White & Broadley 2003; Hirschi 2004). It is present in three forms: covalently bound calcium, loosely bound calcium that is typically associated with fixed and mobile anions, and cytosolic free calcium (Ge et al. 2007). The loosely bound calcium, which could be detected by a potassium antimonite precipitation method, has lower affinity for fixed and mobile anions and functions as an exchangeable form of calcium by transforming into other forms as it is on demand (Wick & Hepler 1982; Zhao et al. 2002; Zhu et al. 2010).

Transient spatial and temporal changes of calcium pools and cytoplasmic Ca²⁺ concentration were the initial responses to external stimulation, which might trigger a physiological and developmental cascade in plants (Knight 1999; Lecourieux et al., 2006). In this study, transmission electron microscopy analysis revealed that abundant calcium precipitates were distributed in cell wall, intercellular space, and vacuole of plants treated with FGDB or CaSO₄ in a dose-dependent manner. A change of free calcium concentration might alter the loosely bound calcium in the cell wall and intercellular space, together with the evidence of high total calcium content found in the oil sunflower leaves might be loosely bound calcium and free calcium, indicating that the entering calcium was partially stored in vacuole or bound to pectins in the middle lamella and maintains the cell wall stability and integrity (Hanson 1984; Palta 1996; Hirschi 2004).

Ca²⁺ signal cascade could be directly triggered by extracellular Ca²⁺ influx and/or released by cytosolic Ca²⁺. In order to detect the role of Ca²⁺-ATPase in Ca²⁺ influx or in discharging cell, the activities of Ca²⁺-ATPase in plasma membrane and tonoplast were measured. Indeed, increasing activities of Ca²⁺-ATPase were detected in plasma membrane and in tonoplast of plants grown in soils added with FGDB or CaSO₄. This clearly evidenced that the FGDB or CaSO₄ induced elevating Ca²⁺ concentration in cell wall and vacuole and was primarily due to cytosolic Ca²⁺ efflux by enhancing Ca²⁺-ATPase activity and re-establishing the low basal free Ca²⁺ concentration in cytosol; this notion was in agreement with the result of our cytosolic Ca²⁺ fluorescence intensity and the findings from other groups (Beffagna et al. 2000; Sanders et al. 2002; Romani et al. 2004).

To test whether the effective compound for improving the plant alkali resistance was CaSO₄ in desulfurization gypsum, a comparison test between FGDB (containing 80% CaSO₄) and CaSO₄ (Analytical grade) was included in this study. As expected, FGDB and CaSO₄ displayed a similar effect on increasing cytosolic Ca²⁺ fluorescence intensity, the distribution of calcium, and Ca²⁺-ATPase activity in the leaves cells of oil sunflower, suggesting that CaSO₄ in desulfurization gypsum was the compound that played an essential role in relieving alkali stress in this study.

Conclusions

The results presented in this report demonstrated a dose-dependent effect of FGDB and CaSO₄ on cytosolic Ca²⁺ concentration, Ca²⁺ distribution, and plasma Ca²⁺-ATPase activity in oil sunflower. The calcium sulfate of FGDB was the effective compound for plant against saline–alkali stress. The alteration of cytosolic Ca²⁺ distribution and concentration induced by FGDB or CaSO₄ might be partially mediated by enhancing Ca²⁺-ATPase activity in the leaf cells of this plant. These findings provided an insight into the molecular mechanism of alkaline soil amendment by supplementation of desulfurization gypsum and warranted the potential application of FGDB in the restoration of alkaline soils in Northwestern China.

Acknowledgments

We thank Drs. Liu HH and Jia JZ (College of Biological Sciences, Agricultural University of China) for their helpful suggestions, technical assistance for ultrathin sections, and transmission electron microscopy. This work was supported by grants from the National Basic Research Program of China (973 program) (2012CB723206) and National Key Technology R&D Program of the Ministry of Science and Technology (2013BAC02B04 and 2011BAC07B03).