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Exogenous foliar application of fulvic acid alleviate cadmium toxicity in lettuce (Lactuca sativa L.)
Ecotoxicology and Environmental Safety 167 (2019) 10–19
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Exogenous foliar application of fulvic acid alleviate cadmium toxicity in lettuce (Lactuca sativa L.) Yanmei Wang, Ruixi Yang, Jiaying Zheng, Zhenguo Shen, Xiaoming Xu
T
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College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Fulvic acid Cadmium stress Lettuce Antioxidant enzymes Mineral nutrition
It was reported that fulvic acid (FA) has a positive effect on enhancing plant tolerance to various environmental stresses, including salinity stress and drought stress and so on. However, there is little study regarding the effects of FA on plants in response to heavy metal stress. Hence, the objective of this study was to investigate the potential effects of fulvic acid (FA) on cadmium (Cd) toxicity alleviation in lettuce seedlings. Our results showed that application of 0.5 g/L FA significantly mitigate Cd-induced toxic symptoms in lettuce seedlings. Cd stress triggered plant growth inhibition, photosynthetic pigment reduction, destruction of the photosynthesis apparatus, reactive oxygen species (ROS) accumulation, and nutrient elemental imbalance. We observed that FA promoted the growth in lettuce under Cd stress, mainly reflected in those alterations that the increase of biomass, chlorophyll content and photosynthesis capacity and reduction of the Cd content and lipid peroxidation in plant tissue. Foliar spraying of FA significantly alleviated these detrimental symptoms and facilitated nutrient element translocation from root to shoot, particularly the absorption of elements involved in photosynthesis, including iron (Fe), zinc (Zn), and manganese (Mn). In summary, foliar application of FA conferred Cd toxicity tolerance to lettuce by increasing ROS-scavenging capacity, inhibiting Cd uptake and the transport of elemental nutrients to shoots, which in turn protected the photosynthetic apparatus and promoted plant growth.
1. Introduction
chloroplast ultrastructure, as well as cell membrane structure (Ali et al., 2013). Various methods have been reported to reduce Cd accumulation in plants, including exogenous application of materials such as plant growth regulators, elemental nutrients, and amino acids; the application of these substances could be an effective strategy to reduce Cd uptake by vegetables destined for human consumption (Parvaiz et al., 2016; Wang et al., 2016; Meng et al., 2009). However, few studies have been conducted on the effects of foliar application on plants exposed to Cd. Humic substances are natural organic compounds comprising organic acids derived from the decomposition of plant and animal residues under the action of microorganisms and geochemistry (Morales et al., 2012). Humic substance can be divided into humin, humic acid, and fulvic acid (FA) according to its solubility at different pH (Suh et al., 2014). Among these substances, fulvic acid (FA) has a relatively low molecular weight and contains a high amount of oxygen-rich and carbon-poor functional groups (Weng et al., 2006). FA confers benefits to plants by enhancing drought resistance, improving nutrient uptake, stabilizing soil pH, and reducing fertilizer leaching (Suh et al., 2014). Adani et al. (1998) revealed that soil application of humic acid could stimulate the growth of tomato plants grown by hydroponics, and
In recent years, heavy metal pollution has attracted increasing attention worldwide, due to its release into the environment through anthropogenic activities such as mining, waste water for irrigation, sewage sludge application, excessive application of phosphate fertilizers, herbicides and pesticides to farmland, and vehicular and industrial activities (Rizwan et al., 2017). Among heavy metals, cadmium (Cd) is a highly toxic, non-essential element for humans, animals, and plants. It is easily taken up by plant roots and transported to shoots (Saidi et al., 2014), Cd toxicity causes various morphological, physiological, biochemical, and ultrastructural changes in plants. Its main symptoms manifested growth inhibition of shoots and roots, leaf chlorosis, browning of root tips and so on (Zhang et al., 2015). A large amount of toxic effects of Cd on metabolism have been reported, such as imbalance of nutritional elements, reduction of chlorophyll biosynthesis, enzymes inhibition, disruption of basic metabolic functioning, including photosynthesis, respiration, and nucleic acid production (Erdal and Turk, 2016; Ali et al., 2013; Santos et al., 2018). Moreover, the burst out of reactive oxygen in plants with Cd stress, which caused oxidative stress and responsible for damage to mitochondria and
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Corresponding author. E-mail address: [email protected] (X. Xu).
https://doi.org/10.1016/j.ecoenv.2018.08.064 Received 18 January 2018; Received in revised form 16 August 2018; Accepted 17 August 2018 0147-6513/ © 2018 Published by Elsevier Inc.
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promote the nutrients uptake, especially Fe. Similar results were found that total yield was raised in tomato by foliar and soil application of humic acid (Yildirim, 2007). In many cases, humic acid is regarded as a plant growth regulator, which has the similar effect with auxins, but we do not confirm whether it contain auxin-like substances or not (Nardi et al., 2002). The explicit physiological mechanism that fulvic acid (FA) enhanced plant resistance against abiotic stresses is seldom known. Current report has exhibited that soil and foliar applied FA has beneficial effects on seedling growth, seed germination, and root weight of wheat plants (Katkat et al., 2009). Tang et al. (2014) reported that FA could also be applied to remove heavy metals from aqueous media. Ali et al. (2015) explored the effects of foliar FA application on wheat plants exposed to chromium (Cr) and found that FA alleviated Cr toxicity by increasing photosynthetic pigments, reducing Cr uptake, and improving antioxidant activities in wheat. However, few studies have examined the effect of FA application to soil to enhance Cd stress tolerance in lettuce. (Haghighi et al., 2013, Horuz et al., 2015). Therefore, this study was conducted to illuminate the effects of foliar application of fulvic acid on lettuce under Cd stress. Lettuce (Lactuca sativa L.) is a common leafy vegetable that is cultivated commercially and in home gardens worldwide (Konstantopoulou et al., 2010; Matraszek et al., 2016). Lettuce is rich in a variety of vitamins, dietary fibers, low in calories as well as a source of carotenoids, which is beneficial for human health. In addition, it contains many nutrient minerals, such as Ca, Fe, Zn, Se etc., which is essential for human body to maintain normal function by serving as electrolytes (Matraszek et al., 2016). Lettuce is valued for its high nutritional value and is often used in salads as a fresh vegetable. It has a high capacity for Cd accumulation from soil without showing visible symptoms of metal toxicity, which poses a potential risk to human health (Cobb et al., 2000). In this study, we use the method of foliar spraying to investigate the effects of FA on lettuce exposed to Cd and try to elucidate its possible mitigation mechanism for two reasons. Firstly, FA can chelate directly with Cd in solution (Tang et al., 2014). Meanwhile, foliar spraying is easy to achieve cultivation in the field. The objectives of this study were to examine (a) whether FA supply ameliorates Cd toxicity in lettuce, (b) the optimal concentration of FA to alleviate Cd toxicity, and (c) the possible mechanism of Cd toxicity defense by FA in lettuce.
Secondly, we have chosen an optimum FA concentration (0.5 g/L) and seen how it influences in Chlorophyll a fluorescence, photosynthetic pigments and gas exchange parameters, oxidative stress, Antioxidant enzymes and nutrient element uptake. Based on preliminary trials, four treatments respectively were Control, 20 μM Cd, Control+FA, Cd+FA. The solutions were renewed every other day. Each treatment was designed with three replicate randomly, and each replicate contain three plants. After treatment 2 weeks later, seedlings were harvested to make the further analysis.
2. Materials and methods
2.5. Gas exchange parameter measurements
2.1. Plant material and growth conditions
The second fully expanded leaves from the top of plants were used to estimate the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (E) using a portable photosynthesis system (LiCOR 6400, Lincoln, NE, USA). The experiment was performed on sunny day from 9:00–11:30 a.m.
2.3. Plant biomass measurements and chlorophyll content measurements Fifteen days after treatment, lettuce seedlings were harvested and photographed. After that, samples were separated into leaves and roots, which fresh weights were determined immediately with electronic balance. Dry weights were estimated after drying samples in an oven at 80 °C until biomass became constant. Chlorophyll content was measured with the help of SPAD-502 chlorophyll meter. Photosynthetic pigment was determined with spectrophotometry, which extracted with 95% ethanol. The details of experimentation depend on the method of Knudson et al. (1977). 2.4. Chlorophyll fluorescence parameter measurements and JIP-test analysis Leaves were adapted in the dark at least 30 min before measurements. Chlorophyll fluorescence parameters were measured using the Handy Plant Efficiency Analyzer (Plant Efficiency Analyzer; Hansatech, UK). The measured light source is a red light with a wavelength of 650 nm and a light intensity of 3000 μmol m−2 s−1 provided by three light-emitting diodes, which continue to record 1 s. An OJIP curve was plotted to normalize the fluorescence data to relative variable fluorescence data, using the following equation: Vt = (Ft – Fo)/(FM – Fo), where Vt is the relative variable fluorescence at time t, Fo is the initial fluorescence, Ft is the fluorescence at time t, and FM is the maximum fluorescence. JIP test parameters (transient fluorescence steps O, J, I, and P) were calculated according to the JIP-test algorithm described by Strasser et al. (2004). The analyzed parameters are described in Supplementary Table 1.
Lettuce (Lactuca sativa L.) seeds, acquired from Jiangsu Agricultural Institutes (Nanjing Province, China) were germinated in a tray filled with vermiculite at 25 ℃ in a climate chamber. Uniformly sized seedlings were selected 5 days later and transplanted into plastic cups (three plants per cup) containing 400 mL a half-strength Hoagland nutrient solution (pH 6.0). The selected seedlings were incubated in the climate chamber at a temperature of 25/20 ℃ (day/night), with a light intensity of 600 μmol m–2 s–1 and a 12-h photoperiod.
2.6. Determination of lipid peroxidation, hydrogen peroxide (H2O2), Superoxide radical (O2·–) and eletctrolyte leakage (EL) The level of lipid peroxidation in leave and roots were estimated by quantifying the content of the thiobarbituric acid reactive substance (TBARS), which determined by the thiobarbituric acid (TBA) based on the method of Hodges et al. (1999) with minor modifications (Zhao et al., 2017). H2O2 content was measured depending on the description of Zhao et al. (2017). O2.− was measured by monitoring nitrite formation from hydroxylamine in the presence of O2.−, according to the method of Jabs et al. (1996) with some modifications. The measurement of EL in leaves was according to the method of Bajji et al. (2002). Fresh leaves (0.1 g) were cut into the same size leaf discs of the same size and put into a tube containing 10 mL of deionized water. The initial electrical conductivity (EC1) was measured after the tubes were placed at 25 ℃ for 4 h. Then, the tubes were put into boiling water bath for 30 min. The second electrical conductivity (EC2) was
2.2. Experimental design Firstly, we have studied which FA dose works better to improve Cd damage by these indicators: the plant growth, chlorophyll content, P.I., photosynthesis, electrolyte leakage and reduction of Cd accumulation. After 10 days of growth, the seedlings in three leaf stage were treated with 20 μM Cd (CdCl2·2.5 H2O) in hydroponics, Meanwhile, the seedlings exposed to Cd were sprayed with 0, 0.1, 0.3, 0.5, 1.0, 2.0 g/L fulvic acid (FA) in the morning every day. FA (> 90%) was purchased from Bio Aladdin (Shanghai, china). Seedling without Cd and fulvic acid treatment were used as the control (Con), All treatments we described as (1) Con, (2) Cd, (3) Cd + 0.1FA, (4) Cd + 0.3FA, (5) Cd + 0.5FA, (6) Cd + 1.0FA, (7) Cd + 2.0FA. 11
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Fig. 1. Effects of different concentrations of FA on biomass accumulation (A), SPAD value (B), PSⅡ performance index (C), photosynthesis (D) and electrolyte leakage (E) in lettuce under Cd stress. Data are shown as means ± SE of five replicates. Bars marked by different letters indicate significant differences by Tukey's test (P ≤ 0.05).
determined after the solution in tubes were cooled at 25 ℃. The EL was calculated with following formula: EL (%) =EC1/EC2 × 100.
2.8. Antioxidant enzymes assays Fresh plant sample (0.5 g) were homogenized in 5 mL pre-cooled extraction buffer containing 1 mM Ethylenediaminetetraacetic acid disodium salt (EDTA-Na2) and 1% polyvinylpyrrolidone (PVP) in 100 mM potassium phosphate buffer (pH 7.0). The homogenate was centrifuged at 12,000 g for 20 min and the supernatant used for the enzyme assays below. All the processes were carried out at 4 ℃. Superoxide dismutase (SOD) activity was assayed based on its ability to restrain the photochemical reduction of nitroblue tetrazolium (NBT) following the method of Giannopolitis and Ries (1977). One unit of SOD activity was defined as the amount of enzyme required to inhibit the reduction of NBT by 50% as monitored at 560 nm. According to the reports of Aebi (1984), Catalase (CAT) activity was determined by monitoring reduce of H2O2 (extinction coefficient:
2.7. Histochemical detection of H2O2 and O2.− H2O2 accumulation in leaves was detected with the 3-diaminobenzidine (DAB) staining procedure, which involved the incubation of the leaves with DAB (1 mg mL−1, pH 3.8) solution in the dark for 6 h (Thordal Christensen et al., 1997). O2.− accumulation in leaves was visually detected by staining with Nitroblue tetrazolium (NBT) using the method as described by Jabs et al. (1996). Leaves were removed from plants, submerged in NBT solution (0.5 mg mL−1, pH 7.8) immediately, and incubated at for 6 h at 25 ℃ in dark. 12
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39.4 mM−1 cm−1) at 240 nm for 1 min. Peroxidase (POD) activity was measured by the means as described by Zhao et al. (2017), which mainly monitor the growing absorbance at 470 nm for 3 min as guaiacol was oxidized. The extinction coefficient was 26.6 mM−1 cm−1. Ascorbate peroxidase (APX) activity was determined by following the oxidation of ascorbic acid (extinction coefficient: 2.8 mM−1 cm−1) at 290 nm for 1 min (Nakano and Asada, 1981).
compared with Cd treatment alone. The ratio of absorption flux per active reaction center (ABS/RC, TRo/RC and DIo/RC) was higher under Cd stress than under co-treatment with Cd and FA, while the opposite trend was observed for electron transport flux (ETo/RC). The absorbance flux (ABS/CSo, TRo/CSo, ETo/CSo, DIo/CSo, and RC/CSo) decreased to different extents according to the Cd stress, and was significantly improved by FA application. Changes in the absorption of photon flux via chlorophyll molecules (ABS/CSm, TRo/CSm, ETo/CSm, DIo/CSm, and RC/CSm) showed a similar pattern to those of ABS/CSo, and similar parameters.
2.9. Elemental analysis
3.3. photosynthetic pigments and gas exchange parameters
After harvest, the fresh seedlings were rinsing several times with deionized water after soaking in EDTA-Na2 solution for 10 min. After that, samples were divided into shoots and roots and dried at 105 °C for 25 min and then at 80 °C in an oven until a constant weight was achieved and maintained. The dried samples were weighed and digested with an acid mixture (HNO3:HClO4, 87:13,v/v) at 180 °C. Cd and nutrient elements concentration were measured by inductively coupled plasma-optical emission spectroscopy (ICP-OES, Perkin Elmer Optima 2100DV) as described by Zhao et al. (2017).
Carotenoid and total chlorophyll content in leaves under Cd stress were reduced by 69.0% and 61.4%, respectively, compared with the control; this decline was reversed following FA foliar spraying, by approximately 20.3% and 29.4%, respectively (Fig. 3A). Under Cd stress, there were apparent declines in net photosynthetic rate (Fig. 3B), stomatal conductance (Fig. 3C) and transpiration rate (Fig. 3D), by 29.2% and 62.5%, and 54.5%, respectively. The application of FA resulted in an improvement in the net photosynthetic rate (22.6%), stomatal conductance (35.5%), and transpiration rate (36.5%), compared with Cd treatment.
2.10. Statistical analysis Data was presented with mean ± SD of at least three replicates. It was analyzed by one-way analysis of various (ANOVA), which was conducted with SPSS 22.0 software. A Tukey's Test was performed to compare significant difference between treatments at P < 0.05. All the figures are.completed with Origin 9.0 software (Origin Lab, Northampton, MA, USA).
3.4. Oxidative stress Cd stress induced ROS overproduction in lettuce. H2O2 and O2.− levels increased by 30.9%/47.2% (Figs. 4C) and 44.9%/59.3% (Fig. 4D), respectively, in leaves (roots) of lettuce seedlings exposed to Cd stress alone; these effects were effectively inhibited by FA application. These results are consistent with leaf histochemical staining results. As presented in DAB staining (Fig. 4A), the brown coloration in leaf co-treated with Cd and FA was less than that in leaf only treated with Cd, which indicated the level of H2O2 in plants co-treated with Cd and FA is lower than that in plants only exposed to Cd stress. NBT staining results were similar to those of DAB staining, a small amount of blue coloration indicated that less O2.− accumulation in leaf (Fig. 4B). Lipid peroxidation (TBARS) was higher in plants under Cd stress than in control plants; this effect was inhibited by FA application (Fig. 4E).
3. Results 3.1. Effects of FA on biomass, chlorophyll content, PSⅡ performance index (P.I.), and photosynthesis and eletctrolyte leakage of lettuce under Cd stress To examine the sensitivity of lettuce seedlings to Cd toxicity, and the effects of different FA amounts on lettuce seedlings exposed to Cd stress, we investigated plant growth, chlorophyll content, performance index (PI), photosynthesis, and electrolyte leakage. Changes in phenotype following Cd and FA treatments are shown in Supplementary Fig. 1. Compared with the control, Cd stress caused a significant decrease in root and shoot fresh biomass, chlorophyll content, PI, photosynthesis, and an increase in electrolyte leakage (Fig. 1). However, almost all doses of FA alleviated Cd toxicity. Among the experimental concentrations, 0.5 g/L FA significantly improved lettuce growth in shoots (79.5%) and roots (53.8%), increased chlorophyll content (60.2%), P.I. (45.3%), photosynthesis (39.9%) and reduced electrolyte leakage (35.0%) in comparison with lettuce exposed to Cd alone. Therefore, we used a treatment of 0.5 g/L FA to investigate the role of FA in the mitigation of Cd-induced lettuce seedling growth inhibition.
3.5. Antioxidant enzymes The antioxidant enzyme activities in shoots and roots are presented in Fig. 5. Superoxide dismutase (SOD) and peroxidase (POD) activities increased by 53.6% (29.8%) and 69.65% (53.3%), respectively, in shoots (roots) under Cd stress. FA application resulted in a decline in shoot SOD and POD activities, to varying degrees, compared with Cd stress alone, whereas there were no significant changes in root SOD or POD activity (Fig. 5A, B). Cd stress induced significant decreases in shoot catalase (CAT) and ascorbate peroxidase (APX) activities, which were enhanced by FA foliar spraying (Fig. 5C, D).
3.2. Chlorophyll a fluorescence 3.6. Effects of FA on Cd and elemental nutrient uptake by lettuce As shown in Fig. 2A, fluorescence intensity significantly decreased in lettuce leaves exposed to Cd for 2 weeks, and this effect was mitigated by foliar spraying with FA. We observed significant changes in the OJIP curve under different treatments, indicating that lettuce was sensitive to Cd (Fig. 2B). We observed an increase in ΔVJ and ΔVI after treatment with Cd; however, these two parameters were lower following co-treatment with Cd and FA than they were with Cd alone (Fig. 2C). JIP-test parameters from chlorophyll fluorescence transient are presented in Fig. 2D. We observed that dVG/dto, dV/dto, and WK increased to various degrees in lettuce exposed to Cd compared with the control; this increase was inhibited by FA application. φEo and φRo values increased to various degrees under Cd and FA co-treatment
To determine the effects of foliar spraying of FA on Cd accumulation in shoots and roots, we evaluated the Cd content of shoot and root tissues. Cd concentrations were approximately 0.31 mg (g–1 dry weight) in shoots and roots (Fig. 6). After FA foliar spraying, the Cd concentration in plants declined. A significant decrease in Cd content in roots and shoots was observed following the application of 0.5 g/L FA, by approximately 29.5% (Figs. 6A) and 37.9% (Fig. 6B), respectively. The impacts of FA application on elemental nutrient uptake by lettuce exposed to Cd are shown in Table 1. Cd stress caused a significant reduction in iron (Fe), zinc (Zn), and manganese (Mn) uptake in shoots, by 84.2%, 30.1%, and 44.1%, respectively. FA application significantly increased the accumulation of these nutrients in lettuce, 13
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Fig. 2. Effects of FA on fast chlorophyll fluorescence curves of lettuce leaves under Cd stress. The fluorescence intensity of original fluorescence kinetic curve (A). The normalized fluorescence data is expressed by "Vt", Vt = (Ft-Fo)/(Fm-Fo) (Ft is the fluorescence value at any time), which is normalized with Fm-Fo (B); The difference of relative chlorophyll a fluorescence of control and treatments (C). Effects of FA on fast chlorophyll fluorescence parameters of lettuce leaves under Cd stress (D).
promoted nutrients uptake slightly but not significantly in roots.
but had no effect on Cu accumulation in shoots. Similar results of the nutrients changes were found in roots. The level of Fe, Zn and Mn accumulation in root under Cd stress respectively declined by 22.4%, 73.7% and 87.2%, respectively. while the level of Cu accumulation in roots is raised when exposed to cadmium. Exogenous FA application
4. Discussion FA is a soluble micromolecular substance and the major constituent 14
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Fig. 3. Effects of FA on chlorophyll and Carotenoid contents (A), gas exchange parameters (B, C, D) of lettuce leaves under Cd stress. Data are shown as means ± SE of five replicates. Treatments conditions are described in Fig. 4. Bars marked by different letters indicate significant differences by Tukey's test (P ≤ 0.05).
2013). In the present study, Cd stress caused Cd accumulation in lettuce. However, foliar application of FA decreased Cd accumulation in lettuce shoots and roots, with the greatest reduction in Cd seen at 0.5 g/ L FA. We therefore selected this FA concentration for further study.
of humic substances (Weng et al., 2006). It has been widely demonstrated that FA can enhance plant tolerance to biotic and abiotic stresses, including drought, water, and salinity stress (Anjum et al., 2011, Aydin et al., 2012, Selim et al., 2012). However, in recent years, there have been fewer reports of Cd stress tolerance enhancement by FA application. In the current study, we elucidated the possible mechanism underlying photosynthetic apparatus defense, reduced oxidative stress, decreased Cd accumulation, and elemental nutrient uptake by FA.
4.2. FA protected photosynthetic apparatus from Cd toxicity It is well-known that photosynthesis is sensitive to Cd stress (Zhang et al., 2015). Consistent with past findings, the present study showed that electron transfer in lettuce was significantly inhibited by Cd stress, as indicated by the increase in △Vj and △Vi. In fact, the reduction of efficiency of excitation energy capture (ψEo) led to the decrease of the quantum yield of PSII electron transport (φEo). In order to consume too much-activated electrons, the quantum yield of dissipated energy (φDo) increased. However, following FA application, these parameters were nearly comparable to the control; this result demonstrated that FA enhanced electron transport capacity on the receptor side of photosystem II (PSII). Wk is a special indicator that values the degree of OEC impaired by stress (Appenroth et al., 2001). After FA application, we found Wk was decreased under Cd stress, suggesting that FA protected OEC from Cd toxicity. Our results also showed that Cd toxicity reduced ABS/CS, TRo/CS, ETo/CS and DIo/CS parameters, possibly due to the degradation or inaction of reaction centers and destruction of antenna pigments, causing a decrease in the trapped energy (TRo/CS) and reduction energy (ET0/CS) of reaction centers (Appenroth et al., 2001). The increase of DIo/CS indicated that the reaction centers start the defense mechanism (heat dissipation) in response to Cd stress. PI (abs) and PI (total), a measure of plant photosynthetic performance, is sensitive to Cd stress (Fig. 2D). Our results indicate that FA protected PSII from Cd toxicity by increasing reaction center density (RC/CS), absorption flux (ABS/CS), trapped energy flux (TR0/CS), electron transport flux (ETo/CS)/dissipated energy flux (DIo/CS) per excited cross
4.1. Cd toxicity was dose-dependently ameliorated by FA The major phenotypes affected by Cd toxicity in plants are biomass reduction and leaf chlorosis (Das et al., 1997). In the present study, we observed that both of these phenotypes were inhibited as a result of Cd stress, which could be explained by a decline in chlorophyll content, performance index of PSII (P.I.), photosynthesis. Moreover, electrolyte leakage (EL), an indicator of membrane permeability also was altered by Cd stress. Various FA treatments had positive effects on these changes caused by Cd toxicity, with the optimal effect being produced by a moderate concentration (0.5 g/L). Thus, FA mitigated Cd stress in lettuce in a dose-dependent manner. The similar results was found in previous research that excess supply of Cd caused growth inhibition, increased Cd accumulation and the accumulation of ROS in lettuce, however, foliar application of exogenous NO alleviated Cd-induced growth suppression, promoted the chlorophyll synthesis and improved photosynthetic efficiency (Xu et al., 2014). Similar effect was also found in wheat grown in calcareous conditions (Katkat et al., 2009). Horuz et al. (2015) revealed that humic acid applied in soil increased the dry matter and reduced Cd uptake in lettuce grown in the soil contaminated with Cd. Another research also shown that 100 mg/L humic acid applied in soil increased growth and fresh weight of lettuce leaves and reduced Cd concentration in lettuce leaves (Haghighi et al., 15
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Fig. 4. The in situ detection of H2O2 (A) and O2·– (B), Bar= 1 cm. Effects of FA on H2O2 content (C), superoxide radical production (D) and thiobarbituric acid reactive substance(TBARS) content (E) in shoots and roots of lettuce. Treatment conditions are described in Fig. 4. Data are shown as means ± SE of five replicates. Bars marked by different letters indicate significant differences by Tukey's test (P ≤ 0.05).
apparatus (Siedlecka et al., 1998). Therefore, we speculate that FA enhanced photosynthesis in lettuce under Cd stress by increasing chlorophyll content, thus improving PSII efficiency.
section. Therefore, FA had clear effects in improving PI (ABS), PI (total) in lettuce under Cd stress. The observed decrease in Pn may be attributed to stomatal closure; hence, CO2 diffusion was limited (Fig. 3B). Meanwhile, the reduction of Pn may as result of non-stomatal limitation (Monteiro et al., 2009). We also observed that Cd caused a significant decrease in chlorophyll content. The same results were observed in the previous study that chlorophylls and carotenoids concentrations in leaves of lettuce were decreased as result of the Cd stress (Xu et al., 2014). It is possible that Cd can replace the magnesium atom (Mg) within chlorophyll to form a chlorophyll–Cd complex, causing damage to the photosynthetic
4.3. FA reduced ROS accumulation and enhanced antioxidant enzyme capacity It has been reported that Cd might cause oxidative stress by generating excess ROS (Cho and Seo, 2005). As the duration of Cd treatment increases, so too does the malondialdehyde (MDA) content in lettuce leaves (Monteiro et al., 2009). Another study has shown that Cd 16
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Fig. 5. Effects of FA on the activities of SOD (A), POD (B), CAT (C) and APX (D) in leaves and roots of lettuce seedlings. Data are shown as means ± SE of five replicates. Bars marked by different letters indicate significant differences by Tukey's test (P ≤ 0.05).
stress increased H2O2 and O2.− accumulation and induced an increase in MDA content in root and leaves of lettuce seedlings (Xu et al., 2015). We observed similar results in the present study, such that TBARS, H2O2 and O2.− content significantly increased in lettuce seedling shoots (Zouari et al., 2016). Generally, higher plants have developed a defense system to overcome oxidative stress caused by Cd toxicity. In the current study, SOD and POD activities significantly increased in lettuce under Cd stress, whereas those of APX and CAT declined. However, FA and Cd co-treatment enhanced APX and CAT activities, such that the two enzymes could effectively scavenge H2O2 in plant tissues. This result is also confirmed in wheat, where the FA improved the activities of APX and CAT under Cr stress (Ali et al., 2015). In contrast, the activities of SOD and POD were reduced after treatment with FA. This result suggests that FA may serve directly as an antioxidant to eliminate
excess ROS, or as a signal molecule to promote antioxidant production. A similar report indicated that FA exhibited auxin-like activity (Nardi et al., 2002). Moreover, a remarkable increase in carotenoid content was observed following FA treatment compared with Cd stress alone. It has previously been reported that ROS led to impaired chlorophyll and PSII function, and that carotenoid acts as an antioxidant, a first line of defense for plants against ROS (Zhang et al., 2015). Therefore, we speculate that FA enhanced photosynthesis and growth in lettuce under Cd stress by reducing ROS accumulation. 4.4. FA decreased Cd content and facilitated elemental nutrient uptake in lettuce exposed to Cd stress Previous studies showed that Cd impaired macronutrient balance
Fig. 6. Effects of different concentrations of FA on Cd accumulation in shoot (A) and root (B) of lettuce under Cd stress. Data are shown as means ± SE of five replicates. Bars marked by different letters indicate significant differences by Tukey's test (P ≤ 0.05). ND: not detected. 17
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Table 1 Effects of FA on nutrient elements uptake by lettuce under Cd stress. Treatments
Elements content (mg g−1 DW) Fe
Shoot Con Con+FA Cd Cd+FA Root Con Con+FA Cd Cd+FA
Zn
Cu
Mn
0.074 0.112 0.012 0.040
± ± ± ±
0.018b 0.029a 0.010d 0.002c
0.080 0.085 0.056 0.073
± ± ± ±
0.008a 0.010a 0.005b 0.021 BCE
0.016 0.016 0.016 0.013
± ± ± ±
0.002a 0.002a 0.002a 0.001a
0.092 0.099 0.052 0.073
± ± ± ±
0.013ab 0.015a 0.001c 0.010b
4.179 4.121 3.242 3.840
± ± ± ±
0.228a 0.757a 0.333b 0.114ab
0.276 0.492 0.072 0.119
± ± ± ±
0.144b 0.134a 0.016c 0.014 BCE
0.049 0.049 0.061 0.060
± ± ± ±
0.007b 0.001b 0.006a 0.004a
0.390 0.542 0.050 0.071
± ± ± ±
0.057a 0.083b 0.010c 0.006c
Treatments conditions are described in Fig. 4. Data are shown as means ± SE of five replicates. Bars marked by different letters indicate significant differences by Tukey's test (P ≤ 0.05).
Appendix A. Supporting information
and accumulation in lettuce (Monteiro et al., 2009; Matraszek et al., 2016; Rizwan et al., 2017). Similar results were observed in the present study, in which Cd stress caused an imbalance of mineral elements in lettuce. Fe content significantly declined in lettuce shoots and roots. Zn and Cu contents were reduced to different degrees in lettuce subjected to Cd stress, perhaps due to the accumulation of Cd in shoots and roots. FA application increased the Fe and Zn contents in lettuce shoots under Cd stress, but had little effect in roots. It is worth noting that FA had a more remarkable effect on Fe uptake than other nutrient elements, and that it improved Fe accumulation to a greater degree in shoots than in roots. Cd stress disturbed the balance of mineral elements may attribute to two ways: (1) inhibition of Fe chelation and loading to xylem and (2) the competition between nutrients and Cd for the same transporters (Tran and Popova, 2013). We found that foliar application of FA significantly decreased Cd accumulation in shoots and roots of lettuce exposed to cadmium. Therefore, we speculate that FA could inhibit Cd uptake and improve Fe translocation from roots to shoots in lettuce.
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2018.08.064. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Ali, B., Wang, B., Ali, S., et al., 2013. 5-Aminolevulinic acid ameliorates the growth, photosynthetic gas exchange capacity, and ultrastructural changes under Cadmium stress in Brassica napus L. J. Plant Growth Regul. 32 (3), 604–614. Ali, S., Bharwana, S.A., Rizwan, M., et al., 2015. Fulvic acid mediates chromium (Cr) tolerance in wheat (Triticum aestivum L.) through lowering of Cr uptake and improved antioxidant defense system. Environ. Sci. Pollut. Res. Int. 22 (14), 601–609. Anjum, S.A., Wang, L., Farooq, M., et al., 2011. Fulvic acid application improves the maize performance under well-watered and drought conditions. J. Agron. Crop Sci. 197 (6), 409–417. Appenroth, K.J., Stöckel, J., Srivastava, A., et al., 2001. Multiple effects of chromate on the photosynthetic apparatus of Spirodela polyrhiza, as probed by OJIP chlorophyll a, fluorescence measurements. Environ. Pollut. 115 (1), 49–64. Aydin, A., Kant, C., Turan, M., 2012. Humic acid application alleviate salinity stress of bean (Phaseolus vulgaris L.) plants decreasing membrane leakage. Afr. J. Agric. Res. 7 (7), 1073–1086. Bajji, M., Kinet, J.M., Lutts, S., 2002. The use of the electrolyte leakage method for assessing cell membrane stability as a water stress tolerance test in durum wheat. Plant Growth Regul. 36 (1), 61–70. Cho, U.H., Seo, N.H., 2005. Oxidative stress in Arabidopsis thaliana, exposed to cadmium is due to hydrogen peroxide accumulation. Plant Sci. 168 (1), 113–120. Cobb, G.P., Sands, K., Waters, M., et al., 2000. Accumulation of heavy metals by vegetables grown in mine wastes. Environ. Toxicol. Chem. 19 (3), 600–607. Das, P., Samantaray, S., Rout, G.R., 1997. Studies on cadmium toxicity in plants: a review. Environ. Pollut. 98 (1), 29–36. Erdal, S., Turk, H., 2016. Cysteine-induced upregulation of nitrogen metabolism-related genes and enzyme activities enhance tolerance of maize seedlings to cadmium stress. Environ. Exp. Bot. 132, 92–99. Yildirim, Ertan, 2007. Foliar and soil fertilization of humic acid affect productivity and quality of tomato. Acta Agric. Scand. 57 (2), 182–186. Adani, Fabrizio, Genevini, Pierluigi, Zaccheo, Patrizia, et al., 1998. The effect of commercial humic acid on tomato plant growth and mineral nutrition. J. Plant Nutr. 21 (3), 561–575. Giannopolitis, C.N., Ries, S.K., 1977. Superoxide dismutases I. Occurrence in higher plants. Plant Physiol. 59 (2), 309–314. Hodges, D.M., DeLong, J.M., Forney, C.F., et al., 1999. Improving the thiobarbituric acidreactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207 (4), 604–611. Horuz, A., Karaman, M.R., Gulluce, M., 2015. Effect of humic acid application on the reduction of cadmium concentration in lettuce(Lactuca sativa L.). Fresenius Environ. Bull. 24 (10), 3141–3147. Jabs, T., Dietrich, R.A., Dangl, J.L., 1996. Initiation of runaway cell death in an Arabidopsis mutant by extracellular superoxide. Science 273 (5283), 1853–1856. Katkat, A.V., Celik, H., Turan, M.A., et al., 2009. Effects of soil and foliar applications of humic substances on dry weight and mineral nutrients uptake of wheat under calcareous soil conditions. Aust. J. Basic Appl. Sci. 3 (2), 1266–1273. Knudson, L.L., Tibbitts, T.W., Edwards, G.E., 1977. Measurement of ozone injury by determination of leaf chlorophyll concentration. Plant Physiol. 60 (4), 606–608. Konstantopoulou, E., Kapotis, G., Salachas, G., et al., 2010. Nutritional quality of greenhouse lettuce at harvest and after storage in relation to N application and cultivation season. Sci. Hortic. 125 (2), 1–5. Haghighi, Maryam, Kafi, Mohsen, Khishgoftarmanesh, Amir, 2013. Effect of humic acid application on cadmium accumulation by lettuce leaves. J. Plant Nutr. 36 (10), 1521–1532.
5. Conclusion We found that Cd exposure have deleterious effect on lettuce, mainly reflecting in the reduction of growth, photosynthetic capacity, ROS accumulation, inhibition of mineral element uptake. Foliar application of FA plays an important role in protecting the photosynthetic apparatus from Cd toxicity. FA inhibited ROS accumulation in plants exposed to Cd by stimulating the antioxidant system, and enhanced the photosynthetic capacity of lettuce by protecting PSII against Cd toxicity, accelerating chlorophyll biosynthesis and promoting translocation of the elemental nutrients from roots to shoots. This effect was especially clear in elements involved in photosynthesis, such as Fe, Zn, and Mn. The ability of FA to alleviate Cd toxicity in lettuce could be due to elimination of excess ROS and suppression of Cd uptake. However, to fully elucidate the role of FA in lettuce Cd-stress response, more detailed molecular analyses will be needed. To sum up, FA application is an effective detoxification method for lettuce under Cd stress and could be explored in phytoremediation.
Acknowledgment This work was supported by National key research and development program of China (2016YFD0800703, SQ2018YFD080066). The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/Nt7iKo
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Fluorescence. Springer, Netherlands, pp. 321–362. Suh, H.Y., Yoo, K.S., Sang, G.S., 2014. Effect of foliar application of fulvic acid on plant growth and fruit quality of tomato (Lycopersicon esculentum L.). Hortic. Environ. Biotechnol. 55 (6), 455–461. Tang, W.W., Zeng, G.M., Gong, J.L., et al., 2014. Impact of humic/fulvic acid on the removal of heavy metals from aqueous solutions using nanomaterials: a review. Sci. Total Environ. 468, 1014–1027. Thordal Christensen, H., Zhang, Z., Wei, Y., et al., 1997. Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley—powdery mildew interaction. Plant J. 11 (6), 1187–1194. Tran, T.A., Popova, L.P., 2013. Functions and toxicity of cadmium in plants: recent advances and future prospects. Doga Turk. J. Bot. 37 (1), 1–13. Wang, W., Cang, L., Zhou, D., et al., 2016. Exogenous amino acids increase antioxidant enzyme activities and tolerance of rice seedlings to cadmium stress. Environ. Prog. Sustain. Energy 36 (1), 155–161. Weng, L.P., Van Riemsdijk, W.H., Koopal, L.K., et al., 2006. Adsorption of humic substances on goethite: comparison between humic acids and fulvic acids. Environ. Sci. Technol. 40 (20), 7494–7500. Xu, L.L., Dong, Y.J., Kong, J., et al., 2014. Effects of root and foliar applications of exogenous NO on alleviating cadmium toxicity in lettuce seedlings. Plant Growth Regul. 72 (1), 39–50. Xu, L.L., Fan, Z.Y., Dong, Y.J., et al., 2015. Effects of exogenous NO supplied with different approaches on cadmium toxicity in lettuce seedlings. Plant Biosyst. 149 (2), 270–279. Zhang, Y., Xu, S., Yang, S., et al., 2015. Salicylic acid alleviates cadmium-induced inhibition of growth and photosynthesis through upregulating antioxidant defense system in two melon cultivars (Cucumis melo L.). Protoplasma 252 (3), 911–924. Zhao, X., Chen, Q., Wang, Y., et al., 2017. Hydrogen-rich water induces aluminum tolerance in maize seedlings by enhancing antioxidant capacities and nutrient homeostasis. Ecotoxicol. Environ. Saf. 144, 369–379. Zouari, M., Ben, A.C., Elloumi, N., et al., 2016. Impact of proline application on cadmium accumulation, mineral nutrition and enzymatic antioxidant defense system of Olea europaea L. cv Chemlali exposed to cadmium stress. Ecotoxicol. Environ. Saf. 128, 195–205.
Matraszek, R., Hawrylak-Nowak, B., Chwil, S., et al., 2016. Macroelemental composition of cadmium stressed lettuce plants grown under conditions of intensive sulphur nutrition. J. Environ. Manag. 180, 24–34. Meng, H.B., Hua, S.J., Shamsi, I.H., et al., 2009. Cadmium-induced stress on the seed germination and seedling growth of Brassica napus L. and its alleviation through exogenous plant growth regulators. Plant Growth Regul. 58 (1), 47–59. Monteiro, M.S., Santos, C., Soares, A.M., et al., 2009. Assessment of biomarkers of cadmium stress in lettuce. Ecotoxicol. Environ. Saf. 72 (3), 811–818. Morales, J., Manso, J.A., Cid, A., et al., 2012. Degradation of carbofuran and carbofuranderivatives in presence of humic substances under basic conditions. Chemosphere 89 (11), 1267–1271. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22 (5), 867–880. Nardi, S., Pizzeghello, D., Muscolo, A., et al., 2002. Physiological effects of humic substances on higher plants. Soil Biol. Biochem. 34 (11), 1527–1536. Parvaiz, A., Abdel, L.A.A., Abd_Allah, E.F., et al., 2016. Calcium and potassium supplementation enhanced growth, osmolyte secondary metabolite production, and enzymatic antioxidant machinery in cadmium-exposed chickpea (Cicer arietinum L.). Front. Plant Sci. 7 (347), 1–12. Rizwan, M., Ali, S., Adrees, M., et al., 2017. A critical review on effects, tolerance mechanisms and management of cadmium in vegetables. Chemosphere 182, 90–105. Saidi, I., Chtourou, Y., Djebali, W., 2014. Selenium alleviates cadmium toxicity by preventing oxidative stress in sunflower (Helianthus annuus) seedlings. J. Plant Physiol. 171 (5), 85–91. Santos, L.R., Batista, B.L., Lobato, A.K.S., 2018. Brassinosteroids mitigate cadmium toxicity in cowpea plants. Photosynthetica 56 (2), 591–605. Selim, E.M., Shedeed, S.I., Asaad, F.F., et al., 2012. Interactive effects of humic acid and water stress on chlorophyll and mineral nutrient contents of potato plants. J. Appl. Sci. Res. 8 (1), 531–537. Siedlecka, A., Samuelsson, G., Gardenstrom, P., et al., 1998. The activatory model of plant response to moderate cadmium stress-relationship between carbonic anhydrase and Rubisco. In: Garab, G. (Ed.), Photosynthesis: Mechanisms and Effects. Kluwer Academic Publishers, Dordrecht, pp. 2677–2680. Strasser, R.J., Tsimilli-Michael, M., Srivastava, A., 2004. Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou, George, Govindjee (Eds.), Chlorophyll a
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Exogenous foliar application of fulvic acid alleviate cadmium toxicity in lettuce (Lactuca sativa L.) Yanmei Wang, Ruixi Yang, Jiaying Zheng, Zhenguo Shen, Xiaoming Xu
T
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College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Fulvic acid Cadmium stress Lettuce Antioxidant enzymes Mineral nutrition
It was reported that fulvic acid (FA) has a positive effect on enhancing plant tolerance to various environmental stresses, including salinity stress and drought stress and so on. However, there is little study regarding the effects of FA on plants in response to heavy metal stress. Hence, the objective of this study was to investigate the potential effects of fulvic acid (FA) on cadmium (Cd) toxicity alleviation in lettuce seedlings. Our results showed that application of 0.5 g/L FA significantly mitigate Cd-induced toxic symptoms in lettuce seedlings. Cd stress triggered plant growth inhibition, photosynthetic pigment reduction, destruction of the photosynthesis apparatus, reactive oxygen species (ROS) accumulation, and nutrient elemental imbalance. We observed that FA promoted the growth in lettuce under Cd stress, mainly reflected in those alterations that the increase of biomass, chlorophyll content and photosynthesis capacity and reduction of the Cd content and lipid peroxidation in plant tissue. Foliar spraying of FA significantly alleviated these detrimental symptoms and facilitated nutrient element translocation from root to shoot, particularly the absorption of elements involved in photosynthesis, including iron (Fe), zinc (Zn), and manganese (Mn). In summary, foliar application of FA conferred Cd toxicity tolerance to lettuce by increasing ROS-scavenging capacity, inhibiting Cd uptake and the transport of elemental nutrients to shoots, which in turn protected the photosynthetic apparatus and promoted plant growth.
1. Introduction
chloroplast ultrastructure, as well as cell membrane structure (Ali et al., 2013). Various methods have been reported to reduce Cd accumulation in plants, including exogenous application of materials such as plant growth regulators, elemental nutrients, and amino acids; the application of these substances could be an effective strategy to reduce Cd uptake by vegetables destined for human consumption (Parvaiz et al., 2016; Wang et al., 2016; Meng et al., 2009). However, few studies have been conducted on the effects of foliar application on plants exposed to Cd. Humic substances are natural organic compounds comprising organic acids derived from the decomposition of plant and animal residues under the action of microorganisms and geochemistry (Morales et al., 2012). Humic substance can be divided into humin, humic acid, and fulvic acid (FA) according to its solubility at different pH (Suh et al., 2014). Among these substances, fulvic acid (FA) has a relatively low molecular weight and contains a high amount of oxygen-rich and carbon-poor functional groups (Weng et al., 2006). FA confers benefits to plants by enhancing drought resistance, improving nutrient uptake, stabilizing soil pH, and reducing fertilizer leaching (Suh et al., 2014). Adani et al. (1998) revealed that soil application of humic acid could stimulate the growth of tomato plants grown by hydroponics, and
In recent years, heavy metal pollution has attracted increasing attention worldwide, due to its release into the environment through anthropogenic activities such as mining, waste water for irrigation, sewage sludge application, excessive application of phosphate fertilizers, herbicides and pesticides to farmland, and vehicular and industrial activities (Rizwan et al., 2017). Among heavy metals, cadmium (Cd) is a highly toxic, non-essential element for humans, animals, and plants. It is easily taken up by plant roots and transported to shoots (Saidi et al., 2014), Cd toxicity causes various morphological, physiological, biochemical, and ultrastructural changes in plants. Its main symptoms manifested growth inhibition of shoots and roots, leaf chlorosis, browning of root tips and so on (Zhang et al., 2015). A large amount of toxic effects of Cd on metabolism have been reported, such as imbalance of nutritional elements, reduction of chlorophyll biosynthesis, enzymes inhibition, disruption of basic metabolic functioning, including photosynthesis, respiration, and nucleic acid production (Erdal and Turk, 2016; Ali et al., 2013; Santos et al., 2018). Moreover, the burst out of reactive oxygen in plants with Cd stress, which caused oxidative stress and responsible for damage to mitochondria and
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Corresponding author. E-mail address: [email protected] (X. Xu).
https://doi.org/10.1016/j.ecoenv.2018.08.064 Received 18 January 2018; Received in revised form 16 August 2018; Accepted 17 August 2018 0147-6513/ © 2018 Published by Elsevier Inc.
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promote the nutrients uptake, especially Fe. Similar results were found that total yield was raised in tomato by foliar and soil application of humic acid (Yildirim, 2007). In many cases, humic acid is regarded as a plant growth regulator, which has the similar effect with auxins, but we do not confirm whether it contain auxin-like substances or not (Nardi et al., 2002). The explicit physiological mechanism that fulvic acid (FA) enhanced plant resistance against abiotic stresses is seldom known. Current report has exhibited that soil and foliar applied FA has beneficial effects on seedling growth, seed germination, and root weight of wheat plants (Katkat et al., 2009). Tang et al. (2014) reported that FA could also be applied to remove heavy metals from aqueous media. Ali et al. (2015) explored the effects of foliar FA application on wheat plants exposed to chromium (Cr) and found that FA alleviated Cr toxicity by increasing photosynthetic pigments, reducing Cr uptake, and improving antioxidant activities in wheat. However, few studies have examined the effect of FA application to soil to enhance Cd stress tolerance in lettuce. (Haghighi et al., 2013, Horuz et al., 2015). Therefore, this study was conducted to illuminate the effects of foliar application of fulvic acid on lettuce under Cd stress. Lettuce (Lactuca sativa L.) is a common leafy vegetable that is cultivated commercially and in home gardens worldwide (Konstantopoulou et al., 2010; Matraszek et al., 2016). Lettuce is rich in a variety of vitamins, dietary fibers, low in calories as well as a source of carotenoids, which is beneficial for human health. In addition, it contains many nutrient minerals, such as Ca, Fe, Zn, Se etc., which is essential for human body to maintain normal function by serving as electrolytes (Matraszek et al., 2016). Lettuce is valued for its high nutritional value and is often used in salads as a fresh vegetable. It has a high capacity for Cd accumulation from soil without showing visible symptoms of metal toxicity, which poses a potential risk to human health (Cobb et al., 2000). In this study, we use the method of foliar spraying to investigate the effects of FA on lettuce exposed to Cd and try to elucidate its possible mitigation mechanism for two reasons. Firstly, FA can chelate directly with Cd in solution (Tang et al., 2014). Meanwhile, foliar spraying is easy to achieve cultivation in the field. The objectives of this study were to examine (a) whether FA supply ameliorates Cd toxicity in lettuce, (b) the optimal concentration of FA to alleviate Cd toxicity, and (c) the possible mechanism of Cd toxicity defense by FA in lettuce.
Secondly, we have chosen an optimum FA concentration (0.5 g/L) and seen how it influences in Chlorophyll a fluorescence, photosynthetic pigments and gas exchange parameters, oxidative stress, Antioxidant enzymes and nutrient element uptake. Based on preliminary trials, four treatments respectively were Control, 20 μM Cd, Control+FA, Cd+FA. The solutions were renewed every other day. Each treatment was designed with three replicate randomly, and each replicate contain three plants. After treatment 2 weeks later, seedlings were harvested to make the further analysis.
2. Materials and methods
2.5. Gas exchange parameter measurements
2.1. Plant material and growth conditions
The second fully expanded leaves from the top of plants were used to estimate the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (E) using a portable photosynthesis system (LiCOR 6400, Lincoln, NE, USA). The experiment was performed on sunny day from 9:00–11:30 a.m.
2.3. Plant biomass measurements and chlorophyll content measurements Fifteen days after treatment, lettuce seedlings were harvested and photographed. After that, samples were separated into leaves and roots, which fresh weights were determined immediately with electronic balance. Dry weights were estimated after drying samples in an oven at 80 °C until biomass became constant. Chlorophyll content was measured with the help of SPAD-502 chlorophyll meter. Photosynthetic pigment was determined with spectrophotometry, which extracted with 95% ethanol. The details of experimentation depend on the method of Knudson et al. (1977). 2.4. Chlorophyll fluorescence parameter measurements and JIP-test analysis Leaves were adapted in the dark at least 30 min before measurements. Chlorophyll fluorescence parameters were measured using the Handy Plant Efficiency Analyzer (Plant Efficiency Analyzer; Hansatech, UK). The measured light source is a red light with a wavelength of 650 nm and a light intensity of 3000 μmol m−2 s−1 provided by three light-emitting diodes, which continue to record 1 s. An OJIP curve was plotted to normalize the fluorescence data to relative variable fluorescence data, using the following equation: Vt = (Ft – Fo)/(FM – Fo), where Vt is the relative variable fluorescence at time t, Fo is the initial fluorescence, Ft is the fluorescence at time t, and FM is the maximum fluorescence. JIP test parameters (transient fluorescence steps O, J, I, and P) were calculated according to the JIP-test algorithm described by Strasser et al. (2004). The analyzed parameters are described in Supplementary Table 1.
Lettuce (Lactuca sativa L.) seeds, acquired from Jiangsu Agricultural Institutes (Nanjing Province, China) were germinated in a tray filled with vermiculite at 25 ℃ in a climate chamber. Uniformly sized seedlings were selected 5 days later and transplanted into plastic cups (three plants per cup) containing 400 mL a half-strength Hoagland nutrient solution (pH 6.0). The selected seedlings were incubated in the climate chamber at a temperature of 25/20 ℃ (day/night), with a light intensity of 600 μmol m–2 s–1 and a 12-h photoperiod.
2.6. Determination of lipid peroxidation, hydrogen peroxide (H2O2), Superoxide radical (O2·–) and eletctrolyte leakage (EL) The level of lipid peroxidation in leave and roots were estimated by quantifying the content of the thiobarbituric acid reactive substance (TBARS), which determined by the thiobarbituric acid (TBA) based on the method of Hodges et al. (1999) with minor modifications (Zhao et al., 2017). H2O2 content was measured depending on the description of Zhao et al. (2017). O2.− was measured by monitoring nitrite formation from hydroxylamine in the presence of O2.−, according to the method of Jabs et al. (1996) with some modifications. The measurement of EL in leaves was according to the method of Bajji et al. (2002). Fresh leaves (0.1 g) were cut into the same size leaf discs of the same size and put into a tube containing 10 mL of deionized water. The initial electrical conductivity (EC1) was measured after the tubes were placed at 25 ℃ for 4 h. Then, the tubes were put into boiling water bath for 30 min. The second electrical conductivity (EC2) was
2.2. Experimental design Firstly, we have studied which FA dose works better to improve Cd damage by these indicators: the plant growth, chlorophyll content, P.I., photosynthesis, electrolyte leakage and reduction of Cd accumulation. After 10 days of growth, the seedlings in three leaf stage were treated with 20 μM Cd (CdCl2·2.5 H2O) in hydroponics, Meanwhile, the seedlings exposed to Cd were sprayed with 0, 0.1, 0.3, 0.5, 1.0, 2.0 g/L fulvic acid (FA) in the morning every day. FA (> 90%) was purchased from Bio Aladdin (Shanghai, china). Seedling without Cd and fulvic acid treatment were used as the control (Con), All treatments we described as (1) Con, (2) Cd, (3) Cd + 0.1FA, (4) Cd + 0.3FA, (5) Cd + 0.5FA, (6) Cd + 1.0FA, (7) Cd + 2.0FA. 11
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Fig. 1. Effects of different concentrations of FA on biomass accumulation (A), SPAD value (B), PSⅡ performance index (C), photosynthesis (D) and electrolyte leakage (E) in lettuce under Cd stress. Data are shown as means ± SE of five replicates. Bars marked by different letters indicate significant differences by Tukey's test (P ≤ 0.05).
determined after the solution in tubes were cooled at 25 ℃. The EL was calculated with following formula: EL (%) =EC1/EC2 × 100.
2.8. Antioxidant enzymes assays Fresh plant sample (0.5 g) were homogenized in 5 mL pre-cooled extraction buffer containing 1 mM Ethylenediaminetetraacetic acid disodium salt (EDTA-Na2) and 1% polyvinylpyrrolidone (PVP) in 100 mM potassium phosphate buffer (pH 7.0). The homogenate was centrifuged at 12,000 g for 20 min and the supernatant used for the enzyme assays below. All the processes were carried out at 4 ℃. Superoxide dismutase (SOD) activity was assayed based on its ability to restrain the photochemical reduction of nitroblue tetrazolium (NBT) following the method of Giannopolitis and Ries (1977). One unit of SOD activity was defined as the amount of enzyme required to inhibit the reduction of NBT by 50% as monitored at 560 nm. According to the reports of Aebi (1984), Catalase (CAT) activity was determined by monitoring reduce of H2O2 (extinction coefficient:
2.7. Histochemical detection of H2O2 and O2.− H2O2 accumulation in leaves was detected with the 3-diaminobenzidine (DAB) staining procedure, which involved the incubation of the leaves with DAB (1 mg mL−1, pH 3.8) solution in the dark for 6 h (Thordal Christensen et al., 1997). O2.− accumulation in leaves was visually detected by staining with Nitroblue tetrazolium (NBT) using the method as described by Jabs et al. (1996). Leaves were removed from plants, submerged in NBT solution (0.5 mg mL−1, pH 7.8) immediately, and incubated at for 6 h at 25 ℃ in dark. 12
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39.4 mM−1 cm−1) at 240 nm for 1 min. Peroxidase (POD) activity was measured by the means as described by Zhao et al. (2017), which mainly monitor the growing absorbance at 470 nm for 3 min as guaiacol was oxidized. The extinction coefficient was 26.6 mM−1 cm−1. Ascorbate peroxidase (APX) activity was determined by following the oxidation of ascorbic acid (extinction coefficient: 2.8 mM−1 cm−1) at 290 nm for 1 min (Nakano and Asada, 1981).
compared with Cd treatment alone. The ratio of absorption flux per active reaction center (ABS/RC, TRo/RC and DIo/RC) was higher under Cd stress than under co-treatment with Cd and FA, while the opposite trend was observed for electron transport flux (ETo/RC). The absorbance flux (ABS/CSo, TRo/CSo, ETo/CSo, DIo/CSo, and RC/CSo) decreased to different extents according to the Cd stress, and was significantly improved by FA application. Changes in the absorption of photon flux via chlorophyll molecules (ABS/CSm, TRo/CSm, ETo/CSm, DIo/CSm, and RC/CSm) showed a similar pattern to those of ABS/CSo, and similar parameters.
2.9. Elemental analysis
3.3. photosynthetic pigments and gas exchange parameters
After harvest, the fresh seedlings were rinsing several times with deionized water after soaking in EDTA-Na2 solution for 10 min. After that, samples were divided into shoots and roots and dried at 105 °C for 25 min and then at 80 °C in an oven until a constant weight was achieved and maintained. The dried samples were weighed and digested with an acid mixture (HNO3:HClO4, 87:13,v/v) at 180 °C. Cd and nutrient elements concentration were measured by inductively coupled plasma-optical emission spectroscopy (ICP-OES, Perkin Elmer Optima 2100DV) as described by Zhao et al. (2017).
Carotenoid and total chlorophyll content in leaves under Cd stress were reduced by 69.0% and 61.4%, respectively, compared with the control; this decline was reversed following FA foliar spraying, by approximately 20.3% and 29.4%, respectively (Fig. 3A). Under Cd stress, there were apparent declines in net photosynthetic rate (Fig. 3B), stomatal conductance (Fig. 3C) and transpiration rate (Fig. 3D), by 29.2% and 62.5%, and 54.5%, respectively. The application of FA resulted in an improvement in the net photosynthetic rate (22.6%), stomatal conductance (35.5%), and transpiration rate (36.5%), compared with Cd treatment.
2.10. Statistical analysis Data was presented with mean ± SD of at least three replicates. It was analyzed by one-way analysis of various (ANOVA), which was conducted with SPSS 22.0 software. A Tukey's Test was performed to compare significant difference between treatments at P < 0.05. All the figures are.completed with Origin 9.0 software (Origin Lab, Northampton, MA, USA).
3.4. Oxidative stress Cd stress induced ROS overproduction in lettuce. H2O2 and O2.− levels increased by 30.9%/47.2% (Figs. 4C) and 44.9%/59.3% (Fig. 4D), respectively, in leaves (roots) of lettuce seedlings exposed to Cd stress alone; these effects were effectively inhibited by FA application. These results are consistent with leaf histochemical staining results. As presented in DAB staining (Fig. 4A), the brown coloration in leaf co-treated with Cd and FA was less than that in leaf only treated with Cd, which indicated the level of H2O2 in plants co-treated with Cd and FA is lower than that in plants only exposed to Cd stress. NBT staining results were similar to those of DAB staining, a small amount of blue coloration indicated that less O2.− accumulation in leaf (Fig. 4B). Lipid peroxidation (TBARS) was higher in plants under Cd stress than in control plants; this effect was inhibited by FA application (Fig. 4E).
3. Results 3.1. Effects of FA on biomass, chlorophyll content, PSⅡ performance index (P.I.), and photosynthesis and eletctrolyte leakage of lettuce under Cd stress To examine the sensitivity of lettuce seedlings to Cd toxicity, and the effects of different FA amounts on lettuce seedlings exposed to Cd stress, we investigated plant growth, chlorophyll content, performance index (PI), photosynthesis, and electrolyte leakage. Changes in phenotype following Cd and FA treatments are shown in Supplementary Fig. 1. Compared with the control, Cd stress caused a significant decrease in root and shoot fresh biomass, chlorophyll content, PI, photosynthesis, and an increase in electrolyte leakage (Fig. 1). However, almost all doses of FA alleviated Cd toxicity. Among the experimental concentrations, 0.5 g/L FA significantly improved lettuce growth in shoots (79.5%) and roots (53.8%), increased chlorophyll content (60.2%), P.I. (45.3%), photosynthesis (39.9%) and reduced electrolyte leakage (35.0%) in comparison with lettuce exposed to Cd alone. Therefore, we used a treatment of 0.5 g/L FA to investigate the role of FA in the mitigation of Cd-induced lettuce seedling growth inhibition.
3.5. Antioxidant enzymes The antioxidant enzyme activities in shoots and roots are presented in Fig. 5. Superoxide dismutase (SOD) and peroxidase (POD) activities increased by 53.6% (29.8%) and 69.65% (53.3%), respectively, in shoots (roots) under Cd stress. FA application resulted in a decline in shoot SOD and POD activities, to varying degrees, compared with Cd stress alone, whereas there were no significant changes in root SOD or POD activity (Fig. 5A, B). Cd stress induced significant decreases in shoot catalase (CAT) and ascorbate peroxidase (APX) activities, which were enhanced by FA foliar spraying (Fig. 5C, D).
3.2. Chlorophyll a fluorescence 3.6. Effects of FA on Cd and elemental nutrient uptake by lettuce As shown in Fig. 2A, fluorescence intensity significantly decreased in lettuce leaves exposed to Cd for 2 weeks, and this effect was mitigated by foliar spraying with FA. We observed significant changes in the OJIP curve under different treatments, indicating that lettuce was sensitive to Cd (Fig. 2B). We observed an increase in ΔVJ and ΔVI after treatment with Cd; however, these two parameters were lower following co-treatment with Cd and FA than they were with Cd alone (Fig. 2C). JIP-test parameters from chlorophyll fluorescence transient are presented in Fig. 2D. We observed that dVG/dto, dV/dto, and WK increased to various degrees in lettuce exposed to Cd compared with the control; this increase was inhibited by FA application. φEo and φRo values increased to various degrees under Cd and FA co-treatment
To determine the effects of foliar spraying of FA on Cd accumulation in shoots and roots, we evaluated the Cd content of shoot and root tissues. Cd concentrations were approximately 0.31 mg (g–1 dry weight) in shoots and roots (Fig. 6). After FA foliar spraying, the Cd concentration in plants declined. A significant decrease in Cd content in roots and shoots was observed following the application of 0.5 g/L FA, by approximately 29.5% (Figs. 6A) and 37.9% (Fig. 6B), respectively. The impacts of FA application on elemental nutrient uptake by lettuce exposed to Cd are shown in Table 1. Cd stress caused a significant reduction in iron (Fe), zinc (Zn), and manganese (Mn) uptake in shoots, by 84.2%, 30.1%, and 44.1%, respectively. FA application significantly increased the accumulation of these nutrients in lettuce, 13
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Fig. 2. Effects of FA on fast chlorophyll fluorescence curves of lettuce leaves under Cd stress. The fluorescence intensity of original fluorescence kinetic curve (A). The normalized fluorescence data is expressed by "Vt", Vt = (Ft-Fo)/(Fm-Fo) (Ft is the fluorescence value at any time), which is normalized with Fm-Fo (B); The difference of relative chlorophyll a fluorescence of control and treatments (C). Effects of FA on fast chlorophyll fluorescence parameters of lettuce leaves under Cd stress (D).
promoted nutrients uptake slightly but not significantly in roots.
but had no effect on Cu accumulation in shoots. Similar results of the nutrients changes were found in roots. The level of Fe, Zn and Mn accumulation in root under Cd stress respectively declined by 22.4%, 73.7% and 87.2%, respectively. while the level of Cu accumulation in roots is raised when exposed to cadmium. Exogenous FA application
4. Discussion FA is a soluble micromolecular substance and the major constituent 14
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Fig. 3. Effects of FA on chlorophyll and Carotenoid contents (A), gas exchange parameters (B, C, D) of lettuce leaves under Cd stress. Data are shown as means ± SE of five replicates. Treatments conditions are described in Fig. 4. Bars marked by different letters indicate significant differences by Tukey's test (P ≤ 0.05).
2013). In the present study, Cd stress caused Cd accumulation in lettuce. However, foliar application of FA decreased Cd accumulation in lettuce shoots and roots, with the greatest reduction in Cd seen at 0.5 g/ L FA. We therefore selected this FA concentration for further study.
of humic substances (Weng et al., 2006). It has been widely demonstrated that FA can enhance plant tolerance to biotic and abiotic stresses, including drought, water, and salinity stress (Anjum et al., 2011, Aydin et al., 2012, Selim et al., 2012). However, in recent years, there have been fewer reports of Cd stress tolerance enhancement by FA application. In the current study, we elucidated the possible mechanism underlying photosynthetic apparatus defense, reduced oxidative stress, decreased Cd accumulation, and elemental nutrient uptake by FA.
4.2. FA protected photosynthetic apparatus from Cd toxicity It is well-known that photosynthesis is sensitive to Cd stress (Zhang et al., 2015). Consistent with past findings, the present study showed that electron transfer in lettuce was significantly inhibited by Cd stress, as indicated by the increase in △Vj and △Vi. In fact, the reduction of efficiency of excitation energy capture (ψEo) led to the decrease of the quantum yield of PSII electron transport (φEo). In order to consume too much-activated electrons, the quantum yield of dissipated energy (φDo) increased. However, following FA application, these parameters were nearly comparable to the control; this result demonstrated that FA enhanced electron transport capacity on the receptor side of photosystem II (PSII). Wk is a special indicator that values the degree of OEC impaired by stress (Appenroth et al., 2001). After FA application, we found Wk was decreased under Cd stress, suggesting that FA protected OEC from Cd toxicity. Our results also showed that Cd toxicity reduced ABS/CS, TRo/CS, ETo/CS and DIo/CS parameters, possibly due to the degradation or inaction of reaction centers and destruction of antenna pigments, causing a decrease in the trapped energy (TRo/CS) and reduction energy (ET0/CS) of reaction centers (Appenroth et al., 2001). The increase of DIo/CS indicated that the reaction centers start the defense mechanism (heat dissipation) in response to Cd stress. PI (abs) and PI (total), a measure of plant photosynthetic performance, is sensitive to Cd stress (Fig. 2D). Our results indicate that FA protected PSII from Cd toxicity by increasing reaction center density (RC/CS), absorption flux (ABS/CS), trapped energy flux (TR0/CS), electron transport flux (ETo/CS)/dissipated energy flux (DIo/CS) per excited cross
4.1. Cd toxicity was dose-dependently ameliorated by FA The major phenotypes affected by Cd toxicity in plants are biomass reduction and leaf chlorosis (Das et al., 1997). In the present study, we observed that both of these phenotypes were inhibited as a result of Cd stress, which could be explained by a decline in chlorophyll content, performance index of PSII (P.I.), photosynthesis. Moreover, electrolyte leakage (EL), an indicator of membrane permeability also was altered by Cd stress. Various FA treatments had positive effects on these changes caused by Cd toxicity, with the optimal effect being produced by a moderate concentration (0.5 g/L). Thus, FA mitigated Cd stress in lettuce in a dose-dependent manner. The similar results was found in previous research that excess supply of Cd caused growth inhibition, increased Cd accumulation and the accumulation of ROS in lettuce, however, foliar application of exogenous NO alleviated Cd-induced growth suppression, promoted the chlorophyll synthesis and improved photosynthetic efficiency (Xu et al., 2014). Similar effect was also found in wheat grown in calcareous conditions (Katkat et al., 2009). Horuz et al. (2015) revealed that humic acid applied in soil increased the dry matter and reduced Cd uptake in lettuce grown in the soil contaminated with Cd. Another research also shown that 100 mg/L humic acid applied in soil increased growth and fresh weight of lettuce leaves and reduced Cd concentration in lettuce leaves (Haghighi et al., 15
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Fig. 4. The in situ detection of H2O2 (A) and O2·– (B), Bar= 1 cm. Effects of FA on H2O2 content (C), superoxide radical production (D) and thiobarbituric acid reactive substance(TBARS) content (E) in shoots and roots of lettuce. Treatment conditions are described in Fig. 4. Data are shown as means ± SE of five replicates. Bars marked by different letters indicate significant differences by Tukey's test (P ≤ 0.05).
apparatus (Siedlecka et al., 1998). Therefore, we speculate that FA enhanced photosynthesis in lettuce under Cd stress by increasing chlorophyll content, thus improving PSII efficiency.
section. Therefore, FA had clear effects in improving PI (ABS), PI (total) in lettuce under Cd stress. The observed decrease in Pn may be attributed to stomatal closure; hence, CO2 diffusion was limited (Fig. 3B). Meanwhile, the reduction of Pn may as result of non-stomatal limitation (Monteiro et al., 2009). We also observed that Cd caused a significant decrease in chlorophyll content. The same results were observed in the previous study that chlorophylls and carotenoids concentrations in leaves of lettuce were decreased as result of the Cd stress (Xu et al., 2014). It is possible that Cd can replace the magnesium atom (Mg) within chlorophyll to form a chlorophyll–Cd complex, causing damage to the photosynthetic
4.3. FA reduced ROS accumulation and enhanced antioxidant enzyme capacity It has been reported that Cd might cause oxidative stress by generating excess ROS (Cho and Seo, 2005). As the duration of Cd treatment increases, so too does the malondialdehyde (MDA) content in lettuce leaves (Monteiro et al., 2009). Another study has shown that Cd 16
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Fig. 5. Effects of FA on the activities of SOD (A), POD (B), CAT (C) and APX (D) in leaves and roots of lettuce seedlings. Data are shown as means ± SE of five replicates. Bars marked by different letters indicate significant differences by Tukey's test (P ≤ 0.05).
stress increased H2O2 and O2.− accumulation and induced an increase in MDA content in root and leaves of lettuce seedlings (Xu et al., 2015). We observed similar results in the present study, such that TBARS, H2O2 and O2.− content significantly increased in lettuce seedling shoots (Zouari et al., 2016). Generally, higher plants have developed a defense system to overcome oxidative stress caused by Cd toxicity. In the current study, SOD and POD activities significantly increased in lettuce under Cd stress, whereas those of APX and CAT declined. However, FA and Cd co-treatment enhanced APX and CAT activities, such that the two enzymes could effectively scavenge H2O2 in plant tissues. This result is also confirmed in wheat, where the FA improved the activities of APX and CAT under Cr stress (Ali et al., 2015). In contrast, the activities of SOD and POD were reduced after treatment with FA. This result suggests that FA may serve directly as an antioxidant to eliminate
excess ROS, or as a signal molecule to promote antioxidant production. A similar report indicated that FA exhibited auxin-like activity (Nardi et al., 2002). Moreover, a remarkable increase in carotenoid content was observed following FA treatment compared with Cd stress alone. It has previously been reported that ROS led to impaired chlorophyll and PSII function, and that carotenoid acts as an antioxidant, a first line of defense for plants against ROS (Zhang et al., 2015). Therefore, we speculate that FA enhanced photosynthesis and growth in lettuce under Cd stress by reducing ROS accumulation. 4.4. FA decreased Cd content and facilitated elemental nutrient uptake in lettuce exposed to Cd stress Previous studies showed that Cd impaired macronutrient balance
Fig. 6. Effects of different concentrations of FA on Cd accumulation in shoot (A) and root (B) of lettuce under Cd stress. Data are shown as means ± SE of five replicates. Bars marked by different letters indicate significant differences by Tukey's test (P ≤ 0.05). ND: not detected. 17
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Table 1 Effects of FA on nutrient elements uptake by lettuce under Cd stress. Treatments
Elements content (mg g−1 DW) Fe
Shoot Con Con+FA Cd Cd+FA Root Con Con+FA Cd Cd+FA
Zn
Cu
Mn
0.074 0.112 0.012 0.040
± ± ± ±
0.018b 0.029a 0.010d 0.002c
0.080 0.085 0.056 0.073
± ± ± ±
0.008a 0.010a 0.005b 0.021 BCE
0.016 0.016 0.016 0.013
± ± ± ±
0.002a 0.002a 0.002a 0.001a
0.092 0.099 0.052 0.073
± ± ± ±
0.013ab 0.015a 0.001c 0.010b
4.179 4.121 3.242 3.840
± ± ± ±
0.228a 0.757a 0.333b 0.114ab
0.276 0.492 0.072 0.119
± ± ± ±
0.144b 0.134a 0.016c 0.014 BCE
0.049 0.049 0.061 0.060
± ± ± ±
0.007b 0.001b 0.006a 0.004a
0.390 0.542 0.050 0.071
± ± ± ±
0.057a 0.083b 0.010c 0.006c
Treatments conditions are described in Fig. 4. Data are shown as means ± SE of five replicates. Bars marked by different letters indicate significant differences by Tukey's test (P ≤ 0.05).
Appendix A. Supporting information
and accumulation in lettuce (Monteiro et al., 2009; Matraszek et al., 2016; Rizwan et al., 2017). Similar results were observed in the present study, in which Cd stress caused an imbalance of mineral elements in lettuce. Fe content significantly declined in lettuce shoots and roots. Zn and Cu contents were reduced to different degrees in lettuce subjected to Cd stress, perhaps due to the accumulation of Cd in shoots and roots. FA application increased the Fe and Zn contents in lettuce shoots under Cd stress, but had little effect in roots. It is worth noting that FA had a more remarkable effect on Fe uptake than other nutrient elements, and that it improved Fe accumulation to a greater degree in shoots than in roots. Cd stress disturbed the balance of mineral elements may attribute to two ways: (1) inhibition of Fe chelation and loading to xylem and (2) the competition between nutrients and Cd for the same transporters (Tran and Popova, 2013). We found that foliar application of FA significantly decreased Cd accumulation in shoots and roots of lettuce exposed to cadmium. Therefore, we speculate that FA could inhibit Cd uptake and improve Fe translocation from roots to shoots in lettuce.
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5. Conclusion We found that Cd exposure have deleterious effect on lettuce, mainly reflecting in the reduction of growth, photosynthetic capacity, ROS accumulation, inhibition of mineral element uptake. Foliar application of FA plays an important role in protecting the photosynthetic apparatus from Cd toxicity. FA inhibited ROS accumulation in plants exposed to Cd by stimulating the antioxidant system, and enhanced the photosynthetic capacity of lettuce by protecting PSII against Cd toxicity, accelerating chlorophyll biosynthesis and promoting translocation of the elemental nutrients from roots to shoots. This effect was especially clear in elements involved in photosynthesis, such as Fe, Zn, and Mn. The ability of FA to alleviate Cd toxicity in lettuce could be due to elimination of excess ROS and suppression of Cd uptake. However, to fully elucidate the role of FA in lettuce Cd-stress response, more detailed molecular analyses will be needed. To sum up, FA application is an effective detoxification method for lettuce under Cd stress and could be explored in phytoremediation.
Acknowledgment This work was supported by National key research and development program of China (2016YFD0800703, SQ2018YFD080066). The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/Nt7iKo
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