УДК 581.1 The Response of Maize Seedlings to Cadmium Stress under Hydroponic Conditions1 © 2013 C. X. Wang*,**, J. Ren*** * College of Environmental Science and Engineering, Taiyuan University of Technology, Taiyuan, China ** State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China *** School of Environmental and Municipal Engineering and Institute of Environmental Ecology, Lanzhou Jiaotong University, Lanzhou, China Received February 2, 2012 The effects of cadmium (Cd) supply level in nutrient solution (0, 12.5, 25, 50, 100, 200, 400, and 800 µM) on growth, Cd accumulation ability, and the related physiological indices of maize (Zea mays L.) seedlings were studied under hydroponic conditions. The results showed that the increments in the shoot height and biomass were stimulated at relatively low external Cd supply levels (<100 µM), while they were inhibited at Cd supply levels over 200 µM. Cd accumulation ability of the maize seedlings also showed the similar stimulation/inhibition pattern as shoot growth, and the Cd contents in the shoots and roots reached the peaks (389.5 and 505.5 mg/kg dry wt, respectively) at 50 µM Cd. The contents of chlorophyll a, chlorophyll b, and carotenoids in the maize leaf blades decreased with increasing external Cd supply level. At the highest Cd supply level (800 µM), the contents of chlorophyll a, chlorophyll b, and carotenoids in the leaf blade were only 38.9, 46.0, and 29.7% of the control plants, respectively. Moreover, chlorophyll b was more sensitive to the Cd stress than chlorophyll a. The increased proline content in the leaf blade of maize seedlings resulted from external Cd stress indicates that maize can adapt to the adversity menace via changing the content of proline. ---------------------------------------- 1 This text was submitted by the authors in English. ---------------------------------------- Corresponding author: Chaoxu Wang. College of Environmental Science and Engineering Taiyuan University of Technology, No.79 West Yingze Street, Wanbailin District, Taiyuan 030024, P. R. China Keywords: Zea mays  cadmium  chlorophylls  carotenoids  proline INTRODUCTION The contamination of soils with metals is a major environmental problem throughout the world [1]. Cadmium (Cd) is a highly toxic metal, and it has been ranked number 7 among the top 20 toxins mainly due to its negative influence on the enzymatic systems of the cells [2, 3]. Large areas of land in many countries have been contaminated by Cd due to the application of sludge or urban composts, pesticides, fertilizers, emissions from waste incinerators, waste water irrigation, residues from metalliferous mining, and the metal smelting industry [4, 5]. Maize is one of the major crops cultivated throughout the world, and China is the second largest maize producer and consumer after the United States [6]. Maize may be exposed to heavy metal stress during their growing period. The accumulation of Cd in the seeds and other aboveground parts of maize became a serious problem for agriculture and human health. Cd retained in stems, leaves, and other crop parts are either used as fodder or recycled when the inedible crop residues are returned to the soil [7]. Cd excess may stimulate the formation of free radicals and ROS, perhaps resulting in oxidative stress [8, 9]. The chlorophyll content is used to assess the impact of environmental stresses on plants. Moreover, proline accumulation, accepted as an indicator of environmental stresses, is also considered to have important protective roles [10]. The objectives of this study were (i) to examine the growth response and Cd accumulation in maize seedlings at different external Cd supply levels under hydroponic conditions; (ii) to determine the effect of external Cd stress on the physiological traits, such as chlorophylls, carotenoids, and proline in the leaf blades of maize seedlings. MATERIALS AND METHODS Plant materials and experimental design. Seeds of maize (Zea mays L.) cultivar were offered by Chinese Academy of Agricultural Sciences. Seeds were germinated in a perlite medium and under intermittent mist until the development of the first true leaf. Then the plants were grown in a greenhouse under natural light and temperature conditions (daytime, ~24°C; night, ~22°C). Seedlings were watered with deionized water or 0.25-strength Hoagland solution [11]. At the two-true-leaf stage, roots were washed free of perlite, and selected plants were transferred to 2 L of 0.25-strength modified Hoagland solution in aerated polyethylene containers. After pre-cultured for 12 days, plants were subjected to full-strength modified Hoagland solution with different cadmium (Cd) supply levels. The treatments were as follows: control (without addition of Cd), 12.5, 25, 50, 100, 200, 400, and 800 µM Cd, and cadmium was supplied as CdCl2 ·2.5 H2O. Each treatment was replicated three times. The nutrient solution was aerated and replaced every four days, and the pH was maintained at 5.8 by daily adjustment with 0.1 M HCl or 0.1 M NaOH. Plants were grown under controlled glasshouse conditions at a temperature of 27 ± 3°C, and relative humidity of 70 ± 3%. The experiment was terminated, and the plants were harvested after they were grown in the metal-containing nutrient solution for 21 days. At harvest, roots of intact plants were rinsed with tap water and immersed in 20 mM Na2-EDTA for 15 min to remove Cd adhering to root surfaces. Roots were then washed for 5 min with deionized water. The plants were divided into two parts: shoots and roots. The lengths of shoots (from the apex of the leaf to the base of the stem) and roots (from the base of the stem to the tip of the taproot) were measured respectively. After fresh weights being determined, the samples were dried in a forced-air oven at 80°C for 48 h, and their dry weights were determined. The analysis of cadmium content in plants. After grinding, portions of shoot and root samples were ashed in a muffle furnace at 500°C for 18 h, and the resulting ash was dissolved in 10 mL of concentrated HNO3 and diluted to a final volume of 50 mL with distilled water. These digested samples were the basis for the determination of Cd in plant tissues. The concentrations of Cd in the solutions were measured using inductively coupled plasma atomic emission spectrometry (ICP-AES). Extraction and estimation of chlorophyll, carotenoid, and proline contents. The chlorophylls and carotenoids were extracted from fresh leaf blades with 80% acetone. Pigments were determined by spectrophotometry according to the procedure described by Lichtenthaler [12] and using the following equations for the determination of concentrations of chlorophylls and carotenoids in the leaves: Ca = 12.25 A663  2.79 A645, Cb = 21.50 A645  5.10 A663, Ca + b = 7.15 A663 + 18.71 A645, where Ca is chlorophyll a content; Cb is chlorophyll b content; Ca + b is total chlorophyll content; Cx + c is carotenoids content; A is absorbance at  (nm). The fresh leaf blade material was also used for free proline extraction. The extraction and determination procedures were carried out according to Bates et al. [13]. Three replicas were carried out for every index. Data analysis. Analysis of variance (ANOVA) for the data was performed on all data sets. Least significant different (LSD) was used for multiple comparisons at p < 0.05 level of probability. Statistical analysis was performed using Statistica software [14]. RESULTS Response of Plant Growth to Different Cd Supply Levels Maize growth was normal at the cadmium (Cd) supply levels >200 µM Cd. However, visual Cd toxicity symptoms with necrosis and browned root tips were observed in the roots of maize grown for 7 days at external Cd level of 400 µM. The toxicity symptoms became more severe with increasing Cd supply level and exposure time. At 800 µM, maize leaves wilted after the plants were grown for 7 days, and the old leaves began to fall off after growth for 14 days. Compared with the control treatment, shoot height was significantly stimulated at relatively low Cd supply levels (<100 µM), while it was inhibited at relatively high Cd supply levels (>200 µM). Shoot fresh/dry weight showed the similar pattern as shoot height. As for the root, the root length decreased significantly (from 247.8 to 118.0 mm) with increasing Cd supply levels (from 0 to 800 µM), and the maximum root length at the treatment with 800 µM Cd was only about 48% of the control (table 1). Overall, Cd stress showed significantly toxic impact on the growth of maize seedlings. Cadmium Content in the Shoots and Roots of Maize Seedlings at Different Cd Supply Levels The Cd content in the shoots and roots of maize seedlings significantly increased with increasing external Cd supply levels and peaked at 50 µM (shoot, 389.5 mg/kg dry wt; root, 505.5 mg/kg dry wt), and then decreased slowly with further increasing Cd levels. Cd content in roots was relatively higher than that in shoots at Cd supply levels of 0, 12.5, 25, 50, 100, and 200 µM, while the pattern was reversed at Cd supply levels of 400 and 800 µM. Over all, the results suggest that maize Cd accumulation ability would be stimulated at relatively low Cd supply levels (<50 µM), while it would be restrained at relatively high Cd supply levels (>100 µM) (fig. 1). Chlorophyll and Carotenoid Contents in the Leaf Blades of Maize Seedlings at Different Cd Supply Levels To explore the toxicity of Cd in maize seedlings, the content of chlorophylls and carotenoids in the leaf blades were determined. At the Cd supply levels of no more than 200 µM, chlorophyll a content in the leaf blades of maize seedlings showed no significant difference from the control plant. However, it was significantly lower than the control at the treatments with 400 and 800 µM Cd, indicating that relatively high Cd concentrations (>400 µM) exerted a more serious impact on chlorophyll a content in the leaf blades of maize seedlings. On the other hand, chlorophyll b content was significantly lower than that of control even at 12.5 µM Cd, indicating that chlorophyll b was more sensitive to the Cd stress compared with chlorophyll a. Both the content of chlorophylls a + b and carotenoids decreased significantly with increasing external Cd supply levels (table 2). Proline Content in the Leaf Blades of Maize Seedlings at Different Cd Supply Levels To illustrate the general biochemical stress defense response of maize seedlings, the proline content in the leaf blades was determined. Proline content significantly increased from 4.43 to 26.6 µg/g fr wt when the plants were subjected to the Cd supply levels from 0 to 400 µM. The increased proline content indicates that maize could adapt to the external toxic stress via changing the content of proline in leaf blades. However, proline content at the treatment with 800 µM Cd was lower than at treatment with 400 µM Cd (fig. 2). The result indicates that excessive external intimidation may destroy the immune system of maize seedlings. DISCUSSION The present study was performed to examine the impact of external cadmium stress on the growth and physiological traits of maize seedlings. Stunting is a commonly observed growth response in a wide range of plants grown under heavy metal stress. It may be due to specific toxicity of the metal to the plant metabolism or antagonism with other nutrients in plants [15]. In our study, the shoot growth of maize seedlings was significantly stimulated at relatively low external Cd concentrations (<100 µM), while it was inhibited at relatively high Cd treatments. However, the growth of root got affected at any external Cd supply levels. In agreement, Elloumi et al. [16] also reported that compared to the shoot, the greater impact of Cd was observed on the root growth, leading to a greater reduction in the root length and fresh/dry weight. Chlorophyll content is often measured in plants in order to assess the impact of environmental stress, as changes in the pigment content are linked to visual symptoms of plant illness and disturbed photosynthetic productivity. Our results showed that the content of chlorophyll decreased gradually with the increase of Cd concentration, and the overabundance of heavy metal resulted in the sharp decline in the pigment (including carotenoid) content in maize seedlings compared with the control. Chlorophyll b was more sensitive to Cd stress than chlorophyll a, and the similar result was also observed by Angadi and Mathad [17]. A decreased chlorophyll content associated with heavy metal stress may be the result of inhibition of the enzymes responsible for chlorophyll biosynthesis. Cadmium was reported to affect chlorophyll biosynthesis and to inhibit protochlorophyll reductase synthesis [18]. In our study, proline accumulation with the increase in the Cd supply level indicates that Cd was effective in inducing proline accumulation, and the similar result was also reported by Chen et al. [19]. The proline content increase in maize was the plant physiological and biochemical response to adversity menace. However, the correlation between proline accumulation and plant abiotic stress tolerance is not always apparent [20]. Barcelo et al. [21] suggested that exposure to heavy metals, especially to Cd, is known to disturb the plant water balance. Proline accumulation in plants under Cd stress is induced by a Cd-imposed decrease of the plant water potential, and proline-mediated alleviation of water deficit stress could substantially contribute to Cd tolerance [22]. In conclusion, external cadmium stress had significant effect on the growth, Cd accumulation ability, and the physiological traits of maize seedlings. Shoot growth was stimulated at relatively low external Cd supply levels, while it was restrained at high Cd levels. Cd accumulation ability of the maize seedlings also showed the similar stimulation/inhibition pattern as shoot growth. All the contents of chlorophyll a, chlorophyll b, and carotenoids in the leaf blades decreased with increasing external Cd supply levels, and chlorophyll b was more sensitive to the Cd stress than chlorophyll a. This research was financially supported by the Talent Introduction Foundation of Taiyuan University of Technology. REFERENCES 1. Zaidi A., Wan, P.A., Khan M.S. Toxicity of Heavy Metals to Legumes and Bioremediation. NewYork: Springer-Verlag, 2012. 2. Al-Khedhairy A.A., Al-Rokayan S.A., Al-Misned F.A. Cadmium Toxicity and Cell Stress Response // Pakistan J. Biol. Sci. 2001. V. 4. P. 10461049. 3. Sanita di Toppi L., Gabrielli R. Response to Cadmium in Higher Plants // Environ. Exp. Bot. 1999. V. 41. P. 105130. 4. McGrath S.P., Zhao F.J., Lombi E. 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Plant Physiol. 1986. V. 125. P. 2734. 22. Costa G., Morel J.L. Water Relations, Gas Exchange and Amino Acid Content in Cd-Treated Lettuce // Plant Physiol. Biochem. 1994. V. 32. P. 561570. Table 1. Effect of Cd supply level on maize seedling growth Table 2. Effect of Cd supply level on chlorophyll and carotenoid contents in maize seedling leaf blades FIGURE CAPTIONS Fig. 1. Cadmium content in the shoots and roots of maize seedlings grown at different Cd supply levels. DW means dry weight. Mean values (n = 3). Data for shoots or roots with the same letter are not significantly different at p < 0.05 level of probability. 1  shoot; 2  root. Fig. 2. Proline contents in the maize seedlings leaf blades grown at different Cd supply levels. Mean values (n = 3). Values with the same letter are not significantly different at p < 0.05 level of probability.