ФИЗИОЛОГИЯ РАСТЕНИЙ, 2013, том 60, № 6, с. 828-833
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
INVOLVEMENT OF NITRIC OXIDE IN ACQUIRED THERMOTOLERANCE
OF RICE SEEDLINGS1
© 2013 L. Song, H. Zhao, M. Hou
School of Ecological Technology and Engineering, Shanghai Institute of Technology, Shanghai, P.R. China
Received November 7, 2012
The role of nitric oxide (NO) in thermotolerance acquired by heat acclimation (38°C) was investigated. Results showed that 38°C acclimation, on the one hand, obviously reduced hydrogen peroxide (H2O2) and MDA content and ion leakage degree in rice (Oryza sativa L.) leaves; however, on the other hand, it increased the survival of rice seedlings under 50°C heat stress. Application of nitric oxide donor, sodium nitroprusside (SNP), prior to 38°C acclimation dramatically increased the acquired thermotolerance. To elucidate the role of endogenous NO in acquired thermotolerance, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, a specic NO scavenger) was used (scavengers are used to control the level of both exogenous and endogenous NO). Results showed that PTIO pretreatment resulted in the elimination of acquired thermotolerance induced by 38°C acclimation in rice seedlings. Nitric oxide (NO) release measurement indicated that there was indeed an abrupt elevation in the NO content in 40 min after 38°C acclimation, proving the involvement of NO in acquired thermotolerance inducement in rice seedling.
Keywords: Oryza sativa — seedlings — nitric oxide — acquired thermotolerance — heat stress
DOI: 10.7868/S0015330313060146
INTRODUCTION
Plants have both inherent (basal) and acquired thermotolerance to short and sublethal high temperature acclimation [1, 2] or gradual increase to lethal high temperature [3]. Previous research indicated that field performance of plants under high temperature agreed with the diversity of thermotolerance acquired by acclimation treatment [4]. Therefore, acquired thermotolerance is considered to be one of the innate mechanisms contributing to plant thermotolerance [5] and has been used to identify thermosensitive and thermotolerant genotypes of monocots, including wheat, pearl millet, sorghum, and dicots, such as sunflower, pea, and groundnut [4, 6, 7].
As an important signaling molecule in plants, nitric oxide (NO) is involved in many physiological processes, such as seed germination, leaf expansion, cell senescence, ethylene emission, stomatal closure, and programmed cell death, and responses to abiotic and
1 This text was submitted by the authors in English.
Abbreviations'. APX - ascorbate peroxidase; CAT - catalase; LOX -lipoxygenase; POD - peroxidase; PTIO - 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; SNP - sodium nitroprusside; SOD - superoxide dismutase. Corresponding author. Lili Song. School of Ecological Technology and Engineering, Shanghai Institute of Technology, Shanghai 201418, P.R. China. Fax. +86-21-6494-3128; e-mail. SongLL713@yahoo.com.cn
biotic stresses, such as drought, salinity, UV-B-radiation, high temperature, disease [8-12]. Earlier works have revealed the protective effect of exogenous NO under heat stress and the function of endogenous NO in basal thermotolerance [13, 14], but its role in acquired thermotolerance is little known. In this study, we provide the evidence of the NO involvement in acquired thermotolerance of rice seedlings.
MATERIALS AND METHODS
Plant material and high temperature treatment. Rice (Oryza sativa L. cv. Zhongyou No. 9801) seeds were surface-sterilized in 2% (v/v) sodium hypochlorite solution for 30 min, rinsed three times with de-ionized water, and then soaked in de-ionized water overnight at 25°C in the dark. After accelerating germination at 25°C for 2 days, germinated seeds were grown in pots containing sterilized soil at 25°C. Light intensity was maintained at 180 ^mol/(m2 s) with a 16-h photoperiod. Ten days later, the seedlings were subjected to various treatments. Freshly prepared chemical solutions (sodium nitroprus-side (SNP, NO donor, 0.02 mM), 2-phenyl-4,4,5,5-tet-ramethyl-imidazoline-1-oxyl-3-oxyde (PTIO, NO scavenger, 0.1 mM), or potassium ferrocyanide (residual product of SNP, 0.02 mM)) were sprayed on the seedling leaves until drips formed. The infiltration period lasted for 45 min at a light intensity of
180 ^mol/(m2 s). Then the seedlings were subjected to 50°C for 2 h or 38°C for 2 h followed by 2 h at 25°C before 2 h at 50°C. These treatments were adopted in order to show significantly different phenotypes between control and treated seedlings (data not shown).
Survival determination. After treatments, the seedlings were transferred to 25°C to recover for seven days, during which the light intensity was kept at 180 ^mol/(m2 s) with a 16-h photoperiod. Then the survival rate was determined by the percentage of survival in all treated seedlings.
Ion leakage. Relative ion leakage was determined according to Song et al. [13]. The rice leaves (10 tips for each sample) were placed in Petri dishes with 10 mL of de-ionized water at 25°C for 2 h. After the incubation, the conductivity in the bathing solution was determined (C1). Then, the samples were boiled for 15 min, and the conductivity was measured again (C2). Electrolyte leakage was calculated according to the equation: relative ion leakage (%) = C1/C2 x 100.
H2O2 production. Hydrogen peroxide (H2O2) contents were determined by the POD-coupled assay protocols described by Vljovic-Jovanovic et al. [15]. About 0.1 g leaves were ground in liquid N2, and the powder was extracted in 2 mL of1 M HClO4 with the presence of insoluble polyvinylpyrrolidone (5%). The homogenate was centrifuged at 12000 g for 10 min, and the supernatant was neutralized with 5 M K2CO3 to pH 5.6 in the presence of 100 mL of 0.3 M phosphate buffer, pH 5.6. The solution was centrifuged at 12000 g for 1 min, and the sample was incubated for 10 min with 1 unit of ascor-bate oxidase to oxidize ascorbate prior to assay. The reaction mixture was composed of 0.1 M phosphate buffer, pH 6.5, 3.3 mM 3-(dimethylamino)benzoic acid, 0.07 mM 3-methyl-2-benzothiazoline hydrazone, and 0.3 units of peroxidase. The reaction was initiated by the addition of sample (200 mL). The ab-sorbance change at 590 nm was monitored at 25°C.
Lipid peroxidation. Lipid peroxidation was measured in terms of malondialdehyde (MDA) content following the method of Heath and Packer [16]. Leaves (0.5 g) were homogenized with a mortar and pestle in 10% TCA, and then the homogenate was centrifuged at 4000 g for 30 min. A 2-mL aliquot of the supernatant was mixed with 2 mL of 10% TCA containing 0.5% thiobarbituric acid. The mixture was heated at 100°C for 30 min. The absorbance of the supernatant was measured at 532 nm, with a reading at 600 nm subtracted from it to account for nonspecific turbidity. The amount of MDA was calculated using an extinction coefficient of 155/(mM cm).
LOX activity assay. Lipoxygenase activity (LOX, EC 1.13.11.12) was determined according to Page et al. [17] with some modifications. LOX activity was assayed
in the citric acid—sodium phosphate buffer (100 mM, pH 4.5) using linoleic acid sodium salt (0.80 mM) as a substrate by following the production of conjugated dienes at 234 nm (s =25/(mM cm)).
NO content. NO content was determined as described by Murphy and Noack [18]. The method is based on the direct reaction between NO and oxyhae-moglobin (HbO2), which yields methaemoglobin (metHb). Oxyhaemoglobin was prepared by the reduction of 25 mg of methaemoglobin to haemoglobin using 4 mg sodium dithionite in 1 mL of 50 mM phosphate buffer (pH 7.4), followed by oxidation. The oxy-haemoglobin solution was desalted by passing it through a Sephadex G-25 column eluted with 50 mM phosphate buffer (pH 7.4), and its concentration was estimated spectrophotometrically at 415 nm using an extinction coefficient of 131/(mM cm). Fresh leaves (0.5 g) from rice seedlings were incubated with 100 units of catalase (CAT) and 100 units of superoxide dismutase (SOD) for 5 min to remove ROS before the addition of3 mL of oxyhaemoglobin (5 mM). After a further 2-min incubation, nitric oxide concentrations were estimated by following the conversion of oxyhaemoglobin (HbO2) into methaemoglobin (metHb) spectrophotometrically at 577 and 591 nm (A577 HbO2 — A591 metHb, As = 11.2/(mM cm)).
Statistical analysis. Each experiment was repeated at least three times. Statistical analysis was performed using the ANOVA test.
RESULTS
Influence of heat acclimation and NO on survival
As shown in fig. 1, the survival under 50°C treatment was 22.6%. Pretreatment at 38°C for 2 h significantly elevated the survival (to 33.1%), suggesting the effect of sublethal high temperature acclimation on thermotolerance triggering. SNP pretreatment followed by further 38 °C acclimation increased the survival rate to 42.3%. However, PTIO pretreatment completely blocked the triggering of thermotolerance by 38°C acclimation or 38°C acclimation combined with SNP pretreatment (fig. 1).
Influence of heat acclimation and NO on ion leakage
Under 50°C, ion leakage of rice leaves increased to 262% of the control. Pretreatment at 38°C for 2 h significantly alleviated membrane injury and the ion leakage decreased to 190%. In the presence of SNP pretreatment in combination with 38°C acclimation, the ion leakage declined to 153% of the control. PTIO pretreatment resulted in the deprivation of the protective effect of 38°C pretreatment or SNP pretreatment plus 38°C acclimation (fig. 2).
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Fig. 1. Effect of heat acclimation and NO on survival of rice seedlings.
0.02 mM SNP, 0.1 mM PTIO, and 0.02 mM potassium ferrocyanide were sprayed on the seedling leaves, respectively. After 45 min, the seedlings were subjected to 50°C for 2 h or 38 °C for 2 h followed by 2 h at
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