INFLUENCE OF CALCIUM ON ANTIOXIDANT SYSTEM AND NITROGEN METABOLISM OF MUSKMELON SEEDLINGS UNDER NUTRIENT SOLUTION HYPOXIA

Two varieties of muskmelon were cultivated in hydroponics culture using the following [Ca] in nutrient solution under hypoxia with 4mmol·L Ca (hypoxia), hypoxia with 10mmol·L Ca (hypoxia+Ca), hypoxia with 0mmol·L Ca (hypoxia-Ca), and normal dissolved oxygen with 4mmol·L Ca was the control. Leaf samples were collected at 0, 2, 4, 6 and 8d from all treatments for fresh weight, reactive oxygen species (ROS) and nitrogen metabolism analyses. The results showed that compared to control treatment, the fresh weights of both varieties were decreased and activities of antioxidant enzymes, productive rate of ROS, contents of MDA and NH4-N, NO3-N were increased under hypoxia. Calcium application treatment alleviated the harmful effects of hypoxia to seedlings, as manifested by the higher levels of fresh weights, activities of antioxidant enzyme, contents of NH4-N, NO3-N under hypoxia, while the ROS productive rate and MDA content were lower. The calcium-deficient treatment aggravated the harmful effects of hypoxia, which were reversed by calcium application. The different levels of response by the two varieties show differences in tolerance to hypoxia. INTRODUCTION Hypoxia in the root-zone is thought to be a major determinant in the adverse effects of nutrient solution culture (Gao et al., 2004b). Oxygen deficiency might first be sensed by the mitochondrial electron transport chain, caused by NADP(H) oxidative blockage and the reduced adenylate energy charge of the cell (Subbaiah and Sachs, 2003). As a second messenger, [Ca] in cytoplasm rapidly increases under external stimuli, which is involved in perception and regulation of response of plants to environmental stress (Pandey et al., 2000). There is ample evidence for the role of Ca in plant growth and development, maintenance and modulation of various cell functions under environmental stress, such as cold (Gao et al., 2004a), drought (Sulochana et al., 2002), anoxia (Wang et al., 2002). Subbaiah et al (2003) demonstrated that Ca is a transducer of low O2 signal, but a detailed mechanism of events and the relationship that exists between exogenous application Ca and plant growth, reactive oxygen species (ROS) and nitrogen metabolism under hypoxia are not clear. In the present study, muskmelon seedlings were used to examine the effects of exogenous application Ca and exclusion Ca in nutrient solution under hypoxia on growth, ROS and nitrogen metabolism and to elucidate the relationship between Ca enhancement of hypoxia tolerance and metabolism of ROS and nitrogen metabolism. MATERIALS AND METHODS Two varieties of muskmelon (Cucumis melo L. var. raticulalus Naud) ‘Xiyu No.1’ and ‘Dongfangxingguang’ were cultivated in autumn in 2003. At the 3 true-leaf stage, seedlings were removed from the nutrient cubes, the roots were rinsed with distilled water, and uniform seedlings were divided into four groups then transferred into Hoagland nutrient solution (pH 6.3±0.1, EC 2.0~2.2) in plastic containers (65L). After 2d culture, the first set of seedlings was transferred into normal Hoagland nutrient solution (4 mmol L Ca) and exposed to normal dissolved oxygen (DO) by using vigorous aeration (30min/h) Proc. IC on Greensys Eds.: G. van Straten et al. Acta Hort. 691, ISHS 2005 322 to keep DO at 8±0.2mg L (Control). The second set of seedlings were cultured in normal Hoagland nutrient solution containing 4mmol L Ca but exposed to reduced levels of dissolved oxygen (hypoxia). The O2 was balanced with N2 by using a DO analyzer (Quantum-25, Quantum Analytical Instruments Inc, American) to keep DO at 2±0.2mg L. The third and fourth groups of seedlings were cultured in DO 2±0.2mg L with exogenous application of Ca 10mmol LCa (Hypoxia+Ca) and without Ca (Hypoxia-Ca) in nutrient solution respectively. Superoxide dismutase (SOD) activity was determined by assaying its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). One unit of SOD activity was defined as the amount of the enzyme to bring about 50% inhibition of the photochemical reduction of NBT (Giannopolitis and Ries 1977). Peroxidase enzyme (POD) activity was measured by measuring the increase in absorbance at 470nm as Guaiacol was oxidized according to the method of Kwak (1996). Catalase (CAT) activity was determined according to the method of Dhindsa (1981) by measuring the decrease in absorbance at 240nm for 1min following the decomposition of H2O2. One unit of CAT activity was defined as decrease of 0.1A value for required enzyme quantity. Hydrogen peroxide (H2O2) content determination was performed by monitoring the increase in absorbance at 550nm according to the method of Matsumura (2002). Superoxide radical ion (O2) productive rate was determined according to the method of Wang (1990) and sodium nitrite was used as standard. Malondialdehyde (MDA) content was determined by reaction of trichloroacetic acid (TCA) and thiobarbituric acid (TBA) according to the method of the method of Dhindsa (1981). Nitrate-nitrogen (NO3-N) and ammonium-nitrogen (NH4-N) content was determined according to the method of Gao (2004a), potassium nitrate and leucine was used as the standards respectively. Statistical analysis of the data was performed by Excel statistical software. Duncan Multiple Range Test (SAS) was used for mean separation. RESULTS Effect of Ca on Fresh Weight of Muskmelon Seedlings under Hypoxia Table1 indicates that after 8d of culture under hypoxia conditions, the fresh weights of seedlings in hypoxia+Ca, hypoxia alone and hypoxia-Ca decreased compared with those of the control treatment. The decrease in fresh weight under hypoxia-Ca was greater than that of hypoxia alone, and under hypoxia+Ca was the least. The fresh weight of ‘Dongfangxingguang’ was higher than that of ‘Xiyu No.1’ seedlings under all treatments. Effect of Ca on the Activity of Antioxidant Enzymes under Hypoxia SOD activity in ‘Xiyu No.1’ and ‘Dongfangxingguang’ seedlings under hypoxia alone and hypoxia+Ca increased significantly, reaching a peak 2d after hypoxia treatment, however, the SOD activity of hypoxia+Ca treatment was higher than those of hypoxia alone (Fig.1). SOD activity with hypoxia-Ca treatment increased slightly but was significantly lower than that control during the hypoxia at 6-8d. SOD activity of hypoxia-Ca and control treatments was lower than those of hypoxia alone and hypoxia+Ca treatment. POD activity of seedlings of both varieties with hypoxia+Ca treatment increased significantly compared to the other three treatments, peaking at 6d of hypoxia treatment (Fig. 1). POD activity of hypoxia alone and hypoxia-Ca treatments increased initially then decreased, but they were both higher than that of the control at peak activity. Similar to SOD and POD activities, CAT activity of both varieties with hypoxia+Ca, hypoxia alone and hypoxia-Ca treatments increased, peaking at 4d after hypoxia treatment (Fig.1). CAT activity with hypoxia+Ca treatment was higher than that of hypoxia alone. In ‘Xiyu No.1’ seedlings, the Ca deficient treatment (hypoxia-Ca) was not different from the control. Generally, antioxidant enzyme activity was consistently higher for ‘Dongfangxingguang’ than for ‘Xiyu No.1’ seedlings for any one treatment and time of determination.