ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 5, с. 662-667
^ ЭКСПЕРИМЕНТАЛЬНЫЕ ^^^^^^^^^^^^
СТАТЬИ
УДК 581.1
EFFECT OF SALICYLIC ACID PRETREATMENT ON DROUGHT STRESS RESPONSES OF ZOYSIAGRASS (Zoysia japonica)1 © 2014 Z. L. Chen*, X. M. Li**, L. H. Zhang*
*School of Environmental Science, Liaoning University, Shenyang, China **College of Chemistry and Life Science, Shenyang Normal University, Shenyang, China
Received October 19, 2013
The present study was carried out to examine the effects of exogenous salicylic acid (SA) on growth, activities of antioxidant enzymes, and some physiological and biochemical characteristics of zoysiagrass (Zoysiajapon-ica Steud.) plants subjected to drought. Aqueous 0.1, 0.5, or 1.0 mM SA solution was sprayed on the leaves of zoysiagrass for 3 days. Drought was induced by withholding watering for 16 days after SA application. Biomass, chlorophyll content, net photosynthetic rate (Pn), activities of antioxidant enzymes (e.g., superoxide dis-mutase (SOD), peroxidase (POD), and catalase (CAT)), MDA and proline contents were determined. Pre-treatments with 0.1 and 0.5 mM SA significantly increased fresh and dry weights and chlorophyll content, while 1 mM SA pretreatment did not show significant change compared to controls. SA pretreatments showed a marked increase in Pn compared with controls from the 7th to 16th day after drought start. Activities of SOD, POD, and CAT were increased by SA pretreatments. MDA and proline contents after 0.1 and 0.5 mM pretreatments were lower than those of controls from the 6th to 12th day of drought, while 1 mM SA pretreatment did not show significant change from the 0th to 9th day of drought. This work suggests that suitable exogenous SA (0.5 mM) helps zoysiagrass to perform better under drought stress by enhancing the net photosynthetic rate and antioxidant enzyme activities while decreasing lipid peroxidation as compared to the controls. SA could be used as a potential growth regulator for improving plant growth under drought stress.
Keywords: Zoysia japonica — drought — salicylic acid — antioxidant enzymes — lipid peroxidation
DOI: 10.7868/S0015330314050054
INTRODUCTION
Drought stress is characterized by the reduction of water content; it is one of the major factors limiting the land plant growth. Researchers have used various indices to categorize the symptoms. These include changes in the net photosynthetic rate (Pn), chlorophyll (Chl) content, dry weight, protein biosynthesis, and solute accumulation [1]. Moreover, when plants are subjected to drought stress, some reactive oxygen species (ROS), such as superoxide (O2- ), hydrogen peroxide (H2O2), hydroxyl radicals ("OH), and singlet oxygen (1O2) are produced [2]. Generation of ROS results in lipid peroxidation, which is one of the most important damaging effects of oxidative stress [3]. To cope with ROS, plants are endowed with enzymatic antioxidant system including SOD, which catalyzes the re-
1 This text was submitted by the authors in English.
Abbreviations'. CAT — catalase; Chl — chlorophyll; NBT — ni-troblue tetrazolium; Pn — net photosynthetic rate; POD — peroxidase; SA — salicylic acid; SOD — superoxide dismutase. Corresponding author. Li Hong Zhang. School of Environmental Science, Liaoning University, Shenyang, 110036 China; fax. +86-024-6220-4818; e-mail. lihongzhang132@163.com
action from O2 to H2O2, and CAT and POD detoxifying H2O2 [4].
Salicylic acid (SA) is a common plant-produced phenolic compound that can function as a plant growth regulator, which plays an important role in seed germination, seedling establishment, cell growth, respiration, stomatal closure, senescence-associated gene expression, enhancement of enzyme activity, and photosynthesis under adverse environmental conditions [5]. More recent studies reported that the externally applied SA increased plant tolerance to several abiotic stresses, including osmotic stress [6, 7], drought [8], salinity [9], ozone or UV light [10], temperature stress [11], and heavy metal stress [12]. These studies suggest that SA may enhance the multiple types of stress tolerance in plants by interactive effects on several functional molecules.
A survey of research indicates that SA plays an important role in providing for plant drought tolerance. Hayat et al. [13] revealed that there was a significant increase in photosynthetic parameters, chlorophyll and proline contents, and antioxidant enzyme activities when tomato was treated with SA. Loutfy et al. [14] reported that SA induced drought tolerance in four wheat cultivars and increased the biomass, leaf
relative water content, and the solute contents, except proline. Moreover, the exogenous application of SA decreased the inhibitory effect of drought on Phillyrea angustifolia L. [15], Cucumismelo L. [16], and Ctenan-the setosa (Rosc.) Eichler [17].
Zoysiagrass (Zoysia japonica Steud.) is the most popular turfgrass in East Asia and widely used for home lawns, golf courses, athletic fields, and parks
[18]. Turfgrass is believed needing more water, so its growth is restricted by drought in the cities lacking water. Some studies reported about the morphological and physiological responses of zoysiagrass to drought
[19]. However, attempt has previously not been made to study how to alleviate the adverse effects of drought on zoysiagrass. Therefore, it is necessary to study how to enhance the drought tolerance of zoysiagrass by a plant growth regulator.
The main objective of this work was to study the interactive effects of SA and drought on some physiological and biochemical parameters of zoysiagrass. For this purpose, we analyzed plant growth as well as photosynthesis, enzyme activities of SOD, POD, and CAT, lipid peroxidation, and proline content.
MATERIALS AND METHODS
Plant material and growth conditions. Zoysiagrass (Zoysia japonica Steud.) plants were collected from one-year-old turfgrass plots at the Ecological Research Center, Liaoning University. They were grown in plastic pots (13 cm in diameter, 14.5 cm in depth) filled with a mixture of topsoil and coarse river sand (1 : 1) in a greenhouse (25/20°C day/night, a 16-h photoperiod, 800 ^mol/(m2 s) PAR, and 75% relative humidity) for one week. Then the pots were transferred to the experimental field and grown under natural conditions at the Liaoning University for one month. During this period, plants were watered to field capacity (>75%) every two days and fertilized, using compound fertilizer once a week. Once the assay started, fertilization was stopped.
Plants were randomized and divided into four groups of60 individuals. Aqueous 0.1, 0.5, or 1.0 mM SA solution was sprayed on the leaves of three groups until run-off three times a day for 3 days. Control plants were sprayed with distilled water. Drought was induced by withholding watering for 16 days after SA application. Thus, the treatments were as follows: (1) control: no SA pretreatment then drought; (2) 0.1 mM SA pre-treatment then drought; (3) 0.5 mM SA pretreatment then drought; and (4) 1.0 mM SA pretreatment then drought. Each treatment was carried out in triplicate.
Estimation of plant biomass. The fresh and dry weights of shoots and roots were recorded. Dry weight was obtained after sample oven-drying at 80°C for 24 h.
Determination of chlorophyll content and net pho-tosynthetic rate. Chl content was quantified using the method of Agrawal and Rathore [20]. Chl was extracted from 0.1 g of leaves with 10 mL of 80% acetone, and
the absorbance of the solution was measured at 663 and 645 nm.
Between 10:00 and 12:00 on each sampling day, gas exchange by leaves (one leaf per plant, three plants per replicate) was measured with a portable photosynthesis system (Li-6400, "Li-Cor", United States). Pn was measured under ambient CO2 (370 ^mol/mol). Pho-tosynthetic photon flux density (PPFD) was set at 800 ^mol/(m2 s) in the cuvette containing the leaf for Pn measurement.
Extraction and assays of antioxidant enzymes.
Fresh leaf sample (0.5 g) was homogenized in 5 mL of extraction buffer (0.1 M phosphate buffer, pH 6.8) with a mortar and pestle on ice. The homogenate was then centrifuged at 12000 g for 15 min at 4°C, and the supernatant was used as the crude extract for SOD, CAT, and POD assays.
SOD activity was detected according to the method of Beyer and Fridovich [21]. SOD activity was assayed by measuring the ability of the enzyme in the crude extract to inhibit the photochemical reduction of ni-troblue tetrazolium (NBT) by superoxide radicals generated photochemically. One unit of SOD was defined as the amount of enzyme required to inhibit the reduction of NBT by 50% at 25°C.
POD and CAT activities were assayed following the method of Chance and Maehly [22] with some modifications. The POD reaction solution contained 50 mM phosphate buffer (pH 7.8), 25 mM guaiacol, 20 mM H2O2, and the enzyme extract. Changes in ab-sorbance of the reaction solution at 470 nm were determined. CAT activity was assayed in a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0, containing 0.1 mM EDTA), 20 mM H2O2, and the enzyme extract. The reaction was started with the addition of the supernatant, and the decomposition rate of H2O2 was followed at 240 nm.
Estimation of lipid peroxidation. Lipid peroxidation of the leaves was measured in terms of MDA content. The MDA content was determined using the method of Fu and Huang [23]. Fresh leaf sample (1.0 g) was homogenized with 4 mL of 0.1% (w/v) trichloroacetic acid (TCA) in an ice bath. The homogenate was cen-trifuged at 12000 g for 20 min, and the supernatant was used for lipid peroxidation analysis. To the 1-mL aliquot of the supernatant, 4 mL of 0.5% thiobarbituric acid in 20% TCA was added. The mixture was incubated in boiling water for 30 min. MDA content was then detected spectrophotometrically at 532 nm and corrected for nonspecific turbidity at 600 nm.
Measurement of proline content. Free proline content
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