ФИЗИОЛОГИЯ РАСТЕНИЙ, 2013, том 60, № 6, с. 813-818
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
EXOGENOUS SPERMIDINE ENHANCES Hydrocharis dubia CADMIUM TOLERANCE1
© 2013 H. Y. Yang*, G. X. Shi**, W. L. Li*, W. L. Wu*
*Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing, China **Jiangsu Key Lab of Biodiversity and Biotechnology, College of Life Science, Nanjing Normal University, Nanjing, China
Received November 12, 2012
The effects of exogenous spermidine (Spd) on arginine decarboxylase (ADC), ornithine decarboxylase (ODC), polyamine oxidase (PAO), and diamine oxidase (DAO) activities, the rate of superoxide radical
(O2 ) generation and polyamine (PA), malondialdehyde (MDA), and H2O2 contents in Hydrocharis dubia
(Bl.) Backer leaves under cadmium (Cd) toxicity were studied after 6-day treatment. Cd stress increased pu-trescine (Put) level and lowered spermidine (Spd) and spermine (Spm) levels. In addition, the activities of ADC, DAO, and PAO were increased, while that of ODC was decreased. Exogenous application of Spd
markedly reversed these Cd-induced effects. It also significantly reduced the generation of O2 and H2O2
and prevented lipid peroxidation. These results suggest that exogenous Spd can enhance the tolerance of H. dubia to Cd. The maintenance of PA homeostasis was necessary for plant metal tolerance.
Keywords: Hydrocharis dubia - cadmium - polyamines DOI: 10.7868/S001533031306016X
INTRODUCTION
Consequent to increased industrialization and geochemical activities, cadmium (Cd) contamination of aquatic ecosystems has become a severe global issue [1]. Cd is a widely spread heavy metal non-essential for plant metabolism. In plants, it is reported that Cd causes severe physiological and morphological effects, such as stunted growth, chlorosis, and decreased reproducibility. At the cellular level, Cd is known to disrupt the plant defense system against naturally occurring ROS [1-3]. ROS react with lipids, proteins, pigments, and nucleic acids and cause lipid peroxidation, membrane damage, inactivation of enzymes, and disruption of DNA strands, thus affecting cell viability.
Aliphatic polyamines (PAs) are a class of ubiquitous compounds found in all prokaryotic and eukary-otic cells, which are involved in various processes, such as cell proliferation, growth, morphogenesis, differentiation, and programmed cell death [4, 5]. In higher plants, the most commonly found polyamines,
1 This text was submitted by the authors in English.
Abbreviations'. ADC - arginine decarboxylase; DAO - diamine oxidase; ODC - ornithine decarboxylase; PA - polyamine; PAO -polyamine oxidase; Put - putrescine; Spd - spermidine; Spm - spermine.
Corresponding author. Hai Yan Yang. Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing, China. E-mail. haiyanyang_025@126.com
e.g., putrescine (Put), spermidine (Spd), and spermine (Spm), may be present in a free soluble form, be conjugated to small molecules, such as phenolic compounds (PS-conjugated PAs), or linked to macromol-ecules, such as proteins or DNA (PIS-bound PAs) [5, 6]. Since PAs were reported to be involved in the regulation of senescence, osmotic adjustment, mineral nutrition, stabilization of membrane, and scavenging of free radicals [2, 4, 7, 8], they were supposed to play a general role in coping with environmental stresses. Besides, PA metabolism is in relation to several environmental stresses, including salt, metal, osmotic, drought, and chilling stresses [2, 5, 7-9]. The first step in PA biosynthesis is the formation of Put, which can be synthesized directly by decarboxylation of ornithine catalyzed by ornithine decarboxylase (ODC), or indirectly by decarboxylation of arginine catalyzed by arginine decarboxylase (ADC) in a pathway involving agmatine and N-carbamoylputrescine as intermediates. Moreover, ADC activity is usually reported to enhance in response to stress, whereas ODC activity is involved in the regulation of plant growth and development [10, 11]. Spd and Spm are synthesized from Put by the addition of two aminopropyl groups (catalyzed by Spd and Spm synthases) [11]. Not only PA formation, but also PA catabolism is associated to stress resistance. Two catabolic enzymes were found in plants. polyamine oxidase (PAO) with high affinity towards Spd and Spm and diamine oxidase (DAO),
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which primarily catalyses Put degradation [3, 11]. Additionally, H2O2 is produced during the degradation of PAs by these two enzymes, which may act as a mediator of different physiological processes, such as signaling, cellular defense, and programmed cell death [7].
Based on the above studies, we chose Hydrocharis dubia (Bl.) Backer as the experimental material and suppose that exogenous Spd may ameliorate toxic effects of Cd on the plant. H. dubia is a kind of floating macrophytes, which is a clonal weedy species native to Asia. For its fast growth and large biomass production, it is a convenient plant material for ecotoxicological investigations. Since no major attempts have been made to improve metal tolerance of aquatic plants, induction of metal tolerance by the application of exogenous PAs continues to be an objective of a great interest. In this research, the influence of exogenous Spd on the Cd-induced changes of three PA levels, not only in free form but also in PS-conjugated and PIS-bound forms has been measured. The activities of some key enzymes in PA metabolism were determined.
MATERIALS AND METHODS
Plant material. Hydrocharis dubia (Bl.) Backer was collected from Tai lake in Suzhou, China. Plants of approximately the same height and weight were selected for experimentation and grown in an aquarium in totally enclosed incubator (Forma 3744, England) at the day/night temperature of 25/20°C, light irradi-ance of 114 ^mol/(m2 s), and a photoperiod of 12 h.
Treatments. After two weeks, plant materials were transferred to glass beakers, each with 1 L of solution. Treatments were as follows: (1) control - 0.1 Hoagland solution and the leaves were sprayed with distilled water; (2) Spd treatment - 0.1 Hoagland solution and the leaves were sprayed with 0.1 mM Spd; (3) Cd treatment - 0.1 Hoagland solution containing 0.04 mM Cd and the leaves were sprayed with distilled water, and (4) (Spd + Cd) treatment - 0.1 Hoagland solution containing 0.04 mM Cd and the leaves were sprayed with 0.1 mM Spd. The sprays of Spd or distilled water (5 mL each time) took place at 07:00 and 19:00 each day. Tween 80 (0.5%, v/v) was used as a surfactant. All solutions were refreshed every two days. After six days, the leaves were sampled and tested. All experiments were performed in triplicate.
Polyamine determination. Plant material (1 g) was homogenized in 4 mL of 6% (v/v) cold perchloric acid (PCA), kept on ice for 1 h, and then centrifuged at 21000 g for 30 min. The pellet was extracted twice with 2 mL of 5% PCA and recentrifuged. The three super-natants were pooled and used to determine the levels of free and PS-conjugated PAs, whereas the pellet was used to determine the levels of PIS-bound PAs. The pellet was resuspended in 5% PCA and hydrolyzed for 24 h at 110°C in flame-sealed glass ampoules after be-
ing mixed with 12 N HCl (1 : 1, v/v). The hydrolyzates were filtered, dried at 70°C, and then resuspended in 1 mL of 5% PCA for analysis of PIS-bound PAs. For PS-conjugated PAs, 2 mL of the supernatant were mixed with 2 mL of 12 N HCl and hydrolyzed under the conditions described above. The supernatant, hy-drolyzed supernatant, and pellet were benzoylated in accordance with the method of Aziz and Larher [12].
The benzoyl derivatives were separated and analyzed by an HPLC system (Agilent 1100, United States) equipped with an UV detector under the following conditions: 200 x 4.6 mm C18 reverse-phase column ("Kromasil", Sweden); particle size, 5 ^m; column temperature, 30°C; mobile phase, 64% (v/v) methanol; the flow rate, 0.8 mL/min; detection wavelength, 254 nm. 1,6-Hexanediamine was used as an internal standard.
Assays of ADC, ODC, DAO, and PAO activities.
The ADC and ODC activities were determined according to Zhao et al. [13], with some modifications. The reaction mixture (1.5 mL) was consisted of 1 mL of the assay buffer with 100 mM Tris-HCl (pH 8.5), 5 mM EDTA, 40 ^M pyridoxal phosphate, 5 mM DTT, 0.3 mL of the ADC (ODC) enzyme extract, and 0.2 mL of 25 mM L-Arg (or Orn). The reaction mixture was incubated at 37°C for 60 min and centrifuged at 3000 g for 10 min, after which 0.5 mL of the supernatant was mixed with 1 mL of 2 mM NaOH, then 10 ^L of benzoyl chloride was added to the mixture and stirred continuously for 20 s. After the reaction proceeded at 25°C for 60 min, 2 mL of saturated NaCl and 2 mL of ether were added to the reaction mixture and stirred thoroughly, then centrifuged at 1500 g for 5 min; 1 mL of the ether phase was collected and evaporated at 50°C. The remainder was dissolved in 0.5 mL of methanol, and its absorption value at 254 nm was measured by an HPLC system (Agilent 1100). A standard curve with Agm (Put) was used to calculate the activity ofADC (ODC). The ADC and ODC activities were expressed as ^mol Agm/(g fr wt h) (U) and ^mol Put/(g fr wt min) (U), respectively.
The DAO and PAO activities were determined according to the procedure described by Gao et al. [14], with some modifications. Fresh samples were homogenized in 100 mM potassium phosphate buffer (pH 6.5). The homogenate was centrifuged at 10000 g for 20 min at 4°C. The supernatant was used for enzyme assay. The reaction mixture contained 2.5 mL of potassium phosphate buffer (100 mM, pH 6.5), 0.2 mL of 4-aminoantipyrine/N,N-dimethylaniline reaction solution, 0.1 mL of horseradish peroxidase (250 U/mL), and 0.2 mL of the enzyme extract. The reaction was initiated by the addition of 0.1 mL of Put (at a final concentration of 20 mM) for DAO determination and 0.1 mL of Spd (at a final concentration of 20 mM) for PAO
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