ФИЗИОЛОГИЯ РАСТЕНИЙ, 2013, том 60, № 2, с. 293-297
КРАТКИЕ ^^^^^^^^^^^^^^ СООБЩЕНИЯ
The Response of Maize Seedlings to Cadmium Stress under Hydroponic Conditions1
© 2013 C. X. Wang", L. TaoA, and J. RenA
a College of Environmental Science and Engineering, Taiyuan University of Technology, Taiyuan, China
b 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 ^M) 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 цМ), while they were inhibited at Cd supply levels over 200 цМ. 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 цМ 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 ^M), 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.
Keywords: Zea mays - cadmium - chlorophylls - carotenoids - proline
The contamination of soils with metals is a major environmental problem throughout the world . 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 . 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
1 This text was submitted by the authors in English.
Corresponding authors: Chaoxu Wang and Jun Ren. College of Environmental Science and Engineering, Taiyuan University of Technology, No. 79 Wfest Yingze Street, Wanbailin District, Taiyuan 030024, P.R. China. E-mail: email@example.com and School of Environmental and Municipal Engineering and Institute of Environmental Ecology, Lanzhou Jiaotong University, No. 88 Anning Wfest Street, Anning District, Lanzhou 730070, P. R. China. E-mail: firstname.lastname@example.org
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 .
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 .
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 hy-droponic 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 Gansu 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
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or 0.25-strength Hoagland solution . 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 ^M 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, caro-tenoid, and proline contents. The chlorophylls and car-otenoids were extracted from fresh leaf blades with 80% acetone. Pigments were determined by spectro-photometry according to the procedure described by Lichtenthaler  and using the following equations for the determination of concentrations of chlorophylls and carotenoids in the leaves:
Ca = 12.25^663 - 2.794 Cb = 21.504« - 5.104
Ca+b = 7.15A663 + 18.71A645,
Cx + c = (1000A470 - 1.82Ca - 85.02Cb)/198,
where Ca is chlorophyll a content; Cb is chlorophyll b content; Ca+b is total chlorophyll content; Cx + c is car-otenoids content; Ax is absorbance at X (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. . 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 atp < 0.05 level of probability. Statistical analysis was performed using Statistica software .
Response of plant growth to different cd supply levels
Maize growth was normal at the cadmium (Cd) supply levels >200 ^M 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 ^M. The toxicity symptoms became more severe with increasing Cd supply level and exposure time. At 800 ^M, 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 ^M), while it was inhibited at relatively high Cd supply levels (>200 ^M). 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 ^M), and the maximum root length at the treatment with 800 ^M 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 ^M (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 of0, 12.5, 25, 50, 100, and 200 ^M, while the pattern was reversed at Cd supply levels of 400 and 800 ^M. Over all, the results suggest that maize Cd accumulation ability would be stimulated at relatively low Cd supply levels (<50 ^M), while it would be restrained at relatively high Cd supply levels (>100 ^M) (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 ^M, chlorophyll a content in the leaf
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