Plant material and experimental designs

A pot experiment was undertaken in growth season in the greenhouse of Taiyuan University of Science and Technology, Taiyuan, China, using the common cultivated cucumber ˈJinyan 4ˈ. Soil was collected from the experimental farm (depth 0–15 cm) in the campus. Air-dried soil was sieved (the mesh diameter is 1 cm) and 4.5 kg of the soil was loaded in a plastic pot (5 L, 20 cm height). The soil in each pot was fertilized with 1 L of basal nutrient solution (BNS). The composition of the BNS was described in Sun et al. (2013). Cd (as CdCl₂) and Se (as Na₂SeO₃) were added to the corresponding pots to form four treatments: (1) control, BNS; (2) Se, BNS+6 µM Se; (3) Cd, BNS+100 µM Cd; and (4) Cd+Se, BNS+100 µM Cd+6 µM Se. There were 3 replicates for each treatment. Healthy cucumber seeds were sown in the above mentioned pots 20 days after application of BNS, Cd and Se. One week after emergence, each pot was thinned to four seedlings. After one month of treatment, plants from both control and treatments were sampled for physiological parameters measurements, and biochemical analysis.

Chlorophyll contents, photosynthesis and chlorophyll fluorescence parameters analysis

The topmost fully expanded leaves from three random plants of each treatment were sampled for measurement of chlorophyll content. Chl was extracted with 80% acetone, analyzed on a spectrophotometer (UV-1750, Shimadzu, Japan) according to the method of Lichtenthale & Wellburn (1983). The net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and intercellular CO₂ concentration (Ci) were measured using a LI-6400 portable photosynthesis system (LI-COR, Lincoln, NE, USA). The chlorophyll fluorescence parameters were measured with an IMAGINGPAM (chlorophyll fluorometer) system (Zhang et al., 2015).

Plant growth, Cd and other microelement determination

After one month of treatment, plants were uprooted and separated into roots, stems and leaves, and plant height of each plant was measured. Roots were soaked in 20 mM Na₂EDTA for 20 min to eliminate adsorbed ions and possible chemical contamination on surfaces and then rinsed in deionized water; then all samples were dried at 70°C until constant weight, and weighed, respectively. Dried samples were powdered and digested in acid mixture (HNO₃:HClO₄, 5:1, v/v). Cd, Zn, Mn and Cu concentrations were determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (SPS 1200 VR, Seiko Co., Ltd., Japan).

Carbohydrate contents measurements

Carbohydrates were extracted from 0.2 g of freeze-dried samples with 50 mL of 80% ethanol (v/v); the supernatant was used for analysis of hexose, sucrose, and total soluble sugars using a modified phenolsulfuric acid method (Buysse & Merckx, 1993). The residue was boiled for 3 hours in 10 mL of 2% HCl (v/v) to hydrolyze starch, and the supernatant was analyzed for soluble sugars released from starch by acid hydrolysis (Yu et al., 2004).

·OH, H₂O₂ and lipid peroxidation determination

The hydroxyl free radical (·OH) and H₂O₂ contents were determined using a ·OH detection kit and a hydrogen peroxide assay kit, respectively (Liu et al., 2015). The level of lipid peroxidation was quantitated by the amount of malondialdehyde (MDA) which was determined by thiobarbituric acid reaction in all treatments (Wu et al., 2003).

Determination of ATPase activities

The frozen cucumber tissues (0.4 g) were homogenized in 8 mL of 50 mM phosphate buffer saline (pH 7.8) using a pre-chilled mortar and pestle; the homogenate was centrifuged for 15 min at 10000 × g at 4 °C, then the supernatant was used to assays of activities of enzymes. ATPase activities were determined by measuring the release of inorganic phosphate (Pi) using the activity assay kit (Jiancheng Bioengineering Institutes, China).

Statistical analysis

Each experiment was repeated three times. All statistical analyses were carried out with Data Processing System (DPS) statistical software package using ANOVA, followed by Duncan’s Multiple Range Test (SSR) with statistical significance of p ≤ 0.05.

Cd accumulation in cucumber seedlings under Cd and Se application

Cadmium concentrations of all tissues are shown in Table 1. In control condition, Cd concentration was lower than 0.1 mg/kg DW, and it was within the safe range. Cd treatment positively affected the cucumber seedlings Cd concentration in all tissues, especially in the roots (Table 1). Contrary to Cd-alone treatment, application of Se negatively affected the Cd concentration, and significantly decreased by 42.8%, 25.7% and 23.5% in leaves, stems and roots, respectively.

Table 1. Effect of external Se addition on microelement concentration in leaves, stems and roots of cucumber seedlings exposed to Cd for one month.

Alleviatory effect of exogenous Se on plant growth, chlorophyll content, photosynthetic and chlorophyll fluorescence parameters under Cd stress

Under non-stress conditions, Se addition had no significant effect on the growth of cucumber plants (Fig. 1). Cd treatment significantly decreased plant height, root, stem and leaf DW in cucumber seedlings. Moreover, under Cd treatment, Se addition (Cd+Se) significantly increased these parameters by 20.3%, 61.3%, 34.7% and 26.2%, respectively, and improved plant growth. In addition, compared with control, Cd treatment significantly decreased both chlorophyll a and b content, also chlorophyll a+b and a/b ratio. Significantly, plants for Cd+Se treatment showed higher chlorophyll a and b content than those for Cd treatment alone. Also, the addition of Se (Cd+Se) greatly alleviated Cd-induced reductions in chlorophyll a+b, which were 25.4% higher than those for Cd alone-treated plants (Fig. 2).

Exposure to Cd stress significantly depressed the values of Pn, Tr and Gs by 44.9%, 39.1% and 27.6% respectively, compared to the control (Fig. 3). The addition of Se (Cd+Se) greatly alleviated the inhibitory effect of Cd toxicity in photosynthetic parameters, such as Pn increased by 39.8% compared with Cd stress merely (Fig. 3a). Furthermore, the Ci increased significantly after Cd treatment, while decreased after addition of Se (Fig. 3d). And there were no significant differences between plants treated with Se alone and the control plants (Fig. 3).

The maximum efficiency of photosystem II photochemistry (Fv/Fm) decreased under Cd stress; however, initial fluorescence (F₀) increased significantly. Addition of Se improved Fv/Fm, and distinctly suppressed Cd-induced increase in the F₀ by 22.5% (Fig. 4a,b). There were no significant changes between Se alone and the control plants. With respect to quantum yield of non-regulated energy dissipation Y(NO), there was no significant difference among the four treatments (Fig. 4c).

Effect of Se and Cd on micro-elements

Compared with control, Cd treatment significantly decreased Zn concentration in root, and Mn concentration in leaf, stem and root. Moreover, these reductions in mineral concentrations were lower in plants in the Cd+Se treatment than Cd-alone treatment, and exogenous Se distinctly brought root Zn and leaf Mn concentration back to the control level. Such as, leaf Mn and root Zn concentration under Cd+Se was 169.5% and 19.7 higher than those under Cd alone treatment. With respect to the stems and roots Mn, the concentrations were intermediate between those of control and Cd treated plants (Table 1).

On the other side, plants under Cd treatment showed higher Zn concentration in leaf and stem, and Cu concentration in all tissues (leaf, stem and root) than those under control. However, exogenous Se (Cd+Se) significantly suppressed Cd-induced increase in stem Zn, leaf, stem and root Cu concentration, e.g. Cu concentrations in Cd+Se being 37.9%, 26.4% and 13.6% in leaves, stems and roots, respectively, lower than the Cd alone treatment (Table 1).

Effect of Se and Cd on carbohydrate contents

Cadmium treatment significantly increased total soluble sugar, sucrose and hexose contents. The change of sucrose was the most significant; the sucrose content for Cd treated plants were 49.1%, 57.9% and 84.8% higher than those for control plants in leaves, stems and roots, respectively (Table 2). Furthermore, the combination of Se and Cd completely inhibited the induction of these three parameters by Cd alone, and brought them back to the control level except for the root hexose which was higher than the control. In terms of starch contents, root starch under Cd treatment was 39.6% lower than control, while exogenous Se markedly increased the content by 23.8% compared with Cd-alone treatment. However, no significant difference was detected in leaf and stem starch among the four treatments (Table 2).

Table 2. Effect of external Se addition on carbohydrate contents (mg/g dry weight) in leaves, stems and roots of cucumber seedlings exposed to Cd for one month.

Effect of Se and Cd on ·OH, H₂O₂ and lipid peroxidation contents

A distinct increase in the accumulation of ·OH was observed in plants under Cd exposure (Fig. 5), Se addition in 100 μM Cd markedly reduced Cd-induced ·OH accumulation, c.f. Se + Cd treatment being 38.4%, 20.4% and 16.2% in leaves, stems and roots, respectively, lower than Cd stress (Fig. 5a-c). The response of leaves and roots H₂O₂ to Cd and Se resembled to that of ·OH (Fig. 5d-f). Cd stress also increased MDA accumulation, Se addition effectively inhibited the Cd-induced MDA accumulation in shoots (c.f. 54.5 % and 29.9 lower in leaves and stems than the Cd-alone treatment, respectively), while there was no significant effect on root MDA content. In addition, no significant change in MDA content was found in seedlings treated with Se (without Cd) in comparison with control (Fig. 5g-i).

Responses of ATPase activities to Cd and Se application

Activities of ATPase under each treatment are shown in Fig. 6. There was a significant difference among different cucumber tissues, and the highest value was observed in the roots, followed by stems and leaves, simultaneously, a significant difference was also found between Cd and Se-mediated effects on ATPase activity. Cucumber seedlings treated with Cd showed significant increase in Na⁺K⁺-ATPase, being 52.1%, 30.1% and 11.1% higher than those in control. Se addition (Cd+Se) counteracted Cd-mediated increase of Na⁺K⁺-ATPase activity, giving values 23.1% in leaves, 12.0% in stems and 21.7 % in roots lower than those in Cd alone (Fig. 5a-c). Similar tendencies were shown in Ca²⁺Mg²⁺-ATPase and stem and root total-ATPase activities. However, Cd+Se and Cd alone treatment induced no significant effects on leaf total-ATPase activity in comparison with control (Fig. 6).

Exogenous Se decreases Cd accumulation

Increasing evidence has indicated that heavy metals such as Cd, As, Hg and Pb toxic symptoms to plants were alleviated by Se via decreasing their uptake (Ebbs & Leonard, 2001; He et al., 2004; Muñoz et al., 2007; Yathavakilla & Caruso, 2007; Lin et al., 2012; Sun et al., 2013; Saidi et al., 2014; Zhang et al., 2014; Liu et al., 2015). And several reports have also demonstrated that Se is poorly translocated to shoots in selenite-treated plants (Hopper & Parker, 1999); similarly, the present experiment demonstrates that most of Cd taken up by plants is accumulated in roots (Table 1). This response implies a low translocation of Cd within the cucumber plant under the Cd+Se treatment, which may be a mechanism of cucumber to defend itself toward Cd toxicity. Meanwhile, cucumber plants grown on Cd+Se medium showed the lower Cd concentration in all tissues (Table 1) in comparison with Cd treatment alone. The reduced Cd uptake might be attributable to the formation of a complex between Se and Cd which may hinder Cd uptake by root cells. Hawrylak-Nowaka et al. (2014) investigated complex formation between Cd and Se in aqueous solutions and found reduced glutathione (GSH) and phytochelatins (PCs) were formed in cucumber plants.

Se alleviates Cd-induced suppression in plant growth, chlorophyll content, photosynthetic and chlorophyll fluorescence parameters

In the present pot experiment, 6 μM Se supply to 100 μM Cd elevated Cd-induced reduction in plant growth as indicated by plant height and biomass (Fig. 1). The study by Terry et al. (2000) has demonstrated that assimilation of Se, which requires reducing power from NADPH and GSH, occurs in the chloroplast and can alter the redox state of chloroplasts, therefore affecting biomass production. Hawrylak-Nowak et al. (2014) reported that the effect of Se on the Cd-stressed plants greatly depended on the proportion of these two elements in the nutrient solution. The current results provide strong evidence that Se can effectively alleviate Cd-induced cucumber seedlings plant growth inhibition, suggesting that low micromoles of Se can exert beneficial effects on plants, although it is toxic at high concentrations (Terry et al., 2000; Lin et al., 2012; Haghighi & da Silva, 2016). On the other hand, it is known that one of the most universal symptoms of Cd stress in plants is leaf chlorosis; furthermore, the reduced pigment could result in photosynthetic capacities decrease. In the present study, chlorophyll a and b content, chlorophyll a+b and chlorophyll a/b ratio significantly decreased under Cd stress comparing with control (Fig. 2). On the other side, cucumber seedlings treated with Cd and Se together showed similar chlorophyll b content with control, the Cd-induced growth inhibition was greatly alleviated compared with those under Cd alone treatment. Meanwhile, addition of Se (Cd+Se) increased chlorophyll a, chlorophyll a+b content. Moreover, significant decrease in Pn was simultaneously accompanied by marked reduction of Tr and Gs (Fig. 3), nevertheless, the Ci level was significant higher under Cd stress compared with control. Addition of Se (Cd+Se) recovered cucumber photosynthetic ability, which might likely link to protective mechanisms maintain the integrity of the photosynthetic machinery. Similar results were reported by Haghighi & da Silva (2016), treatments Se1 (2 mg/L) and Se2 (4 mg/L) improved photosynthesis in Cd1 (5 μM) in cucumber (Cucumis sativus cv. 4200) at an early growth stage in a hydroponic system.

Recently, chlorophyll a fluorescence measurements have been used to rapidly estimate the operating quantum efficiency of electron transport through PSII in leaf plants (Feng et al., 2010), and it can be as a probe of heavy metal stress in plants, as it can also reflect photosynthetic activities in a complex manner (Mateos-Naranjo et al., 2008). In the present experiment, considerable increased Fo and reduced Fv/Fm were observed in cucumber under Cd stress. Similar results were observed in rice, soybean, tobacco and barley (Kao et al., 2003; Cai et al., 2011; Wang et al., 2011; Liu et al., 2015), however, there was no significant different in Y(NO) under Cd stress in comparison with control (Fig. 4). Generally, F₀ is also fluorescent when the reaction center of PSII is open, and the increase in F₀ under Cd stress indicates the injury of PSII (Kitajima & Butler, 1975). The decrease in Fv/Fm is a sign of photoinhibition; on the other side, the decrease of Fv/Fm under Cd stress also indicates that the photochemistry of PSII and its ability to reduce the primary acceptor Q_A are also affected by Cd; this reduction under Cd exposure may be attributed to the inhibition of chlorophyll content biosynthesis or due to an altered stoichiometry between PSI and PSII (Babani & Lichtenthaler, 1996; Ding et al., 2008). A non-considerable change of Y(NO) under Cd stress indicates that there is no severe irreversible photodamage to PSII (Kramer et al., 2004). On the contrary, addition of Se (Cd+Se) significantly enhanced Fv/Fm, but decreased Fo under Cd stress (Fig. 4a,b), indicating that cucumber plants under Cd stress could keep higher activity of reaction center by the addition of Se, the effect of Se has also been observed in Cd-treated tobacco (Liu et al., 2015). Taken together, these results suggested that Se mediated photosynthesis improvement partly because of the protection of PS II reaction centers and the increase of chlorophyll synthesis, which are important for improving Cd tolerance.

Exogenous Se counteracts Cd-induced changes in micro-elements

Absorption and distribution of essential microelements, and the interaction with Cd has been reported, although there are some contradicting results (Zhang et al., 2002; Wu et al., 2005). In present research, Cd treatment elevated leaf/stem Zn and leaf/stem/root Cu, however decreased root Zn, leaf/stem/root Mn concentration. Se addition markedly down regulated Cd induced increase in stem Zn and Cu concentration in all tissues (Table 1), which leads to mineral deficiencies and imbalance in cucumber. Se application on Cd-treated plants reversed the tendency and counterbalanced Cd induced decrease in root Zn and leaf/stem/root Mn concentration, indicating Se could mitigate Cd toxicity through balance elements metabolism in plants. The possible reason for the conflicting results could be the differences in the methods of growth, plant species, and interactions between cultivars and metals (Lin et al., 2012).

Se balances Cd-induced changes in carbohydrate contents

Photosynthesis is also limited by inorganic phosphate supply to chloroplasts, which is accompanied by triose-phosphate transport and also sucrose metabolism. Therefore, we determined the effect of Se and Cd on carbohydrate metabolism. In this study, cucumber seedlings under Cd stress had higher total soluble sugar, sucrose, hexose, especially in roots, accompanied by lower root starch as compared with those under control condition (Table 2). These results suggested that Cd might regulate carbohydrate metabolism at transcriptional levels. Application of Se resulted in the decrease of above carbohydrate contents while increase root starch. These results indicated that there is an equilibrium shift between photosynthesis and respiration as previously observed under Cd excess condition (Kováčik et al., 2011), and demonstrated that Se could increase starch accumulation, thus promotes plant growth.

Se counteracts Cd-induced changes in ·OH, H₂O₂ and MDA contents

The Cd-induced accumulation of leaf ·OH, H₂O₂ and MDA in all tissues was markedly reduced by Se addition, especially leaf O₂^•– and ·OH (Fig. 4), Se-subdued ·OH, H₂O₂ and MDA formation were supported by several recent reports (Chen et al., 2010; Lin et al., 2012; Sun et al., 2013; Liu et al., 2015), which have illustrated Se could regulate ROS production and quenching, this control in plants may be a key mechanism for counteracting environmental stress.

Se alleviates Cd-reduced ATPase activities in cucumber

ATPase could facilitate ions, organic and inorganic solutes transport in and out of the plant cells. Theoretically, preventing Cd ions from entering the cytosol through the plasma membrane is one of the best defense mechanisms (Lin et al., 2012). Duby & Boutry (2009) reported Ca²⁺-ATPase was involved in the homeostasis of Ca²⁺ signal in plant cells and was very sensitive to many abiotic stresses. In current study, Cd treatment significantly increased leaf/stem/root Na⁺K⁺-, Ca²⁺Mg²⁺- and stem/root total ATPase activities (Fig. 5). Similar results were found in Cd stressed (Cao et al., 2014b) and salt/drought-stressed barley (Ahmed et al., 2013), Cd stressed maize (Li et al., 2016). Exogenous Se mitigated these Cd-induced increased in ATPase activities, which indicated that Se ameliorated Cd toxicity by modulating ATPase activity. Nevertheless, further studies should be carried out to understand the mechanism by which Se alleviates Cd toxicity by regulating ATPase activity.

In summary, our results illustrated that exogenous Se application may protect cucumber against Cd toxicity and partially alleviate its destructive physiological effects on plants. The mechanism involved in the prevention of Cd toxicity is mainly linked to the decreased Cd concentration in leaves, stems and roots and dramatically depressed ·OH, H₂O₂ and MDA accumulation in cucumber seedlings in comparison with Cd treatment alone. In addition, the results also suggested that Se could activate protective mechanisms which can ameliorate Cd-induced reduction in chlorophyll content, Fv/Fm ratio, and repair photosynthetic machinery. Furthermore, the alleviating effect of Se was associated with improved ATPase activity, and balanced nutrients and carbohydrate contents in cucumber plants. However, detailed studies on molecular mechanism of Se-mediated tolerence to Cd stress, such as identify Cd tolerant related proteins and genes, and their post translational modification may facilitate a better understanding of the mechanisms involved in Cd-tolerence of cucumber.