ФИЗИОЛОГИЯ РАСТЕНИИ, 2010, том 57, № 6, с. 841-848
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
SALT STRESS RESPONSES OF A HALOPHYTIC GRASS Aeluropus lagopoides AND SUBSEQUENT RECOVERY
© 2010 Hamid Sobhanian*, **, ***, ****, Nasrin Motamed*, Ferdous Rastgar Jazii**, Khadija Razavi**, \&hid Niknam*, Setsuko Komatsu***
*School of Biology, College of Science, University of Tehran, Tehran, Iran **National Research Center for Genetic Engineering and Biotechnology, Tehran, Iran ***NationalInstitute of Crop Science, Tsukuba, Japan ****Payame Noor University, Tehran, Iran Received February 25, 2009
To investigate the salt tolerance mechanisms, Aeluropus lagopoides (L.) Trin. as a halophytic plant was used. Plants were treated with 0, 150, 450, 600, and 750 mM NaCl and harvested at 0, 4, 8, and 10 days after treatment and 1 day and 1 week after recovery. Optimal growth, measured as fresh and dry weights, occurred at 150 mM NaCl, but it was suppressed by 450, 600, and 750 mM NaCl. Recovery significantly increased fresh and dry weights only in 750 mM NaCl-treated plants. Water content was decreased after NaCl treatment and increased after recovery. Na+ and proline contents and activity of superoxide dismutase (SOD) were increased after NaCl treatment and decreased after recovery in all treated plants. In contrast, K+ content and ascorbate peroxidase activity decreased after NaCl treatment and increased after recovery in all treated plants. Catalase (CAT) was activated only in 750 mM NaCl-treated plants. Total content of soluble protein was slightly changed after NaCl treatment. It was concluded that proline accumulation for osmotic adjustment,
SOD activation for O2 scavenging, and CAT activation at the higher level of salt stress to detoxify produced H2O2 were main A. lagopoides strategies under salt stress. A. lagopoides salt tolerance was not based on the restriction of Na+ uptake.
Key words: Aeluropus lagopoides — antioxidant enzymes — growth — halophyte — ion content — proline — recovery — salt stress
INTRODUCTION
The halophytic plant, Aeluropus lagopoides from Poaceae family, is a stoloniferous perennial grass that has a C4 type of photosynthesis. It is distributed in the regions with intermediate salinity and semi-desert climate on Iranian plateau and is one of the animal forage plants [1]. Vegetative propagation is one of the advantages of A. lagopoides to produce identical plant lines, which are needed for investigation of molecular and physiological aspects of adaptation to harsh environmental conditions. The patterns of ion secretion by A. lagopoides as a powerful strategy to cope with salt stress were reported [2]. A. lagopoides is very important plant because it can tolerate high salt and drought stress levels; it belongs to Poaceae, which is an important family of crops, including wheat and rice. Colmer et al. [3] have reported that it is possible to use wild relatives to improve wheat salt tolerance. Wei et al. [4]
Abbreviations'. APX — ascorbate peroxidase; CAT — catalase; SOD — superoxide dismutase.
Corresponding author. Nasrin Motamed. School of Biology, College of Science, University of Tehran, Tehran 14155-6455, Iran. Fax. +98-21-6640-5141; e-mail. motamed2@khayam.ut.ac.ir
have reported the transfer of salt tolerance to wheat from A. littoralis, which is its wild and remote relative. To investigate the salt tolerance transfer to hybrid clones, proline content and the Na+/K+ ratio were analyzed, and these two parameters were increased in hybrid clones under salt stress [4].
NaCl inhibits plant growth by osmotic or water-deficit effect and salt-specific or ion-excess effect [5]. Several roles for proline in plant are osmotic adjustment, stabilizing subcellular structures, scavenging free radicals, and buffering the cell redox potential under stress conditions [6]. Proline can also induce expression of salt stress-responsive genes, which have proline-responsive elements in their promoters [7]. Earlier, it has been shown that proline content in A. lagopoides was dramatically increased under drought stress [8].
The effect of salt stress on plant growth depends on the plant species. In Salicornia rubra, fresh and dry weights increased at 200 mM NaCl, but a further increase in the NaCl concentration suppressed growth [9]. High Na+ uptake competes with the uptake of other nutrient ions, especially K+, and this decreases the
K+ content, leading to K+ deficiency [5]. Salt stress imposes a water deficit and leads to oxidative stress as the secondary effect of salinity. Plants can synthesize various antioxidant enzymes to neutralize ROS under stresses. The metalloenzyme superoxide dismutase
(SOD) converts O2- into H2O2, catalase (CAT) and a variety of peroxidases catalyze the breakdown of H2O2
[10]. The mechanisms of salt tolerance are not fully investigated.
The aim of present study was to get some insight into the physiological bases of A lagopoides responses to salt stress and consequent recovery. This can help to understand salt tolerance mechanisms and to identify salt-inducible genes in this halophyte plant from Poaceae family in the future.
MATERIALS AND METHODS
Plant material and NaCl treatments. Spikelets of A. lagopoides (L.) Trin. were harvested from a population on the vegetation zone of Kashan Kavir in the central regions of Iran. The collection site has 34°25' latitude, 53°15' longitude, an altitude of1209 m above sea level, and 90 mm of annual rainfalls. Seeds were separated from inflorescence and stored at 4°C; then about 30—40 seeds were germinated in pots (250 ml in volume) containing the mixture of sand and vermicu-lite (1:2) and placed in the phytotron (Model BDW120, "Conviron", Canada) under a 16-h photo-period (white fluorescent lamps; 46 ^mol/m2), a day/night temperature of 25/20°C, and a mean humidity of 60%. Every pot was gently irrigated every day with 150 ml of Hoagland nutrient solution (pH 6.0). After 1 month, plants were treated with Hoagland nutrient solution (pH 6.0) containing 0, 150, 450, 600, and 750 mM NaCl. Electrical conductivity of drained water from every pot was estimated to prevent salt accumulation in pots. For recovery, every pot was gently irrigated every day again with 150 ml of Hoagland nutrient solution without NaCl. Plants were harvested at 0, 4, 8, and 10 days after NaCl treatment and 1 day and
1 week after removing NaCl treatment for recovery. Three independent experiments were carried out, and 200 plants were used in each experiment.
Dry weight, water content, and proline content measurements. For dry weight measurement, fresh shoots from control and treated plants were oven-dried at 70°C for 24 h. Percentage ofwater content was calculated as (water weight/fr wt) x 100. Proline content was quantified using the acid ninhydrin method
[11]. A portion (500 mg) of liquid nitrogen-powdered shoots was homogenized in 10 ml of 3% aqueous sul-fosalicylic acid, and the homogenate was centrifuged at 2000 g for 5 min. The extract (2 ml) was mixed with
2 ml of acid ninhydrin and 2 ml of glacial acetic acid and kept for 1 h at 100°C. The reaction mixture was extracted with 4 ml of toluene. The chromophore-containing toluene was separated, and the absorbance
was recorded at 520 nm. High-speed J2-21M centrifuge ("Beckman", United States) and UV-visible spectrophotometer ("Shimadzu", Japan) with 10-mm quartz cells were used for centrifugation of the extracts and determination of the absorbance, respectively.
Determination of K+ and Na+ contents. Fresh leaves were washed with water to remove ions secreted by the glands. Dried samples (20 mg) were digested with 8 ml of 70% perchloric acid at 300°C until their color converted to black, and then they were cooled to colorless again [12]. Then the volume of samples was adjusted to 50 ml with distilled water, and the contents of K+ and Na+ were determined with a flame photometer ("Jen-way", United Kingdom).
Protein extraction. For determination of antioxi-dant enzyme activities, a portion of shoots (1 g) was homogenized in a chilled mortar with 5 ml of 0.1 M Tris-HCl buffer (pH 7.0) containing 5 mM EDTA and 5 mM dithiothreitol. The homogenate was centri-fuged at 13000 g for 5 min at 4°C. The supernatant was centrifuged again at 13000 g for 5 min at 4°C and then transferred to Eppendorf tubes, and the samples were kept on ice at 4°C [13, 14]. The extracts were used to assay the total content of soluble protein and activities of SOD and CAT; for assay of APX, 5 mM ascorbate was added to the extraction buffer [15]. Total content of soluble protein was measured at 595 nm by the method of Bradford [16] using BSA as a standard. High-speed centrifuge and UV-visible spectropho-tometer with 10-mm matched quartz cells were used for centrifugation of the extracts and determination of the absorbance, respectively.
Determination of catalase activity. CAT activity was measured according to the method of Mishra and Kar [17] by a decline in the extinction of H2O2 at 240 nm. The assay mixture (2.86 ml) consisted of 45 mM Tris-HCl buffer (pH 7.0), 0.3 ml H2O2, and 60 ^l of the crude enzyme. The absorbance was recorded at 240 nm. CAT activity was expressed in units per mg protein.
Determination of ascorbate peroxidase activity.
APX activity was analyzed following the method of Nakano and Asada [18] by measuring the increase in absorbance at 290 nm. The reaction mixture (2.5 ml) contained 2 ml ofphosphate buffer (pH 7.0), 0.2 ml of 3% H2O2, 4 mM ascorbate, and 100 ^l of the crude enzyme. The absorbance was recorded at 290 nm. APX activity was expressed in units per mg protein.
Determination of superoxide dismutase activity.
SOD activity was analyzed by measuring its ability to inhibit the photochemical reduction of nitroblue tet-razolium, as described by Beauchamp and Fridovich [19]. The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 ^M nitroblue tetrazolium, 0.1 mM EDTA, 2 ^M riboflavi
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