= ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
EFFECTS OF EXOGENOUS NITRIC OXIDE ON PHYSIOLOGICAL CHARACTERISTICS OF PERENNIAL RYEGRASS UNDER CADMIUM
AND COPPER STRESSES1 © 2015 X. Y. Bai, Y. J. Dong, L. L. Xu, J. Kong, S. Liu
College of Resources and Environment, Shandong Agricultural University, Tai'an, Shandong, P.R. China
Received June 18, 2014
The effects of sodium nitroprusside (SNP, a donor of NO) on cadmium (Cd) and copper (Cu) toxicity in ryegrass seedlings (Loliumperenne L.) were studied. Plant growth, chlorophyll content, the accumulation of superoxide anion (O2 ), lipid peroxidation, and the activities of antioxidant enzymes were asesed. Cd, Cu, and
especially Cd+Cu caused serious chlorosis, inhibited the growth of ryegrass seedlings, and increased dramatically the accumulation of Cd and/or Cu in both shoots and roots. However, the addition of 100 цМ SNP alleviated significantly the toxic effects induced by Cd or Cu, and especially by Cd+Cu, which was manifested in the increased plant growth, chlorophyll content, and the activation of antioxidant enzymes. Moreover, exogenous NO improved effectively the absorption of mineral elements. In addition, exogenous NO diminished markedly the accumulation of reactive oxygen species (ROS), malondialdehyde (MDA), and proline, but increased the content of ascorbic acid (AsA) and inhibited the translocation of Cd and Cu from roots to shoots. These data also suggested that the alleviating effect of NO may be better when the metal stresses are more serious. NO might act as one of the potential antioxidants to improve plant resistance to the Cd and/or Cu stress.
Keywords: Lolium perenne - Cd stress - Cu stress - nitric oxide - SNP - antioxidant enzymes - mineral content
Cadmium (Cd) is one of the most toxic environmental pollutants in the atmosphere, soil, and water, and its excessive amount can cause serious problems to all organisms. The metal may eventually reach the food chain and ultimately affect human health. The main sources of Cd in agricultural soils are phosphate fertilizers, dispersal of sewage sludge, mining, and atmospheric deposition of industrial emission . Numerous experimental studies mainly focus on the highly toxic effects of Cd on rice , wheat , and some other crops. The main symptoms of Cd toxicity in plants are growth retardation, low biomass, and leaf chlorosis . In plants, Cd toxicity was found to interfere with electron transport chains or block antioxidant enzyme structures, the inhibition of enzyme activities, the alteration of nutrient levels, and negative
1 This text was submitted by the authors in English.
Abbreviations: AsA - ascorbic acid; CAT - catalase; O2 - superoxide anion radical; POD - peroxidase; SNP - sodium nitroprusside; SOD - superoxide dismutase.
Corresponding author: Y. J. Dong. College of Resources and Environment, Shandong Agricultural University, Tai'an, 271018 Shandong, P.R. China; fax: (+86) 0538-824-2250, e-mail: email@example.com
effects on chlorophyll metabolism and photosynthesis leading to oxidative damage, membrane leakage, and finally cell death . To survive under stress, plants have evolved protective mechanisms involving morphological changes, and physiological and biochemical adaptations.
As distinct from Cd, copper (Cu) is an essential mi-cronutrient, which is necessary for plant growth and development, but at elevated concentrations it can also be as toxic as other known metals. Recently, Cu has become increasingly hazardous to plants due to the applications of mining, smelting, industrial waste disposal, sewage sludge application to agricultural soils, the use of fertilizer and pesticide, and so on . Cu released to environment by anthropogenic activities may have a significant impact on the biodiversity of plants, such as rice  and tomato . When it is absorbed in excessive amounts, Cu can be considered as a toxic element, leading to the total inhibition of growth, with impact on a wide range of biochemical and physiological processes . To counteract the toxicity of Cu, plants have developed various strategies: exudation of organic acid, the retention of Cu in roots, and immobilization in the cell wall.
Nitric oxide (NO) is involved in the regulation of multiple responses to a variety of abiotic and biotic
Table 1. The experimental design
Treatment Treatment composition
CK Hoagland nutrient solution
SNP 100 ^M SNP
T1 100 ^M Cd
T2 200 ^M Cu
T3 100 ^M Cd + 200 ^M Cu
T4 100 ^M Cd + 100 ^M SNP
T5 200 ^M Cu + 100 ^M SNP
T6 100 ^M Cd + 200 ^M Cu + 100 ^M SNP
T7 100 ^M Cd + 200 ^M Cu + 100 ^M light-inactivated SNP
All the treatments (CK, SNP, and T1-T7) in other tables and figure are in accordance with the description in table 1. The light-inactivated SNP was exposed to light for one week.
stresses in plants . Recently, an increasing number of articles have reported the effects of exogenous NO on alleviating the effects of heavy metals, such as Cu , Pb , and Cd . Furthermore, exogenous NO was described as an effective substance for the alleviation of Cd toxicity in many plants, such as rice  and wheat . In addition, some studies also showed that NO can mitigate Cu stress in rice , tomato seedlings , wheat seeds , and so on. Many reports indicate that the application of exogenous NO in the form of sodium nitroprusside (SNP), as a NO donor, improves plant tolerance to heavy metal stresses . Our previous study also showed that SNP supplied at low concentrations alleviated Cd toxicity in perennial ryegrass . However, up to now, few studies have been done on the effects of NO on physiological characteristics of perennial ryegrass under Cd and Cu stresses.
Perennial ryegrass can adapt well to temperate climate as an important cool-season turfgrass . It is widely used for livestock, fiber products, improving soil contaminated with heavy metal, habitats for wildlife populations, recreation, and beautification . It also can accumulate metals in its biomass, and commonly used as a suitable species for re-vegetation of metalliferous wastes. In addition, our previous studies have demonstrated that perennial ryegrass has the potential for rehabilitation of Cd stress . Based on the above studies, it is supposed that NO may ameliorate toxic effects of excess Cd and/or Cu on ryegrass. The specific objectives of this study were to examine (a) which is a more severe poisoning of alone or compound metal stresses and the poisoning mechanism of Cd and Cu in ryegrass; (b) whether exogenous NO has the alleviating effect on Cd, Cu, or Cd+Cu toxicity; and (c) to understand the mechanism of NO protecting ryegrass from Cd and/or Cu toxicity.
MATERIALS AND METHODS
Plant material and culture conditions. Ryegrass (Lolium perenne L.) seeds were first sterilized with 5% sodium hypochlorite for 15 min and washed extensively with distilled water, then germinated on moist filter paper in the dark at 26°C for 3 days. Initially, seedlings of uniform size were transferred to plastic pots (volume of 500 mL) filled with perlite (50 plants per pot) and watered with half-strength Hoagland nutrition solution for 7 days. The seedlings were then watered with full-strength Hoagland solution. Three-week-old uniform seedlings were transferred into 1000 mL black plastic containers with 50 seedlings per container. The nutrient solution was renewed every 2 days. These treatments were: CK -Hoagland solution (control); SNP - 100 |M SNP; T1 - 100 |M Cd; T2 - 200 |M Cu; T3 - 100 |M Cd + + 200 |M Cu; T4 - 100 |M SNP + 100 |M Cd; T5 -100 |M SNP + 200 |M Cu; T6 - 100 |M SNP + + 100 |M Cd + 200 |M Cu; T7 - 100 |M light-inactivated SNP + + 100 |M Cd + 200 |M Cu. The experimental design is given in table 1.
The treatments were arranged in a randomized block design with four replicates. The experiment was carried out under a controlled-environment chamber at a 14-h photoperiod and photon flux density of 150 |mol/(m2 s) at the leaf level, day/night temperature of 25/18°C and 65 ± 5% relative humidity. After two weeks of growth at the above conditions, the plants were harvested and the roots and leaves were separated and washed with 5 mM CaCl2 first and then repeatedly washed with deionized distilled water. For the estimation of plant dry matter, Cd, Cu, and mineral nutrient contents, plants were dried at 80°C for 48 h. For the enzyme determination, fresh plant material was frozen in liquid nitrogen and stored at -70°C until use.
Determination of plant growth, cadmium, copper and mineral element contents. Seedlings heights were determined immediately after harvesting. At harvest time, fresh and dry weights were measured. And the root/shoot ratios of fresh weight and dry weight were calculated. The dried tissues were weighed and grinded into powder for the determination of cadmium and mineral element contents, which was measured by flame atomic absorbance spectrometry (SHI-MADZU AA-6300, Japan) after digestion with the mixture of acids (HNO3 + HClO4, 3 : 1, v/v) .
Determination of chlorophyll content. The chlorophyll content was determined according to the method of Knudson et al. . Fresh ryegrass leaves (0.5 g) were extracted with 2 mL of 95% ethanol for 24 h in the dark, and the extracted solution was analyzed. The amounts of chlorophylls a and b and carotenoids were determined spectrophotometrically (SHIMADZU UV-2450, Japan) by reading the absorbance at 665, 649, and 470 nm. The chlorophyll content results were expressed in mg/g fr wt.
Determination of O^ generation rate and MDA content. Fresh leaves (0.2 g) were homogenized in 1 mL of 50 mM phosphate buffer (pH 7.8), and the homoge-nate was centrifuged at 10000 g for 10 min. Then 0.5 mL of the supernatant was added to 0.5 mL of50 mM phosphate buffer (pH 7.8) and 0.1 mL of 10 mM hydroxy-lamine hydrochloride. After 1 h reaction at 25°C, the mixture was added to 1 mL
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