Introduction

Cadmium(Cd) is a toxic metal pollutant and can enter soil-plant ecology system due to discharge of industrial waste water, the excessive use of phosphate fertilizer and atmospheric sedimentation. Cadmium is not an essential element for plant growth, but it is easily taken up by plant roots and transported to above-ground tissues (Shamsi et al. 2008). High concentration of Cd is strongly phytotoxic and inhibits plant growth (Meng et al. 2009). Cadmium toxicity may be ascribed to the deterioration of many physiological processes, such as the reduction in intercellular spaces and the number of chloroplasts (Sandalio et al. 2001), inhibition of chlorophyll synthesis and photosynthesis (Hsu and Kao 2007), and generation of free radicals and reactive oxygen species (ROS) (Milone et al. 2003; Hassan et al. 2008). At high ROS concentrations, ROS promotes oxidative stress and triggers signaling associated with cell death (Alvarez et al. 1998). To avoid ROS-caused oxidative damage, plants have evolved protective mechanisms of mitigating and repairing the ROS damages (Edreva 2005). The ROS-scavenging system includes an enzymatic system consisting of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and glutathione reductase (GR), and a non-enzymatic system consisting of reduced glutathione (GSH) and ascorbic acid (Yin et al. 2008). SOD catalyzes dismutation of superoxide anions to H2O2 which is subsequently catalyzed by CAT and POD to water and O2.

Under normal physiological conditions, plants keep the balance between ROS production and quenching activity of antioxidants. However, under environmental stress, the balance is upset, often resulting in oxidative damage (Weckx and Clijsters 1997). It is important to develop practical techniques for alleviating the Cd stress. Hassan et al (2008) reported that nitrogen affected Cd uptake and accumulation in rice. The application of sulfur helped reducing Cd toxicity in mustard (Anjum et al. 2008). Some species or genotypes such as durum wheat (Penner et al. 1995), rice (Cheng et al. 2008) and barley (Wu and Zhang 2004) are Cd tolerant and are used for phytoremediation as they can accumulate high amounts of heavy metals. Alternatively, crops that do not accumulate Cd in grains or fruits can be planted on Cd-contaminant soil (DalCorso et al. 2008). Hydrogen peroxide (H2O2) pretreatment alleviated Cd toxicity in rice seedlings (Hu et al. 2009). Plant growth regulators, such as jasmonic acid (JA), abscisic acid (ABA), gibberellins (GA) and salicylic acid (SA) can also alleviate Cd toxicity (Guo et al. 2007; Meng et al. 2009; Metwally et al. 2003).

Biologically active oligosaccharides act as signal molecule, regulating growth and development as well as defense mechanisms in plants by regulating gene expression (Albersheim and Darvill 1985). Alginate-derived oligosaccharides (ADO) with 4-deoxy-Lerythro-hex-4-ene pyranosyluronate structures function as endogenous elicitors (Akimoto et al. 1999). Many researchers have reported that ADO enhances seed germination, shoot elongation, and root growth (Hu et al. 2004; Natsume et al. 1994). Akimoto et al (1999) had reported that ADO also promoted the production of certain enzymes. However, no information is available about effects of ADO on wheat seedlings stressed by Cd. Therefore, in the current research, we investigated the growth, chlorophyll content, photosynthetic rate, lipid peroxidation and the activities of some antioxidant enzymes (SOD, POD and CAT) in Cd stressed wheat seedlings.

Materials and methods

Plant culture and treatments

The experiments were carried out with wheat (Triticum aestivum L. Liaochun10) seeds, surface sterilized in 2% sodium hypochlorite solution for 10 min and subsequently triple-rinsed in sterile distilled water. Seeds were soaked for 5 h either in 1,000 mg L−1 ADO (supplied by Ocean University of China) solution or in distilled water. A preliminary study showed that 1,000 mg L−1 ADO solution was optimum for enhancing Cd tolerance in wheat seedlings. Plants were grown hydroponically on Hoagland’s nutrient solution in a growth chamber at a day/night cycle of 12 h/12 h, at 25°C/20°C, respectively, a relative humidity 65% and a light intensity of 800 μmol m−2 s−1. When the second leaf of wheat seedlings was fully expanded, CdCl2 was added into the solution to obtain two Cd levels, 0 and 100 μM. Hence, four treatments were established, including a control (neither ADO nor Cd-stressed), Cd stressed, ADO treated and ADO-Cd stressed (pretreatment with 1,000 mg L−1 ADO and then 100 μM Cd stressed). There were three replications for each treatment. The nutrient solution was renewed every 3 days.

Growth parameters

Plants were harvested after 10 day of Cd exposure. Shoot and root lengths and fresh weight were determined immediately after harvesting. Samples were oven-dried at 105°C for 30 min and kept at 70–80°C for 24 h to obtain a constant dry weight. The data were obtained from three replicates (10 plants in each).

Chlorophyll content and photosynthetic rate

After 10 day of Cd exposure, the second fresh leaves were collected for the determination of photosynthetic pigments. Samples (0.1 g) were extracted in 10 ml 80% acetone and ethanol (v/v = 1:1) for 24 h under dark conditions. The absorbance of the supernatant was recorded at 645 (A645) and 663 (A663) nm, respectively. Photosynthesis rate (P n ) was measured with a portable photosynthesis system (LI-6400, Lincoln, NE, USA) in the open circuit mode. Atmospheric conditions consisted of photosynthetic photon flux density of 800 ± 50 μmol CO2 m−2 s−1 and CO2 concentration of 400 μmol CO2 mol−1 air. Chlorophyll measurements were taken for three independent experiments.

Antioxidant enzyme activities and lipid peroxidation

After 5 and 10 days of Cd exposure, enzymes were extracted at 4°C from about 0.5 g tissue from the second leaves and the roots, using a mortar and pestle, with 5 ml of extraction buffer, containing 0.1 M phosphate buffer (pH 7.8), 0.1 mM EDTA, 1 g PVP. Extracts were centrifuged at 10,000 × g for 15 min, and the supernatants were used for the assays. SOD activity was assayed by the inhibition of the photochemical reduction of β-nitro blue tretrazolium chloride (NBT). One unit of SOD was defined as the amount of enzyme, which produced a 50% inhibition of NBT reduction at 560 nm (Costa et al. 2002). CAT activity was determined by the decomposition of H2O2 that was followed by the decline in absorbance at 240 nm (Cakmak and Horst 1991). POD activity was measured with guaiacol as the substrate. Increase in the absorbance due to oxidation of guaiacol was measured at 470 nm. Protein concentration was estimated by the method of Lowry et al. (1951) using bovine albumin as standard. MDA content was determined as 2-thiobarbituric acid (TBA) reactive metabolites according to Heath and Packer (1968). The level of lipid peroxidation was measured as nmol g−1 fresh weight by using an extinction coefficient of 155 mM cm−1. The results presented are the mean of three independent experiments.

Statistical analysis

Differences among treatments were analyzed taking P < 0.05 as significance according to Duncan’s multiple range test. Statistical procedures were carried out with the software package SPSS11.0 for windows.

Results

Plant growth and biomass accumulation

Shoot and root lengths and biomass were significantly inhibited under cadmium exposure (P < 0.05) (Fig. 1). However, growth inhibition was considerably alleviated (P < 0.05) when plants were pretreated by 1,000 mg L−1 ADO before Cd stress. A similar trend was also observed for fresh (Fig. 1c) and dry weight (Fig. 1d). The alleviation of root growth was more evident than that of shoot growth.

Fig. 1
figure 1

Effect of ADO pretreatment on growth parameters of wheat seedlings. The data of shoot (a) and root (b) length as well as fresh (c) and dry (d) weight are mean ± SE from three independent experiments. Different letters indicate significant differences at P < 0.05. CK: plants were grown in neither ADO nor Cd; Cd: plants were stressed by 100 μM CdCl2; ADO: plants were pretreated with 1,000 mg L−1 ADO; ADO-Cd: plants were pretreated with 1,000 mg L−1 ADO and then stressed by 100 μM CdCl2

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Chlorophyll content and photosynthetic rate

Under Cd stress, Chl-a and Chl-b (Fig. 2) content were significantly reduced (P < 0.05). The application of 1,000 mg L−1 ADO resulted in an increase of the Chl-a and Chl-b contents of plants grown either in the 0 μM Cd control or in the 100 μM Cd medium. The Chl-a content increase was more apparent than that of Chl-b. Under Cd stress, photosynthetic rate was significantly reduced (P < 0.05) compared to the control (Fig. 3). However, ADO had a significant (P < 0.05) effect on the photosynthetic rate of plants under Cd stress, where ADO increased the photosynthetic rate (Fig. 3).

Fig. 2
figure 2

Effect of ADO pretreatment on Chlorophyll content of wheat leaves. Each value represents the mean ± SE. Different letters indicate significant differences at P < 0.05. CK: plants were grown in neither ADO nor Cd; Cd: plants were stressed by 100 μM CdCl2; ADO: plants were pretreated with 1,000 mg L−1 ADO; ADO-Cd : plants were pretreated with 1,000 mg L−1 ADO and then stressed by 100 μM CdCl2

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Fig. 3
figure 3

Effect of ADO pretreatment on photosynthetic rate of wheat leaves. Each value represents the mean ± SE. Different letters indicate significant differences at P < 0.05. CK: plants were grown in neither ADO nor Cd; Cd : plants were stressed by 100 μM CdCl2; ADO: plants were pretreated with 1,000 mg L−1 ADO; ADO-Cd : plants were pretreated with 1,000 mg L−1 ADO and then stressed by 100 μM CdCl2

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Activities of antioxidant enzymes

Cadmium (Cd) stress induced higher SOD activity in leaves and roots than that in the control (Fig. 4a, b). SOD activity was higher in the leaves after 10 days Cd stress compared to the SOD activity after 5 days Cd stress. However, SOD activity in roots showed the opposite trend. Pretreatment with ADO further resulted in an increase of SOD activities in leaves and roots both in the control and in Cd stressed plants.

Fig. 4
figure 4

Effect of ADO pretreatment on activities of SOD (leaf, a; root, b), POD (leaf, c; root, d), CAT (leaf, e; root, f). Each value represents the mean ± SE. CK: plants were grown in neither ADO nor Cd; Cd : plants were stressed by 100 μM CdCl2; ADO: plants were pretreated with 1,000 mg L−1 ADO; ADO-Cd : plants were pretreated with 1,000 mg L−1 ADO and then stressed by 100 μM CdCl2

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Cadmium (Cd) stressed plants had higher POD activity in leaves and roots than the control (Fig. 4c, d). Under ADO-Cd stress, POD activity in leaves and roots increased significantly. Furthermore, POD activity increased with increasing ADO-Cd stress time.

When comparing the Cd stressed plants with the control, there was obvious decrease in leaf CAT, while it showed no significant difference on root CAT activity (Fig. 4e, f). CAT activity was increased (P < 0.05) when seeds were soaked with 1,000 mg L−1 ADO.

Lipid peroxidation

Lipid peroxidation levels in leaves and roots of wheat plants were determined as the content of MDA. MDA content in leaves (Fig. 5a) and in roots (Fig. 5b) was increased significantly to nearly double responding to 100 μM Cd stressed without ADO pretreatment. MDA content of ADO pretreated plants decreased in both the control and Cd stressed plants. In comparison with Cd stressed plants, MDA content in leaves and roots of ADO-Cd stress decreased significantly.

Fig. 5
figure 5

Effect of ADO pretreatment on MDA content (leaf, a; root, b). Each value represents the mean ± SE. CK: plants were grown in neither ADO nor Cd; Cd : plants were stressed by 100 μM CdCl2; ADO: plants were pretreated with 1,000 mg L−1 ADO; ADO-Cd : plants were pretreated with 1,000 mg L−1 ADO and then stressed by 100 μM CdCl2

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Discussion

In the present study, the growth parameters of Cd stressed plants which were indicated by the height of seedling, the root length, fresh weight and dry weight, were significantly reduced compared with the control (Fig. 1). This is in agreement with the literature reports (Kumar et al. 2008). However, these parameters increased markedly when seeds were soaked in 1,000 mg L−1 ADO solution before Cd stress. Growth increase is clearly ascribed to the promotion of carbon fixation as reflected by chlorophyll content (Fig. 2) and photosynthetic rate (Fig. 3). The same results had been reported by Tomoda et al. (1994) in barley.

Accumulation of active oxygen species (AOS), including superoxide radical (O ·−2 ), hydroxyl radical (·OH) and hydrogen peroxide (H2O2) are the major reason of Cd injury to plants, leading to the occurrence of oxidative stress (Romero-Puertas et al. 2004; Shah et al. 2001). In general, plant cells may develop the defense mechanisms against AOS. One of the protective mechanisms is the enzymatic antioxidant system, which involves the sequential and simultaneous action of a number of enzymes. SOD, POD and CAT are major enzymes used to demonstrate the status of antioxidant capacity. SOD is a key enzyme which catalyzes dismutation of superoxide anions to H2O2. In the present study, SOD activity increased in wheat leaves (Fig. 4a) but decreased in roots (Fig. 4b) when under Cd stress. This result is in accordance with that of Molina et al. (2008), but differs from the report of Hu et al. (2009). Shah et al. (2001) reported that increased SOD activity protected the plants from oxidative damage. Produced H2O2 is subsequently scavenged by CAT which is a key enzyme for the defense responses against oxidative stress (DalCorso et al. 2008). In the present study, CAT activity decreased in Cd stressed leaves and roots (Fig. 4e, f). POD also eliminates H2O2. POD activity increased both in leaves and in roots of Cd stressed plants (Fig. 4c, d). MDA, which is a peroxidation product of plant cell membrane exposed to stress, is an indicator of lipid peroxidation and demonstrates the extent of oxidative stress in plants (Chaoui et al. 1997). The results of the present study indicated that MDA content of Cd stressed plants significantly increased in comparison with that of the control, indicating the existence of oxidative stress (Fig. 5). Plant growth was eventually inhibited (Fig. 1).

The adverse symptoms caused by Cd can be partially corrected by applying exogenous chemicals (Anjum et al. 2008; Meng et al. 2009) and by phytoremediation (Cheng et al. 2008). So, the plant-biotechnology research has great implications both from theoretical and practical perspectives. In the present study, we firstly used wheat to show that ADO application markedly ameliorated Cd-caused plant growth inhibition. Similar to plant growth regulators (PGRs) which were used to improve plant defense by acting as a signaling molecule, ADO resembles an endogenous elicitor that functions as a signal to trigger the synthesis of the enzymes and to activate stress-response gene expression. When seeds were soaked in 1,000 mg L−1 ADO before Cd stress, SOD, POD and CAT activity was significantly increased (Fig. 4), and MDA content (Fig. 5) showed a remarked reduction in leaves and roots of the seedling. This showed that the oxidative stress caused by Cd addition may be alleviated, thus allowing the plants to adapt by actively increasing antioxidant enzyme activity and decreasing MDA content.

Taken together, our study confirmed the positive effects of ADO in alleviating Cd toxicity, which were reflected by increased fresh weight, dry weight, Chlorophyll content and photosynthetic rate, higher antioxidant enzyme activities and less MDA content in leaves and roots.