Herein, a link has been established between cold acclimation and Cd uptake in Spirogyra aequinoctialis. The water samples supplemented with cadmium (0.1, 0.5, and 1.0 mg/L) were used for plant growth at two different temperatures. Cold-acclimated plants accumulated high concentrations (0.40 ± 0.02 µg/dry biomass) of Cd as compared to non-acclimated plants (0.30 ± 0.08 µg/dry biomass). The negative effects of Cd on the biomass, carotenoids, and chlorophyll contents in non-acclimated plants were more pronounced in non-acclimated plants as compared to the cold-acclimated plants. High concentrations of free proline and total phenolics in cold-acclimated plants were observed as compared to the non-acclimated plants. About 13 different phenolic compounds were identified using high-pressure liquid chromatography. Cold acclimation as well as the presence of Cd in water highly increased the concentrations of polyphenolics, while synergistic increase was recorded with the combination of cold acclimation and Cd treatments. The highest increase in the concentration of polyphenolics was recorded for syringic acid. Significant positive correlations of total phenolics were noted with dry biomass (R 2 = 0.51) and Cd accumulation (R 2 = 0.59) in plant tissues. Free proline concentration also showed positive correlations with dry biomass, Cd accumulation, and chlorophyll contents (R 2 = 0.80).
Phytoremediation refers to the removal of pollutants from water and soil using plants [1,2]. Algae produce high biomass as compared to higher plants, are considered an important source of biofuel, and can phytoremediate high amounts of pollutants . They have been used commercially for the production of biofuel and biomass at low cost [1,4]. This high biomass production in algae can be exploited for the remediation of polluted water.
Cadmium is among the most toxic heavy metals of great environmental concern . The main sources of Cd pollution in water and soil are from industrial effluents, application of phosphate fertilizers, pesticides, lead–Cd batteries, mining, automobile emissions, and sewage effluents [6,7]. Cadmium is a potential mutagenic and carcinogenic agent in animals . Consumption of Cd-polluted food or water affects vital organs in humans, such as the kidneys, heart, and liver [9,10]. Absorption of excess cadmium by plants leads to the inhibition of certain enzymes involved in photosynthesis and other important metabolic process, and also result in the imbalance of mineral nutrients and water in plant tissues [11,12]. Several green plants have the potential to absorb and accumulate toxic metals from soil and water. The use of green plants for the restoration of polluted soil and water is termed phytoremediation. It is an affordable, environmentally friendly, and effective technological approach for the purification/remediation of polluted water. Plants producing high biomass and accumulating high concentrations of toxic heavy metals are most suitable for phytoremediation. Algae are a group of plants that mostly grow in water and many of them have the ability to tolerate high concentrations of heavy metals in water [13,14]. Heavy metals often decrease growth and biomass in algae . Several factors, such as nutrient status, temperature, and pH of media, greatly influence the uptake and accumulation of Cd in plants . Plants' exposure to non-freezing low temperatures for a certain period is known as cold acclimation, which induces tolerance in plants against dehydration stress [16,17]. Low temperatures can activate several stress-related genes in plants and some of these genes (such as DREB/CBF) were also reported to express under Cd stress [5,18]. Cadmium has been reported to induce physiological drought in plants . Cold acclimation causes an increase in the ABA concentration , changes in the lipid composition of the membrane , production and accumulation of osmolytes, and an increase in antioxidants . Under Cd stress, the concentrations of certain metabolites, such as proline and phenolic compounds, increase in plant tissues . Many investigators reported proline accumulation during stress conditions . Proline helps sustain cell turgidity, stabilize membrane integrity, and reduce electrolyte leakage [24,25]. Similarly, polyphenol acts as an antioxidant by direct scavenging of reactive oxygen species (ROSs) . ROSs destroy DNA, proteins, and chlorophyll . The high production of polyphenol in plants on exposure to heavy metals has been reported [28,29]. Although algae have been used for the accumulation of heavy metals, they have not been exploited for cadmium uptake. Also, under cold acclimation, the effect on this plant for the accumulation of heavy metals has not been studied. The effect of accumulated metal on phenolic production has also not been reported before.
In the present study, we have evaluated the effect of cold acclimation on free proline, polyphenol, chlorophyll, and carotenoid contents in Spirogyra. Aequinoctialis (algae) and investigated the relationship of these compounds with Cd accumulation under cold acclimation treatment for possible improvement in the process of phytoremediation of Cd-contaminated water.
2 Materials and methods
2.1 Algae sampling
The algae S. aequinoctialis was collected from the freshwater pond located near River Swat, north of Pakistan. The algal filaments were brought to the laboratory in cool insulated packing. For acclimatization to the laboratory conditions, filaments were cultured in 5 L of water in a bottom aerated flask, for 5 days at 25°C with 14/10 h of light/dark period. An equal weight of algae was cultured in controls and treatment flasks.
2.2 Cold acclimation and metal treatments
The flasks containing water samples were labeled as treatments (T1, T2, and T3) and controls (C and C1), as given in Table 1. An aqueous solution of Cd [cadmium acetate/Cd (CH3CO2)2] was prepared in sterilized distilled water and added to the treatment flasks (T1, T2, and T3) to achieve different concentrations (0.1, 0.5, and 1.0 mg/L) of Cd in the flasks. Cold acclimated plants were kept at 4°C while non-acclimated plants were kept at 25°C and allowed to grow for 10 days under a 14/10 h light/dark period. Six replicate (n = 6) flasks were used for each control and each treatment concentration.
|Treatment codes||Treatments used during the experiment|
|C||Tap water and algae|
|C1||Algae and water (sample collected area water)|
|T1||Water + 0.1 mg Cd/L|
|T2||Water + 0.5 mg Cd/L|
|T3||Water + 1.0 mg Cd/L|
The same treatments were used for both acclimated and non-acclimated plants.
2.3 Cadmium analysis in water
Water samples (each 50 mL) were mixed with 1 mL of nitric acid (concentrated) and boiled on a hot plate until the volume was reduced to 20 mL. The solutions were cooled, filtered, and diluted to 50 mL each by the addition of distilled water (APHA 1985). The digested water samples were analyzed for Cd concentration using an atomic absorption spectrophotometer (Hitachi Z-8000, Japan).
2.4 Metal analysis in S. aequinoctialis
The dried samples of S. aequinoctialis were ground to a powder form and then subjected to acid digestion. From each powdered sample, 0.25 g was taken in a separate flask (50 mL) and 6.5 mL of three acid mixture (sulfuric acid [1.0 mL], nitric acid [5.0 mL], and perchloric acid [0.5 mL]) was added and boiled on a hot plate till complete digestion of the plant material. Each flask was kept on hot plates until complete digestion. The samples were filtered into a clean flask and distilled water was added to increase the final volume to 50 mL. Each filtrate sample was then stored in two prelabeled plastic bottles. The samples were then analyzed for cadmium concentration using an atomic absorption spectrophotometer (Hitachi Z-8000, Japan).
2.5 Identification and estimation of individual polyphenol compounds in S. aequinoctialis
Polyphenol compounds in acclimated and non-acclimated plants were analyzed by high-pressure liquid chromatography (HPLC) from shade-dried samples of S. aequinoctialis. From finely powdered samples, 1.0 g was dissolved in 10 mL of methanol water . The mixture was kept in a shaking water bath for 10–20 h. The solution was filtered and centrifuged for 10 min at 4,000 rpm (at room temperature). After centrifugation, the solution was filtered again and then subjected to HPLC analysis. The high-pressure liquid chromatograph (Agilent 1260 Infinity system) consisted of a degasser, auto-sampler quaternary pump, and a diode array detector, and separation was made with Agilent rapid resolution Zorbax Eclipse plus C18 (4.6 mm × 100 mm, 3.5 μm) column at 25°C. The gradient system consisted of solvents A (deionized water/acetic acid/ methanol, 88:2:10) and B (deionized water/acetic acid/methanol, 8:2:90). The gradient program used was 100% A at 0 min, 85% A at 5 min, 50% A at 20 min, 30% A at 25 min, and 100% B from 30 to 40 min. Identification was made using available standards, their retention times, and UV spectra, and quantification of compounds was based on the percent peak area.
2.6 Analysis of carotenoids in S. aequinoctialis
From each fresh sample of leaves, 200 mg was crushed in acetone (90%) and centrifuged for 5 min at 1,000 rpm. Pellets were discarded and the supernatants in each tube were transferred to clean test tubes. The final volume of supernatants was increased to 6 mL by the addition of acetone (90%). Absorption (at λ = 480 nm) of each solution was measured using a UV-spectrophotometer (Shimadzu UV-2450), while acetone (90%) was used as a reference .
2.7 Free proline estimation in plant tissues
Proline content in S. aequinoctialis was determined using the method of Bates et al. (1973). The fresh sample (100 mg) was crushed in 2 mL tubes containing 1.5 mL of 3% aqueous solution of sulfosalicylic and then centrifuged for 5 min (13,000 rpm) at room temperature. Then, 300 μL of clean supernatant was transferred into clean tubes containing 2 mL of acid ninhydrin (prepared by heating 1.25 g of ninhydrin in 20 mL of phosphoric acid (6 M) and 30 mL of glacial acetic acid until dissolved completely). The tubes were kept in a water bath for an hour at 100°C and then immediately dipped into ice. Toluene (1 mL) was added to each tube and shaken vigorously for 1 min. Toluene containing the chromophore layer was transferred to covet and absorbance of each sample was measured at a 520 nm wavelength using a spectrophotometer. Toluene was used as blank/reference.
2.8 Chlorophyll estimation in S. aequinoctialis
The sample (200 mg) was thoroughly mixed in 80% acetone (2 mL) and then pulverized. Then, it was centrifuged (at 1,000 rpm) for 5 min at room temperature (25°C). Pellets were discarded. The supernatant was transferred into another test tube. Acetone (80%) was added and the volume was increased to 6 mL. Chlorophyll contents were measured using a UV-spectrophotometer (Shimadzu UV-2450). Chlorophyll a and b contents were analyzed at 663 and 645 nm wavelengths, respectively. The 80% aqueous acetone was used as a reference . Chlorophyll a and b in all samples were determined.
2.9 Statistical analysis
Mean values and standard deviations were calculated using MS Excel (version 2013), and Analysis of variance (ANOVA) and Tukey’s HSD test (P ≤ 0.05) were performed using SPSS 16.
3.1 Effect of cold acclimation and Cd treatments on dry biomass and Cd contents in S. aequinoctialis
The dry biomass of S. aequinoctialis decreased as the concentration of Cd was increased in the growth medium and the decrease was significant in T2 (0.5 mg/L Cd in media) and T3 (1 mg/L Cd in media) plants when compared to control C plants (Figure 1a). Cold acclimation significantly increased the plant dry biomass in controls (C and C1) and T1 (0.1 mg/L Cd in growth media), as compared to their non-acclimated member plants (Figure 1a).
The concentration of Cd and its accumulation in plant tissues are shown in Figure 1b and 1c. It was noted that a high concentration of Cd in growth media (C1 < T1 < T2 < T3) significantly increased the concentration and accumulation of Cd in plants (i.e., T3 > T2 > T1 > C1). Cold acclimation increased Cd contents in plant tissues as compared to the non-acclimated plant; the increases were statistically significant in treatments T2 and T3 while non-significant in C1 and T1 (by comparing acclimated and non-acclimated plants of the same treatment).
3.2 Effect of Cd and cold acclimation on polyphenolics in S. aequinoctialis
Thirteen different phenolic compounds were separated and identified in Spirogyra (Figure 2). The variation in the concentration of the phenolic compounds under Cd stress and cold acclimation is given in Table 2. The highest concentration among the phenolic compounds was shown by gentisic acid (5049.0 µg/g) and isorhamnetin-3-hydroxyferuloylglucoside-7-glucoside (4915.0 µg/g) in control plants, i.e., plants grown in media without Cd under non-acclimation conditions.
|Peak #||Retention time (min)||Absorption spectra (nm)||Phenolic compounds||−Cd, −CA||−Cd, +CA||+Cd, −CA||+Cd, −CA|
|Concentration (µg/g)||Concentration (µg/g)||Times increase||Concentration (µg/g)||Times increase||Concentration (µg/g)||Times increase|
|3||9.0||330, 290||Rosmarinic acid||833.1||1501.8||180.3||1164.2||139.7||2374.2||285.0|
|4||11.1||265||Hydroxy benzoic acid||984.5||3487.2||354.2||1068.3||108.5||2025.7||205.8|
|5||18.5||300, 250||Gentisic acid||5049.0||7961.2||157.7||6609.6||130.9||7618.1||150.9|
|10||26.7||326, 288||Ferulic acid-hexoside||3550.4||4323.6||121.8||4244.2||119.5||7461.1||210.1|
Cd: cadmium; CA: cold acclimation; +: presence of a condition; −: absence of a condition.
Cold acclimation increased the concentration of all the phenolic compounds as compared to the control plants and the highest increase was noted in the concentration of hydroxyl benzoic acid (354.2%). Similarly, Cd in media also increased the concentration of all the identified phenolics in plants as compared to the control plants, while the highest increase was observed in kaemferol-3-feruloylsophoroside-7-glucoside (302.0%). The combined effect of Cd and cold acclimation was most positive on syringic acid (709.0%) and spinacetine-3-O-B-d-glucopyranosyl-glucopyranoside (503.4%) as compared to the control plants (Table 2).
3.3 Carotenoids and free proline production in S. aequinoctialis under cold acclimation and cadmium treatments
Variations in the concentrations of free proline, carotenoids, and chlorophyll in S. aequinoctialis under the effect of Cd treatments and cold acclimation are shown in Figure 3(a–e). A decrease in the concentration of the bio-chemicals was observed (i.e., C1 > T1 > T2 > T3) in both acclimated and non-acclimated plants when the concentration of Cd in growth media was increased gradually (i.e., C1 < T1 < T2 < T3). Cold-acclimated plants were found to have high concentrations of free proline, carotenoids, and chlorophylls as compared to their non-acclimated member plants in the same treatment and control (Figure 3a–e). Free proline concentration was increased in T3 plants under cold acclimation (Figure 3a). In Figure 3b, control C1 showed a significant increase in the concentration of carotenoids as compared to control C.
3.4 Correlations among different parameters
Figure 4(a)–(i) shows correlations of dry biomass with Cd concentration, total phenolics, and free proline as well as the correlations of total phenolics and free proline with chlorophyll a, chlorophyll b, and Cd accumulation. All the correlations were positive, except for the negative correlation between the dry biomass and Cd concentration in plant tissues. The positive correlations of phenolics with Cd accumulation (R 2 = 0.59), and free proline with chlorophyll b (R 2 = 0.80) were statistically significant.
In the present investigation, S. aequinoctialis was investigated for its Cd phytoextraction potential. The algae, S. aequinoctialis, was found to be a good candidate for the purification/phytoextraction of Cd-polluted water. The algae was collected from pond water containing a low concentration of Cd (0.0118 mg/L) and showed normal growth at low concentrations of cadmium. High concentrations of Cd in growth media decreased the biomass of the algae. The decrease might be due to the production of ROS, and the direct inhibitory effect of Cd on many metabolic processes, especially photosynthesis . The concentration of photosynthetic pigments in plants is considered an indicator of growth rate . Chlorophyll contents in S. aequinoctialis decreased due to Cd toxicity. It has been found that Cd disturbs the electron transport chain and biosynthesis of plant hormones, and sometimes damages the cell membrane [8,33,34]. Cadmium negatively affects the photosynthetic pigments by replacing the Fe2+ and Mg2+ with the cation Cd in chlorophyll molecules and thus decreases the efficiency of chlorophyll molecules .
The present results demonstrate that cold acclimation treatment increased the biomass even under Cd stress. Repkina et al.  reported that cold acclimation induces the production of free proline, glutathione, and other phytochelatins in wheat plants, and enhances the plant tolerance to Cd stress. Phytochelatins bind to Cd and protect the cellular components by decreasing the concentration of free Cd ions. It was found that the low-temperature treatment induces anti-oxidative defense in plants against ROS . Polyphenolic compounds are components of antioxidative defense in plants. The S. aequinoctialis showed high concentrations of different polyphenolic compounds in its tissues under Cd stress and low-temperature treatments. The increase in polyphenols might be an adaptive character for the abiotic stress tolerance in plants and could play a key role in ROS scavenging and reduction of oxidative stress [37,38]. Similarly, the concentration of free proline was also noted to increase in algal tissues under the effect of Cd stress and low temperatures. Proline prevents the inactivation of important enzymes and the main function of proline might be the protection of the key enzyme, osmoprotectant , and the protection of plants from oxidative impairment . The combined treatment of cadmium and cold acclimation showed a synergetic effect on the concentrations of free proline and polyphenolic compounds.
In the present findings, the total phenolic content increased in cold-acclimated Spirogyra in contrast to non-acclimated Spirogyra plants under Cd stress. Polyphenol works as an anti-oxidant due to its redox ability, which allows them to act as reducing agents, ROS quenchers, hydrogen donors [40,41], membrane stabilizers , and metal ion chelators . The use of algal plants (Spirogyra) for the removal of heavy metals from water is considered to be a suitable approach due to its high biomass and unpalatable nature to reduce the entry of metals directly into the human body during the phytoremediation of contaminated water.
In this study, cold acclimation and heavy metal exposures were simultaneously evaluated in Spirogyra aequinoctialis. Under room and refrigerator temperatures, the selected plant was grown in water samples containing cadmium ranging from 0.1 to 1.0 mg/L. Under cold, comparatively more cadmium was accumulated (0.40 ± 0.02 µg/dry biomass). At the same time, in cold-acclimated plants, the negative effects of Cd on biomass production, carotenoids, and chlorophyll were not so drastic as compared to non-acclimated plants. By HPLC analysis, 13 different phenolic compounds were also identified in cold-acclimated plants. From the results, it can be inferred that cold acclimation not only improves the phytoremediation of heavy metals but also enhances biomass production and phytochemical constituents. Further experimentation on other plants is mandatory to confirm the observed results of this study.
Funding information: The Authors extend their appreciation to the Deputyship for Research and Innovation, “Ministry of Education” in Saudi Arabia for funding this research (IFKSUOR3-020-2).
Author contributions: Conceptualization: C.B., N.A., M.Z., and F.H.; methodology: C.B., N.A., A.Z., and F.H.; formal analysis: R.U., E.A.A., and A.B.S.; writing of the paper and revisions: C.B., N.A., M.Z., R.U., E.A.A., A.B.S., and F.H.
Conflict of interest: The authors declare no conflict of interest.
Ethical approval: The conducted research is not related to either human or animal use.
Data availability statement: All data generated or analyzed during this study are included in this published article.
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