Studies On Calcium Signal System On The Physiological And Biochemical Mechanism Of Cold-Resistance In Citrus

Low temperature is one of the most important factors limiting the growth and distribution of plants in the world. Citrus is an important commercial fruit crop in southern China, as well as in the world. However, periodic freezing injury seems to be serious problem in citrus industry, which is far from being solved in the world. There were mainly two effective ways to minimize the freezing injury, one is breeding new cultivars with high cold-resistance, and the other is taking available practical measures to prevent freezing injury.Calcium is proved to be a second message involved in responding and adapting to outside environment. Ca²⁺ plays an important role in message transduction of the environment stress involving low temperature. But as to the studies on the role of plant cold-resistant regulated by Ca²⁺ signal system, researchers paid more attention on annual herbals, not on the perennial woody plants such as citrus. Study the regulation mechanism of Ca²⁺ on cold-resistant in citrus could offer the theoretic and practical base about cold-resistant breeding and improving the culture ways for citrus.In this paper, the effects of five concentrations (0, 3, 5, 10 and 15mmol/L) of Ca²⁺, Ca²⁺ chelating agent EGTA (ethylene glycol-bis-β-aminoethylether-N,N'-tetraacetic acid) and calmodulin antagonist TFP (trifluoperazine) on some physiological and biochemical indexes in three citrus calli, which included lime (Citrus aurantifolia Swingle), Page tangelo (C.reticulata Blanco×C. grandis Osb.cv.Page) and Guoqing No.1 (C.reticulata Blanco cv. Guoqing No. 1) under low temperature were studied, and effects of different Ca²⁺ salts on the cold-hardiness of the leaves of Guoqing No.1 satsuma mandarin in the field were investigated. The main results of the research work are as follows:1) The content of membrane fatty acid in citrus could be affected by Ca²⁺ signal system. The assay by gas chromatography demonstrated that the composition of membrane fatty acids of citrus calli included palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. Their composition did not change in despite of being cultured in the media with different Ca²⁺ concentration under low temperature. Among six species of fatty acid, the relative percent of oleic acid was the highest, the percent of palmitic acid, linoleic acid and linolenic acid were in the middle, the less percent was stearic acid and palmitoleic acid. Exogenous Ca²⁺ (5 mmol/L) significantly increased IUFA (index of unsaturated fatty acid) in citrus calli membrane fatty acid, especially sensitive to linoleic acid and linolenic acid. There were significant correlation between the content of linoleic acid and IUFA, and between the content of linolenic acid and IUFA. Supplement with EGTA and TFP in the media, IUFA of citrus calli could decrease, therefore reduced the cold-resistance of citrus.2) Exogenous Ca²⁺ (5 mmol/L) could markedly decrease membrane injury rate and MDA (malondialdehyde) accumulation during low temperature stress. Moreover, it could raise SOD (superoxide dismutase) activity and increase POD (peroxidase) activity. Exogenous calcium could strengthen the cold-resistance of citrus, compared with Ca-deficient treatment. But such effects were inhibited by addition of Ca²⁺ chelating agent EGTA or calmodulin antagonist TFP in the culture medium. Consequently, it is suggested that Ca²⁺ signal system may be involved in the membrane lipid peroxidation and protective enzyme system of cold-resistant citrus under low temperature.3) 5 mmol/L Ca²⁺ treatment could markedly increase the content of soluble protein, soluble sugar and proline in citrus calli, compared with Ca-deficient treatment. In the contrary, addition of EGTA or TFP could markedly decrease the content of soluble protein, soluble sugar and proline. Therefor it could be suggested that Ca²⁺ signal system may be regulated by change of some osmolytes in citrus under low temperature.4) Low-temperature induced protein in citrus callus could be affected by extraneous Ca²⁺ treatment. This experiment investigated the effect of different concentrations of Ca²⁺ on low-temperature induced protein in Page tangelo callus under low temperature. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that these proteins, including 77.8 kD, 51.5 kD, 4.2 kD and 38.2 kD, always existed in Page tangelo callus, and 65.3 kD and 17.1 kD proteins being induced only depended on low temperature treatment time, and 21.1 kD, 23.2 kD, 25.8 kD and 97.6 kD protein being induced depended not only on low temperature treatment time, but also on Ca²⁺ concentrations.5) Spraying calcium salts to the canopy of satsuma mandarin trees could increase its cold resistance. The abilities to increase the resistance depend on the type of Ca²⁺ salts. After sprayed the leaves of Guoqing No.1 satsuma mandarin (C.reticulata cv. Guoqing No.1) with 20 mmol/L CaCl,Ca(NO₃)₂ and CaAc₂, the effects on half lethal temperature (LT₅₀) and some physiological and biochemical indexes under low temperature (-9℃) stress were studied. The results showed that LT₅₀ treated with calcium salts was lower than with the distilled water which was the control, but the enhancing degree were different among calcium salts. LT₅₀, treated with Cl^-salt was only 0.54℃lower than control. Whereas, LT₅₀ treated with CaAc₂ or Ca(NO₃)₂, was 1.34℃and 1.35℃lower than control respectively. Moreover, treatment with CaAc₂ or Ca(NO₃)₂ could improve the activities of antioxidant enzyme, such as SOD and POD, and increase the content of osmotic substances including soluble protein, soluble sugar and proline; and alleviate the accumulation of MDA in the leaves of Guoqing No. 1 satsuma mandarin, compared with that of CaCl₂. Hence, we'd better come to the conclusion that effect on the cold-hardiness of the leaves of Guoqing No.1 satsuma mandarin with Ca(NO₃)₂ treatment or CaAc₂ treatment was better than CaCl₂ treatment.