ФИЗИОЛОГИЯ РАСТЕНИЙ, 2011, том 58, № 3, с. 397-402
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
y%K 581.1
Polyamine Metabolism and Physiological Responses of Potamogeton crispus Leaves under Lead Stress
© 2011 Y. Xu, G. X. Shi, C. X. Ding, X. Y. Xu
College of Life Science, Nanjing Normal University, Nanjing, P. R. China Received May 12, 2010
Aquatic macrophytes were found to be the potential scavengers of heavy metals from aquatic environment. In this study, influences of ladder concentration of lead (Pb) on the leaves of Potamogeton crispus Linn were studied after 7 days of treatment. The accumulation of Pb, nutrient element contents, the generation rate of superoxide radical (O2 ), MDA, proline, and polyamine (PAs) contents, as well as the activities of diamine oxidases (DAO), polyamine oxidases (PAO), arginine decarboxylase (ADC), and ornithine decarboxylase (ODC) in P. crispus leaves were investigated. The result indicated that Pb treatment decreased the activity of DAO, whereas the proline content, MDA content, the generation rate of O2 and the activity of ODC increased in different degrees. Meantime, Pb treatment significantly increased the free putrescine (Put) level and made other PAs levels dynamic changes. The activities of PAO and ADC were declined firstly and then enhanced with the increase in the Pb concentration.
Keywords: Potamogeton crispus — lead toxicity — polyamine metabolism — proline — malondialdehyde — superoxide radical
INTRODUCTION
Heavy metal pollution of environment is one of major ecological concerns that due to its impact on human health through the food chain and its high persistence in the environment. Lead, which is a heavy metal and a potentially hazardous environmental pollutant, is one of the most abundant, ubiquitously distributed toxic elements. Its contamination results from mining and smelting activities, lead containing paints, paper and pulp, gasoline and explosives as well as from the disposal of municipal sewage sludge enriched with Pb [1]. Pb is known to cause oxidative stress resulting in increased ROS production in plants and also to induce phyto toxic symptoms, such as growth retardation, degradation of photosynthetic pigments, antioxidant enzyme activity and ultrastructure changes [2].
Spermidine (Spd), spermine (Spm), and their diamine precursor, putrescine (Put), are major PAs in plant cells. PAs often occur as free molecular bases (free PAs), but they can also be associated with small molecules, such as phenolic acids (perchloric acid-soluble conjugated PAs, PS-conjugated PAs) and with various macromolecules, like proteins, and with cell walls (perchloric acid-insoluble bound PAs, PIS-
Abbreviations: ADC—arginine decarboxylase; DAO—diamine oxidases; ODC—ornithine decarboxylase; PAO—polyamine oxidases; PAs—polyamines; Put—putrescine; Spd—spermidine; Spm-sper-mine; TBA—thiobarbituric acid; TCA—trichloroacetic acid. Corresponding author: Guoxin Shi. College of Life Science, Nanjing Normal University, Nanjing, 210046 P.R. China. Fax: +8625-8359-8257; e-mail: gxshi@njnu.edu.cn
bound PAs). PAs modulate several biological processes in plants, including cell division, differentiation, and senescence, and it had been suggested that they participate in scavenging free radicals [3]. In plants, Put was synthesized from arginine or ornithine by ADC or ODC, respectively. The following addition of two ami-nopropyl groups to Put (catalyzed by Spd synthase and Spm synthase) led to the synthesis of Spd and Spm [4]. Meanwhile, PA content was also regulated by their degradation through the action of PAO, which oxidizes Spd and Spm at their secondary amino groups [5]. PAs are involved in various biochemical and physiological processes related to plant growth and development [4, 6—8]. Until now, most PA studies focused on terrestrial plants [3, 7, 8]. Little is known about the roles PAs play in aquatic plants, and the investigations about the activity of some key enzymes in PA metabolism under heavy metals stress were also infrequent.
Aquatic plants are well known to accumulate heavy metals, and they took up metals from the water, causing an internal concentration severalfolds greater than in their surroundings [9]. Potamogeton crispus Linn, a rooted submerged macrophyte with fast growth and high biomass, can accumulate considerable amounts of toxic metals in polluted waters (Fe, Pb, Ni, Mn, Hg, and Cu) and show its great phytoremediation potential [10]. To our knowledge, few reports are available on PA metabolism in P. crispus under Pb stress. In the present investigation, Pb toxicity was assayed using such parameters as nutrient status, PA contents, proline content, MDA content, and the generating rate of
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02 . Moreover, PA metabolism in the plant was investigated, including the activities of ornithine decarboxylase (ODC), arginine decarboxylase (ADC), polyamine oxidases (PAO), and diamine oxidases (DAO). This study will be helpful in elucidating the key mechanisms that lead to acute toxicity of Pb in freshwater macrophytes.
MATERIALS AND METHODS
Plant material and metal treatment. The Potamogeton crispus Linn was collected from Taihu River in Dongshan, Jiangsu, China early November in 2009. Plants were cultured in aquaria in totally enclosed incubator (Forma, 3744, United Kingdom) at the temperature of 25/18°C (day/night) and a photoperiod of 12 h (70 |M/(m2 s)). One week before experimentation, the plant material was transferred to 0.1-strength Hoagland solution [11]. Similar fronds were treated with Pb (lead nitrate) in the concentrations of 10, 20, 30, 40, and 50 |M in the culture solution in 2-l glass beakers. After 7 days, the fully expanded, morphologically similar leaves were cut and sampled. All solutions were refreshed every 2 days, and all experiments were performed in triplicate.
Elemental analysis. The accumulation of lead and nutrient element concentrations were analyzed by Inductively Coupled Plasma Atomic Emission Spec-trometry (ICP-ES, Leeman Labs, Prodigy, United States).
Measurement for MDA content and the generating
rate of 02-. Lipid peroxidation was measured by the level of MDA, a product of lipid peroxidation, using a reaction with thiobarbituric acid (TBA) as described by Hodges et al. [12]. About 0.5 g leaf segments were homogenized in 10 ml of10% TCA, and centrifuged at 12000 g for 10 min. After that, 2 ml 0.6% TBA in 10% TCA was added to an aliquot of 2 ml of the supernatant. The mixture was heated in boiling water for 30 min and then quickly cooled in an ice bath. After centrifu-gation at 10000 g for 10 min, the absorbance of the supernatant at 450, 532, and 600 nm was determined (Thermo Scientific Genesys 10 UV, United States).
P. crispus leaves (0.5 g) were homogenized on ice with mortar and pestle in 50 mM phosphate buffer. Solid phase was separated centrifugally at 10000 g for 20 min, and the supernatant was analyzed. The generating rate of 02- was etermined by using the hydroxy-lamine chloride method [13].
Proline extraction and determination. Proline was determined by the ninhydrine method [14]. Briefly, 100 mg of plant material was homogenized in 1 ml of distilled water at room temperature and transferred to 10-ml tubes. The tubes were incubated for 20 min in a boiling water bath to extract hot-water-soluble compounds, subsequently cooled to room temperature and centrifuged at 2000 g for 5 min. 300 |l of distilled water
and 2 ml of ninhydrine reagent were added to an aliquot of 200 |l ofwater extract. The mixture was boiled for 60 min, and the reaction was stopped in an ice bath. The chromophore obtained was extracted with 6 ml of toluene by vigorous shaking for 20 s. Absor-bance of the resulting organic layer was measured at 520 nm with Varian UV-VIS spectrophotometer. Calibration was made using L-Pro as a standard.
Measurement for the content of different form polyamines. Plant material (3 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 supernatants 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 resus-pended in 5% PCA and hydrolyzed for 24 h at 110°C in flame-sealed glass ampoules after being 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, hydrolyzed supernatant, and the pellet were benzoylated in accordance with the method of Aziz and Larher [15].
The benzoyl derivatives were separated and analyzed by a HPLC (Agilent 1100 system, United States). Ten microliters of methanol solution of ben-zoyl polyamines was injected into a 20 ml loop, loaded onto a 4.6 x 200 mm, 5 ^m particle size C18 reversephase column (Kromasil, Sweden). Column temperature was maintained at 30°C. Samples were eluted from the column with 64% methanol at a flow rate of 1 ml/min. Polyamine peaks were detected with a UV detector at 254 nm. 1,6-hexanediamine was used as an internal standard.
Assays of ADC, ODC, PAO, and DAO activities.
ADC and ODC activities were determined according to Birecka et al. [16], with some modifications. Plant material (1.5 g) was homogenized in 50 mM potassium phosphate buffer (pH 6.3) containing 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 40 |M pyridoxal phosphate (PLP), 5 mM DTT, 20 mM ascorbic acid, and 0.1% polyvinylpyrrolidone. The homogenate was centrifuged at 12000 g for 40 min at 4°C and the supernatant was dialyzed at 4°C, against 3 ml of 10 mM potassium phosphate buffer (pH 6.3) containing 0.05 mM PLP, 1 mM DTT, 0.1 mM EDTA for 24 h in darkness. The dialyzed extract was used for enzyme
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