Effect of Salt Stress on Ammonium Assimilation Enzymes of the Roots of Rice (Oryza sativa) Cultivars Differing in Salinity Resistance (不同耐盐性水稻幼苗根氨同化酶对盐胁迫的反应)

Effects of NaCl-stress on ammonia assimilation enzymes and the related parameters were determined in the roots of three rice (Oryza sativa L.) cultivars differing in salt tolerance. The results showed that the activities of glutamine synthetase (GS) and NADH-dependent glutamate synthase (NADHGOGAT), as well as the levels of soluble protein decreased under high concentration salt. The influence extent was in accordance with Zao-hua 2 (salt-sensitive), Jin-zhu 1 (normal cultivar) and Jin-dao 779 (saltresistant), which was consistent with their salt-tolerance. Nevertheless, under the stress of high salt concentration, NADH-dependent glutamate dehydrogenase (NADH-GDH) activity of Zao-hua 2 and Jin-zhu 1 was induced significantly, but that of Jin-dao 779 did not increase remarkably. The salt stress led to the accumulation of total soluble sugar (TSS) in the root of Jin-zhu 1 and Jin-dao 779 in different degree. The level of TSS in Zao-hua 2 changed with different NaCl concentrations. Among the cultivars observed, content of proline increased in different degree, but under high salt concentrations, it had a more marked rise in the roots of salt-sensitive cultivars. Key word: glutamine synthetase (GS); glutamate synthase (GOGAT); glutamate dehydrogenase (GDH); rice; salt stress Salt stress has important effects upon plant growth and development. It causes the plant tissue to absorb water difficultly, breeds physiological disorder, and destroys cell structure, resulting in the decrease in yield and quality of crops. Under the adverse stress, the production of nitrogen-metabolizing enzymes in plants is affected, which results in NH4 accumulation and thereby poisons tissue cells. Because of its critical role in absorption of water and inorganic mineral, root is often the first organ experiencing salt stress (Chen and Plant, 1999). It is well-known that some plant genotypes have relatively stronger tolerance to salt stress. It is of significance to understand their salt-resistant mechanisms for improving crop yield and quality. The assimilation of inorganic nitrogen into carbon skeleton in the form of ammonium (NH4) for the production of amino acid is one of the most important biochemical processes in plants. In higher plants, glutamine synthetase (GS, EC 6.3.1.2) is the main enzyme of ammonia assimilation, with energy provision by ATP. The enzyme catalyses the combination of ammonia and glutamate into glutamine, and transfers inorganic nitrogen into organic nitrogen. The circle reaction consisting of GS and glutamate synthase (GOGAT) is the main pathway of ammonium assimilation of plants (Lam et al., 1996). Previous results show that GSra and GSrb are two kinds of GS isozymes existing in the root of rice, and production of GSrb is induced by external nitrogen (Zhang et al., 1997). Immunological assay showed that both GSra and GSrb are the cytosolic forms (Lin et al., 2000). Glutamate dehydrogenase (GDH, EC 1.4.1.2) catalyzes the amination of glutamate and its catabolism. Having high Km to NH4, the role of GDH in the NH4 assimilation is still unclear (Lam et al., 1996). Some researches have shown that with increasing of NH4 concentration in tissues, the amount and activity of GDH enzyme increase significantly to defense the harm of ammonia (Lin et al., 1996; Lutts et al., 1999). Osmotic adjustment is one of important mechanisms by which plant adapts salt and drought stress. Sugar and proline are the main substances of osmotic adjustment in plants under salt stress. Their contents increase significantly (Delauney and Verma, 1993; Soussi et al., 1999; Zhao et al., 2001). To investigate the relationship among ammonium assimilation enzymes and the levels of NH4, total soluble sugar (TSS) and proline, and to explore the mechanisms by which the salt-resistant rice plant keeps higher activity of GS, the enzyme activities and the relative parameters of rice cultivars with different resistance to salt were measured under the salt stresses with different NaCl concentration. Acta Botanica Sinica 植物学报 Vol.46 No.8 2004 922 1 Materials and Methods 1.1 Plant materials and culture Rice (Oryza sativa L.) Jin-zhu 1 (normal cultivar), Jin-dao 779 (salt-resistant) and Zao-hua 2 (salt-sensitive) seeds were provided by Prof. LIU Jing of Rice Research Department at Tianjing Agriculture Science and Technology Research Institute. Seeds were first surface sterilized with 0.1% HgCl2 and washed thoroughly with double-distilled water. The seeds were soaked in distilled water and placed in an incubator (30 °C) for 24 h and then were transferred to a petri dish with several layers of paper towel with small amount of water, and the petri dish was placed inside an incubator for another 48 h. The germinated seeds were sown on the nylon net floating on nutrient solution (Yoshida et al., 1976) in porcelain pots and cultivate them with photoperiod 14 h (with fluorescent illumination of 100 μmol· m· s), relative humidity 60%-70%,temperature 30°C/25°C (day/night), after 7 d, the plants were subjected to salt stress by adding NaCl to nutrient solution (0, 50, 100 and 150 mmol/L, respectively), the roots of seedlings were harvested at 72 h after adding salt, they were directly used into the extraction of enzymes or stored in the low temperature refrigerator . 1.2 Extraction and activity determination of enzymes Enzymes were extracted from the roots of rice seedlings according to the method introduced by Zhang et al. (1997). One gram sample was ground to a fine power in precooled mortar with sand and pestle, and then homogenized in extraction buffer (3 mL/g FW). The homogenates were centrifuged 19 000g for 20 min. The supernatant parts were stored at -70 °C prior to assays. The crude extracts were used for determination of activities of GS, NADH-GOGAT and GDH. GS activity in extracts was determined according to Zhang et al. (1997). One unit of GS activity was the enzyme amount catalyzing the formation 1 μmol γ-glutamylhydroxamate/min at 37 °C. The activity of NADH-GOGAT was determined as described by Lin et al. (1996). The total reaction volume was 3 mL. Glutamine was added to initiate reaction as soon as enzyme extraction was added. The change of absorption value was detected at 340 nm in 3 min. One activity unit of NADH-GOGAT was defined as reducing 1 μmol NADH in reaction mixture/min at 30 °C. NADH-GDH activity was determined according to Loulakakis et al. (1990). One unit of GDH activity was defined as oxidation of 1 μmol NADH/min at 30 °C. 1.3 Determination of root GS isozymes The separation of GS isozymes in crude extract of rice roots was made according to Davis (1964). GS isozyme activity bands on the native gel were displayed by adding acidic FeCl3 solution (Zhang et al., 1997). 1.4 Determination of ammonium level NH4 concentration was measured by high performance liquid chromatography (HPLC) according to the method introduced by Husted et al. (2000). In the method, the reaction between NH4 and o-phthalaldehyde (OPA) was performed at 63°C and at neutral pH with 2-mercaptoethanol as reducing agent (FN). The flow rate of the carrier stream was 1 mL/min and the sample volume used was 5 μL. The derivative was detected at an excitation wavelength of 410 nm and an emission wavelength of 470 nm. 1.5 Analysis of soluble protein content The content of soluble protein was measured according to the method of Bradford (1976). 1.6 Determination of TSS The content of TSS was determined according to the method introduced by Irigoyen (1992). 1.7 Determination of proline content The content of proline was determined according to the method of Bates (1973) and the absorbance of the toluene phase was measured at 520 nm.