ФИЗИОЛОГИЯ РАСТЕНИЙ, 2011, том 58, № 2, с. 195-201
EFFECT OF SALINITY ON OSMOTIC ADJUSTMENT CHARACTERISTICS
OF Kandelia candel © 2011 Z. Zhu*, Z. M. Pei*, **, H. L. Zheng*
*Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University,
Xiamen, PR China **Department of Biology, Duke University, Durham, USA Received February 12, 2010
To elucidate the osmotic adjustment characteristics of mangrove plants, inorganic ion and organic solute contents of intermediate leaves were investigated in 3-month-old Kandelia candel (L.) Druce seedlings during 45 days of NaCl treatments (0, 200, and 500 mM NaCl). The contents of Na+, Cl-, total free amino acids, proline, total soluble sugars, pinitol and mannitol increased to different degree by salinity, whereas, K+ content decreased by salinity compared with control. NaCl treatment induced an increase of inorganic ion contribution while a decrease of organic solute contribution. It was concluded that accumulating a large amount of inorganic ions was used as the main osmotic adjustment mechanism under salinity treatment. However, accumulation of organic osmolytes might be considered to play much more important role in osmoregulation under severe salinity (500 mM NaCl) than under moderate salinity (200 mM NaCl), thus the damage caused by high toxic ions (Na+ and Cl-) concentration in K. candel leaves could be avoided.
Keywords: Kandelia candel — salt — osmotic adjustment — inorganic ions — organic solutes
Salinity is the major environmental factor limiting plant growth and productivity . It is difficult for plants to absorb water from saline environment, thus they can not easily survive. Osmotic adjustment is an effective mechanism for plants, especially for halo-phytes, to be resistant to salt environment. Two intracellular processes contribute to osmotic adjustment: uptake of inorganic ions and accumulation of organic solutes . Ions uptake consumes less energy compared with synthesizing organic solutes , and halo-phytes tend to absorb inorganic ions for their osmotic adjustment, mainly Na+ and Cl-. Inorganic ions can be sequestered into the vacuole, so that the damage of cell function and structure caused by the toxic ions (particularly Na+ and Cl-) can be avoided . Because of the accumulation of inorganic ions in the vacuole, the cytoplasm will be laid between two stresses: internal stress from the accumulated ions in the vacuole and external stress . So, plants accumulate organic solutes as compatible solutes, such as amino acids (e.g., proline), soluble sugars (e.g., sucrose), polyols (e.g., mannitol), and betaines (e.g., glycinebetaine), in the cytoplasm to balance the osmotic pressure inside
Abbreviations'. AA — total free amino acids; GB — glycine betaine; Ys — osmotic potential; SS — total soluble sugars. Corresponding author. H. L. Zheng. Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361005, PR China. Fax. 86-592-218-1015; e-mail. firstname.lastname@example.org
the cells and they do not interfere with normal biochemical reactions [6, 7].
Mangroves are the characteristic intertidal plant formations of sheltered tropical and subtropical coastlines . The most striking feature of mangroves is their ability to tolerate NaCl to seawater level (ca. 500 mM NaCl) , and they are regarded to be more salt tolerant than any other species. Therefore, mangroves are considered as potential models for studying the mechanisms of salt tolerance . Under natural conditions, different mangrove species do not have the same osmotic solute accumulation patterns . Moreover, some osmotic solutes, including the total sugar, the total amino acids, and proline, increased in various degree under NaCl treatment . However, the mechanism of osmotic adjustment in mangroves has not been well explained, and how osmotic adjustment contributes to salt adaptation of mangrove plants remains obscure.
In this study, Kandelia candel (L.) Druce, which is the most widely spread species in the coastal wetland of China, was used to analyze osmotic adjustment characteristics grown under greenhouse condition. In a number of studies, it was shown that suitable salinity for K. candel ranged from 0 to 260 mM NaCl, while the growth was inhibited by 430 mM NaCl and above [13, 14]. Leaf area, plant height and dry weight were greater in the culture treated with 85—260 mM NaCl [13, 14]. Among all the osmotic solutes determined in previous studies, the contents of Na+, Cl-, soluble sugars, and proline increased by salinity treatment and
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K+ content decreased after NaCl application [13, 14]. In natural environment, pinitol or mannitol accumulated in K. candel leaves . Accumulation of these solutes in K. candel leaves might involve in osmotic adjustment. However, most studies only considered the changes of the solute content under salt stress, few works take a broader look at more than one or two solutes at a time, and they rarely analyze and systematically discuss about the contribution of individual solutes to osmotic adjustment. Therefore, the purpose of this paper was (a) to assess the influence of different salinity on the content of some inorganic ions (Na+, Cl-, and K+) and organic solutes (free amino acids, proline, soluble sugars, and polyols); (b) to evaluate what kind of roles in osmotic adjustment is played by each inorganic ion and organic solute; and (c) to analyze the similarities and differences of osmotic adjustment mechanism when K. candel responses to different saline environment.
MATERIALS AND METHODS
Plant materials and treatment conditions. Viviparous seeds of Kandelia candel (L.) Druce were collected from the Nature Reserve for Mangrove in Estuary of Jiulong River, Fugong Town, Longhai County, Fujian Province, China (24°29' N, 117°55' E). The average seawater salinity in the nature reserve was around 17%c . Viviparous seeds were planted in plastic pots filled with washed river sand. The plastic pots were 14 cm tall, 12 cm diameter, and with holes at the bottom. Three plastic pots were placed into one plastic tank, which was 15 cm tall and 30 cm diameter. 1.5 l of culture solution was added into each plastic tank. Viviparous seeds were raised with tap water in the greenhouse (29 ± 2°C, 65 ± 5% relative humidity, and an irradiance of 400-800 ^mol/(m2 s)). One-month-old healthy seedlings were irrigated with half-strength Hoagland nutrient solution. After the third pair of leaves had expanded (about three months after germination), healthy seedlings of uniform size were divided into three groups for salinity treatment: (1) 0 mM NaCl; (2) 200 mM NaCl; (3) 500 mM NaCl. The saline solutions with different NaCl concentration were prepared with half-strength Hoagland solution. At the initiation of the experiment, NaCl concentration of all treatments was gradually increased by 100 mM every day. Solutions in the plastic tanks were renewed each week and restored to a constant salinity from evaporation losses by adding water. Salinity level was monitored every day by a hand-held refractometer (Model S-10E, "ATAGO", Japan). The second pairs of leaves from the apex of the growing shoots were harvested after 0, 7, 15, 30, and 45 days of treatment for assay.
Determination of total osmotic potential (¥s), inorganic ions and organic solute content. For the measurement of ¥s, leaf blades were placed in a sealed plastic bag and frozen in liquid nitrogen. Frozen leaves were put into a syringe to thaw at room temperature.
The liquid squeezed from the leaves was put into a vapor pressure osmometer model 5520 ("Wescor", United States) to determine total osmotic potential. Mean value of three replicates was used.
All leaf samples were dried at 80°C for 48 h and the ground samples were ashed in a muffle oven at 500°C for 4 h. Ashes were dissolved in 0.1 M HNO3 and stored for Na+ and K+ analysis. Na+ and K+ were analyzed by an inductively coupled plasma mass spectrometry (ICP-MS) (Model ELAN DRC-e, "Perkin-Elmer", United States). Cl- extracts were prepared by hot-water extraction from ashes, and Cl- was estimated by titration against silver nitrate using potassium chromate as an indicator following the method of Cle-sari et al. . All the ion concentrations were expressed on a tissue water basis.
The frozen leaves were ground in 10% acetic acid, and total free amino acids were estimated following the method of Moore and Stein  using ninhydrin reagent. Proline was extracted from dry leaves using 3% sulfosalicylic acid. Determination of free proline content was according to the method of Bates et al. . Total soluble sugars were extracted from ground dry leaves after boiling with distilled water for 15 min twice. Concentrations of total soluble sugars were determined by anthrone-sulfuric acid method of McCready et al. . Ground dry leaf samples were used for pinitol and mannitol determination. Samples were dispersed in 100 ml of Mili-Q water and kept in a 90°C water bath for 3 h. The mixture was subsequently sonicated for 30 min, filtered through a 0.22-mm Millex-GS syringe driven filter, and then concentrated to 1 ml. Pinitol and mannitol contents were determined by capillary electrophoresis (Model Biofocus 3000, "Bio-Rad", United States) . All the organic solute concentrations were expressed on a tissue water basis.
Calculation of estimated osmotic potential and solute contribution to ¥s. The estimated osmotic potential of each inorganic ions and organic solutes were calculated according to the van't Hoff equation :
¥s = -nRT,
where R = 0.0083143 l MPa/(mol K), T = 298.15 K, and n is the concentration of the solute, whose value is based on measurements of mmol/l of plant tissue water. The contribution of the solute to was determined as the ratio
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