ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 2, с. 249-256
= ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ =
EFFECTS OF HIGH SALINITY ON PHYSIOLOGICAL AND ANATOMICAL INDICES DURING THE EARLY STAGES OF Populus euphratica GROWTH1
© 2015 V. D. Rajput***, Y. Chen*, M. Ayup*
* State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi, China ** University of Chinese Academy of Sciences, Beijing, China Received June 3, 2014
The effects of salinity stress on stomatal aperture and density, xylem vessels, the activities of antioxidant enzymes, such as superoxide dismutase (SOD) and peroxidase (POD), and xylem embolism (PLC values) in Populus euphratica in the arid ecosystem of China were studied. Pot experiment was conducted at different concentrations of salt (50, 100, 150, and 200 mM NaCl) contained in the water used for irrigation during three months. The POD activity increased with the increase in the severity of NaCl stress, but SOD activity varied at different salt levels. Results indicated that salt treatment reduced stomatal aperture and thus, evidently, leaf photosynthetic capacity. The significant reduction in the stomatal area, in the length of stomata openings, and an increase in stomata density were noticed. Salinity stress affected water transport, reducing native PLC value, whereas xylem vessel section areas were also decreased. Presented results open the possibility of genetic improvement for selecting the salt-tolerant Populus spp. to reclaim salinized lands.
Keywords: Populus euphratica - PLC value - salinity - POD - SOD - xylem anatomy
The Tarim River and the Heihe River are two most important and largest inland rivers in Western China. Their downstream areas are known as a green corridor covered with lush riparian forests several miles wide. World's 54% area of Populus euphratica vegetation is spread in this basin  and mainly distributed on river-banks or areas with deep water tables . Salinity is one of the major abiotic stresses limiting plant growth and productivity, particularly in arid land. Researchers showed that salinity caused some anatomical and physiological modifications in plants viz. changes in tracheary element density, lumen volume, vessels diameter [3-5], stomatal density, shape, and size , plant growth, and activities of antioxidant enzymes [7-9]. The effect of abiotic stress on hydraulic traits in riparian plants is highly correlated with plant anatomy viz. changes in fibers, pith membrane, etc. [10, 11]. Vessels of halophytic trees are more numerous and narrower than those of mesophytic ones.
1 This text was submitted by the authors in English.
Abbreviations: PLC% - percentage loss of hydraulic conductivity; POD - peroxidase; SOD - superoxide dismutase. Corresponding author: Chen Yaning. State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China; e-mail: firstname.lastname@example.org
Plants protect themselves from ROS-induced oxidative damage through both enzymatic and non-enzymatic defense mechanisms , including the activation of antioxidant enzymes, such as superoxide dismutase (SOD) and peroxidase (POD) [7-9]. The degree of oxidative stress experienced by the cell is determined by the levels of superoxide, H2O2, and hy-droxyl radicals generated. The antioxidant activities are crucial for suppressing toxic ROS levels within cells . SOD and POD have been considered as two key enzymes in ROS elimination . The estimation of antioxidants is necessary to know the adaptability of P. euphratica under salinity stress conditions.
Plants adjust their xylem hydraulic traits under various stress conditions through developing xylem more resistant to drought-induced cavitation , reducing root hydraulic conductance , maintaining the high hydraulic efficiency in stems and roots , and also by increasing the whole plant leaf specific conductivity  and changes in the xylem sap. Hydraulic conductivity and embolism are the important factors constraining plant survival and its productivity. These factors are correlated with the xylem structure and functions .
Various studies have been made to understand the variations in xylem anatomical structures and hydraulic functions at the inter-specific, intra-specific, and intra-plant level . According to Zwieniecki et al. , the
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ionic content of the xylem sap has a significant effect on the plant hydraulic conductivity. The hydraulic stress adaptation of P. euphratica involves cell wall modifications and suppresses xylem development . Many researchers performed their study in hydroponics and controlled conditions. There are hardly any reports about physiological and anatomical effects of salinity in early stages of P. euphratica life under natural conditions.
Therefore, it is important to further understand the salinity effect on P. euphratica, especially on stomatal aperture, xylem state, and their relation to hydraulic conductivity and antioxidants. Present study deals with the effects of salinity stress on xylem vessels, antioxidant enzyme activities, stomatal aperture, and hydraulic conductivity (PLC, %) at early stage of P. euphratica growth to gain more information for assessing its capacity to adapt to high saline environment.
MATERIALS AND METHODS
Plant source and maintenance. One-year-old Populus euphratica seedlings have been selected from the nursery located at downstream of Tarim River (41.68° N, 86.06° E), Xinjiang Uygur Autonomous Region, Northwest China. The average height of seedlings above the soil was 60 ± 2.68 cm. There were no leaves on seedlings at a time of planting. Plants were planted into pots containing grey desert soils (organic matter 6.26 ± 1.56 g/kg, total nitrogen 0.11 ± ± 0.04 g/kg, phosphorous 0.17 ± 0.07 g/kg, potassium 21.18 ± 0.19 g/kg, and total salts 4.73 ± 0.05 mg/g, the pH and electrical conductivity of soils were 7.8 and 0.93 (mS/cm), respectively) for experimentation at research station situated at Urumqi, Xinjiang, China (43.46° N, 87.36° E). Each pot contains 23.48 ± ± 0.17 kg soil on dry weight basis. Pots were immediately irrigated with tap water and seedlings were rooted within a month and well survived in 45 days. Thereafter, well survived plants were treated with NaCl with irrigation water. After three months of such irrigation, soils were analyzed and salt accumulation was calculated. Salt concentrations were increased due to the accumulation of NaCl from 4.73 (0 mM) to 59.88 mg/g (200 mM).
Salt treatment. Salt treatment was imposed by irrigation with 1 L of tap water with 50, 100, 150, and 200 mM NaCl once in a week for three months. We irrigated control plants with 1 L of water containing no salt. Each treatment was performed in 10 replicates. Experimental pots were kept in natural conditions facing low precipitation.
Sample collection. Samples were collected after three months of salt treatments along with control ones and immediately transported in the laboratory for analyses. Healthy leaves were collected for the observation of stomatal aperture (density, area, and size) and antioxidant enzyme activities (SOD, POD),
whereas stems of similar diameter were collected for anatomical and hydraulic trait measurements.
Measurement of stomatal aperture. Fresh leaf samples were obtained and prepared for scanning electron microscopy (SEM) to observe stomatal aperture. Leaf samples were re-cut into 5-10 mm length segments, kept in increasing order of ethanol (50, 70, 90, and 100%) for 30 min each for dehydration and air-dried for 12 h at room temperature. Sample segments were fixed to aluminum stubs with electron conductive carbon cement ("Neubauer Chemikalien", Germany), and stomatal aperture and density were observed by SEM. Ten images were obtained from different sites of each sample. Images from SEM were processed by ImageJ software and the observation of stomatal density was performed by circling 1 mm2 area.
Anatomical measurements. Cross sections were obtained from P. euphratica stems 5.50-6.50 mm in diameter and immediately fixed in a FAA solution consisting of formalin, acetic acid, and ethanol (5 : 5 : 90, v/v/v). Samples were immediately dehydrated with alcohol for better slices, and paraffin section method was used for making these sections . Thin cross sections (7-9 ^m) were cut by slide microtome, double-stained with 1% safranin and 1% fast green dye, and observed under a light Olympus, BX51 microscope ("Olympus", Japan) equipped with a digital camera (Olympus U-TV0.5XC-3). Images were analyzed with ImageJ software. We mainly focused on the vessel section area in this context. The average of vessel section area was calculated on the basis of 50 vessels for each treatment.
Extraction and assay of antioxidant enzymes. Leaves were harvested, quickly frozen in liquid nitrogen, and further stored at -80°C until assays of antiox-idant enzyme activities. For enzyme extraction and assays, 0.20 g of leaves were frozen in liquid nitrogen and then ground with a mortar and pestle in 4 mL of the solution containing 50 mM phosphate buffer (pH 7.0) and 1% (w/v) polyvinylpyrrolidone (PVP) under low temperature maintained by ice-tray and centrifuged at 15 000 rpm for 15 min at 4°C, and the supernatant was collected for enzyme assays .
The activity of SOD (EC 18.104.22.168) was measured as described by Giannopolities and Ries . The assay medium contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 ^M p-nitro blue tetrazolium chloride (NBT), 1.30 mM riboflavin, 0.10 mM EDTA-Na, and 100 mL of the enzyme extract. Riboflavin was added last; the test tubes were placed in the reaction chamber with fluorescent light (4000 lx), and the reaction was terminated after one minute by turning off the light. The absorbance was read at 560 nm.
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