ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 1, с. 99-105
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
EFFECT OF LOW DOSE OF SPERMIDINE ON PHYSIOLOGICAL CHANGES IN SALT-STRESSED CUCUMBER PLANTS1 © 2014 R. Radhakrishnan, I. J. Lee
School of Applied Biosciences, Kyungpook National University, Sangyuk-Dong, Buk-gu, Daegu, South Korea
Received March 19, 2013
The present study was aimed to assess the ameliorative potentiality of exogenously applied low dose of spermidine (Spd) (4.0 mL of 0.1 mM) against salt stress in cucumber plants (Cucumis sativus L.). Salt stress inhibited plant growth, while Spd increased the shoot length and dry weight of leaves in salt-stressed plants. Chlorophyll, carotenoids, and sucrose contents were lower, and the accumulation of superoxide radical was higher in salt-affected plants than in controls, and these detrimental effects were mitigated by Spd treatment. Moreover, salinity diminished the reduced glutathione and total polyphenols and inhibited the activities of catalase, peroxidase, and polyphenol oxidase as compared with controls, and Spd treatment increased all antioxidant activities in salt-injured plants. NaCl-induced oxidative stress caused a significant decrease in GA4 and GA5 contents. Spd treatment ameliorated these salt stress effects by increasing the quantities of GA4. In addition, sodium content was higher and calcium content was lower in salt-treated plants, while Spd treatment reduced the sodium accumulation and increased the calcium level in plants exposed to NaCl. The results suggest that exogenous application of low Spd dose can ameliorate the salt stress effects on cucumber by modulating the components of photosynthetic pigments, antioxidants, gibberellins, and minerals.
Keywords: Cucumis sativus - antioxidants - gibberellins - minerals - NaCl - spermidine
DOI: 10.7868/S0015330314010126
INTRODUCTION
Polyamines are secondary messengers in stress signaling pathways; they play an important role in plant response to environmental stress conditions [1]. A number of polyamines (PAs) has been detected in living organisms; putrescine (Put), spermidine (Spd), and spermine (Spm) are major PAs present in plants. Among three major PAs, Spd has been more closely associated with stress tolerance of plants [2]. Spd is involved in several physiological, histochemical, and biochemical activities of plant cells. Exogenous application of Spd altered the activities of scavenging enzymes and ROS in water-stressed cucumber plants [3]. Still now, proteomics and genomics studies could not elucidate the physiological mechanisms of PA action in plants directed against abiotic and biotic stresses [4].
1 This text was submitted by the authors in English.
Abbreviations'. CAT - catalase; GA - gibberellic acid; GSH - reduced glutathione; NBT - nitro blue tetrazolium; POD - peroxidase; PAs - polyamines; PPO - polyphenol oxidase; Put - putrescine; Spd - spermidine, Spm - spermine, O2 - superoxide radical.
Corresponding author. In Jung Lee. School of Applied Biosciences, Kyungpook National University, Sangyuk-Dong, Buk-gu, Daegu, 702-701 South Korea. Fax. +82-53-958-6880; e-mails. ramradhakrish@gmail.com; ijlee@knu.ac.kr
The uptake of macro- and micronutrients by roots is an important process for plant growth and development. However, soil salinity induces toxic ion accumulation in plant cells. The interaction between soil salinity and plant mineral nutrition remains poorly understood [5]. ROS are more abundant in plants exposed to salt stress than in controls. Chloroplasts, mitochondria, and peroxisomes are major sources of ROS production in plant cells [6]. ROS comprise both free radical and non-radical forms, which damage chlorophyll, proteins, DNA, lipids, and other macro-molecules. In addition, the general function of the an-tioxidant defense system is to prevent the oxidative damage by ROS scavenging. Plants have the efficient antioxidant enzymes, such as catalase, superoxide dis-mutase, ascorbate peroxidase, and glutathione reduc-tase [3, 7], and non-enzymatic antioxidants, such as ascorbic acid, reduced glutathione, total polyphenols, and phenolic substances. To date, no study has explored the action of low concentrations of Spd on crop plants; thus, we used the low quantity of Spd (4.0 mL of 0.1 mM) for cucumber plant treatments. The aim of the present investigation was to improve the cucumber plant growth under salt stress conditions by the low dose of Spd and to evaluate their efficacy through the regulation of antioxidants and phytohormones.
MATERIALS AND METHODS
Spermidine and NaCl treatments of cucumber plants. Cucumber (Cucumis sativus L., Chun-pung-chung-jang) seeds were surface sterilized with 60% ethanol and washed with distilled water. The seeds were sown in artificially formulated substrate. The seedlings were grown in a growth chamber at 28 ± ± 3/18 ± 3°C (day/night) and 60-75% relative humidity. After three weeks, 4.0 mL of 0.1 mM Spd was applied three times at two days intervals and followed by 150 mL of 0.1 M NaCl after four weeks. The growth parameters, shoot length and dry weight of leaves, were measured in cucumber plants at 10 days. Plants were collected and stored at -70°C until use. Each treatment was replicated five times in a randomized block design, and each replicate included 6 plants. All biochemical experiments were performed in triplicate.
Determination of photosynthetic pigment and sucrose contents. For photosynthetic pigment analysis, leaves were ground with 80% acetone, and chlorophylls and carotenoids were measured by the method of Arnon [8] and Lichtenthaler [9], respectively. Sucrose was extracted with 80% ethanol [10] and quantified by HPLC. In brief, freeze dried plant samples were homogenized with 80% (v/v) aqueous ethanol for three times. The supernatants were evaporated under vacuum, dissolved in water, and then filtered through the filter paper. HPLC analyses were carried out in a Waters system (Millipore, "Waters Chromatography", United States) comprising sugar-pak column (300 mm), a model 600 controller, and a Waters 410 refractive index detector. Mobile phase was deionized water (50 mg Ca-EDTA/1 L of water). Samples of 0.1 mL were injected and chromatographed under the flow rate of 0.5 mL/min at 90°C. Sucrose was quantified on the basis of peak areas and comparison with a calibration curve obtained with the corresponding standard.
Determination of superoxide and antioxidant systems. The superoxide ( O2-) was measured on the base of its ability to reduce nitro blue tetrazolium (NBT) [11] at 580 nm and the O^ content was expressed as an increase in the absorbance/0.1 g dry wt. Fresh leaves were ground with a mortar and pestle with 5% (v/v) TCA. The homogenates were centrifuged at 10000 rpm for 15 min at 4°C. The supernatants were used for the assays of GSH content. Reduced glu-tathione content was measured according to the method of Ellman [12]. Total polyphenols were determined by a Folin-ciocalteau colorimetric method [13]. Their content was expressed in mg/g fr wt (gallic acid equivalents). Leaf samples were homogenized in 50 mM Tris-HCl buffer (pH 7.0) containing 3 mM MgCl2, 1 mM EDTA, and 1.0% PVP and then centrifuged at 10000 rpm for 15 min at 4°C; the obtained supernatant was used for enzyme analysis. All parameters were expressed as activity per mg protein. The protein con-
centration in each fraction was determined by the method of Bradford [14], using BSA as a standard. Catalase (CAT) activity was assayed by the method of Aebi [15], and one unit of CAT was defined in ^g of H2O2 released/(mg protein min). Peroxidase (POD) and polyphenol oxidase (PPO) activities were measured by the method of Kar and Mishra [16]. One unit of POD or PPO was defined as an increase in the 0.1 units of absorbance.
Determination of GAs. The lyophilized samples were used to extract and quantify the endogenous gib-berellins according to the method of Lee et al. [17]. The lyophilized sample (0.5 g) was used for GA analysis. The extracted GAs were fractionated on 3.9 x 300 m Bondapak C18 column ("Waters") and eluted at 1.5 mL/min with the following gradient: 0-5 min, iso-cratic 28% MeOH in 1% aqueous acetic acid; 5-35 min, linear gradient from 28 to 86% MeOH; 35-36 min, 86100% MeOH; and 36-40 min isocratic 100% MeOH. Forty-eight fractions of 1.5 mL of each sample were collected. The fractions were then prepared for gas chromatography/mass spectrometry (GC/MS) with selected ion monitoring (SIM) (6890N Network GC System and 5973 Network Mass Selective Detector; "Agilent Technologies", United States). GA4 and GA5 contents were calculated from the peak area ratios of 284/286 and 416/418, respectively. The concentrations of GAs were calculated by data obtained from the peak area and were expressed in ng/g dry wt.
Determination of minerals. Minerals of sodium and calcium were analyzed by the method of Rodushkina et al. [18]. Nitric acid was added to plant samples, heated up to complete dry, and allowed to cool at room temperature. The digested sample was diluted by double distilled water, filtered through 0.5 ^m filters and injected to Inductively Coupled Plasma Mass Spec-troscopy (VG Elemental, PlasmaQuad 3, "Perkin Elmer", United States). The quantity of minerals was calculated using known standard values.
Statistical analysis. The results of morphological and biochemical analysis were expressed as mean ± SE, and SPSS software version 11.5 was used to compare the statistical difference based on Duncan's multiple range test (DMRT) at a significance level of P < 0.05.
RESULTS AND DISCUSSION
Cucumber is sensitive to environmental stresses. Plant treatment with PAs is an effective method to improve plant tolerance to abiotic stresses [2, 3, 19]. Moreover, some of the previous reports have documented that the high concentration of Spd (0.1 to 0.5 mM) improved tolerance of cucumber under stress conditions [2, 3, 7]; and this suggests that more research is needed to r
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