Physiological Responses And Ca²⁺ Messenger Changes Under Chilling Stress In Walnut

Walnut (Juglans) is a kind of thermophilic fruit tree. Cold injury occurs sometimes. This study appraised the cold resistance of the dormancy stage branches and leafing date leaves of common walnuts in the north of China. The applied indexes about cold resistance were ascertained. The changes of antioxidases activity, osmotic adjustment matters content, and ca²⁺ messenger localization under chilling stress were investigated with the leafing date leaves of three cultivars. One is "Hartley" of the strong chilling resistance. The other is "Jinlong No.1" of common resistance. The third is "Jinlong No.2" of. the weak resistance. In addition, the functions of Ca²⁺ messenger system on the chilling resistance of walnut in leafing date were studied with CaCl₂, ethylene glycol- bis (β- aminoethyl ether) -N, N, N', N'-tetra acetic acid (EGTA, calcium chelator), trifluoperazine (TFP, calmodulin inhibitor), and LaCl₃ (Ca²⁺ channel blocker). The main results were as follows:1. There was a significant difference of the cold resistance of walnut germplasms in dormancy stage. The interspecific difference was large. The order was J. nigra L., J. mandshuria Maxim., J. hopeiensis Hu., and J. regia L. according to the cold resistance. Electric conductivity, rate of branch or bud browning, and degree of 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining, which all could be indexes of cold resistance of annual branch in dormancy stage. The electric conductivity of annual branches could indicate the cold resistance in dormancy stage more exactly and simply measured at the temperature near LT₅₀ of most walnut germplasms. The rate of cork and cortical layer thickness in annual branch could be a morphological structure index of cold resistance.2. There was much difference of the cold resistance of walnut germplasms in leafing date by conductivity method. The interspecific difference of cold resistance of walnuts was little. The electrical leakage conductivity of leaves could indicate the cold resistance in leafing date more exactly and simply measured at the temperature near LT₅₀ of most walnut germplasms. The thicknesses of palisade tissue and leaves could be morphological structure indexes of cold resistance. It was not the same level in cold resistance between the branch in dormancy stage and leaves in leafing date.3. The changes of superoxide dismutase (SOD) and peroxidase (POD) activities were consistent before and after chilling stress. The enzyme activities were highest and the superoxide anion content was lowest in "Hartley" leaves at all time. On the contrary, the enzyme activities decreased fastest and the superoxide anion content was the highest in "Jinlong No.2" leaves under chilling stress for 72 hours. SOD was sensitive to low temperature and could be the key enzyme of antioxidases. The much lower temperature was need for POD activated. The amylase activity and soluble sugar content were higher in "Hartley" leaves than in "Jinlong No.2" under chilling stress.4. There were more fusion phenomenons of two chloroplasts in walnut mesophyll cells under chilling stress. The fusion phenomenons occured earlier and more frequently in the cells of "Jinlong No.2" of the least cold resistance. The number of mitochondria in mesophyll cells increased under chilling stress. The mitochondria and nucleus were much stabler than chloroplasts and plasma membrane. The mesophyll cells ultrastructure of the chilling resistant cultivar "Hartley" was obviously stabler than that of "Jinlong No.2" under chilling stress.5. Ca²⁺ mainly localized in vacuole and intercellular spaces in the leafing date leaves. Ca²⁺ concentration increased rapidly in the mesophyll cells under chilling stress. Chloroplasts could act as temporary Ca²⁺ pools under chilling stress. Ca²⁺ could be pumped into chloroplasts, which occurred earlyer in the chilling resistant cultivar. The activity of the Ca²⁺-pump was stable under chilling stress. Mitochondria could act as Ca²⁺ pools under chilling stress too, which absorbed Ca²⁺ fastly in the mesophyll cells. The calmodulin (CaM) content increased at initial stage in the mesophyll cells under chilling stress. The CaM content decreased remarkably in "Jinlong No.2" cells in late stage of chilling stress.6. There were significant differences of proline contents in the leaves treated with 5 kinds of reagents in late stage of chilling stress. The proline content was the highest in the leaves treated with EGTA and the lowest treated with CaCb. It took second place in the leaves treated with water. The contents of soluble protein is low in leaves treated with CaCl₂ and water, while the contents is higher in leaves treated with three kinds of inhibitors of Ca²⁺ messenger system, particularly EGTA. The contents of soluble sugar were higher in leaves treated with CaCb, EGTA, LaCl₃ and TFP than that treated with water under chilling stress.7. There was positive correlation between the heavy concentration Ca²⁺ and chilling injury of the cells. It improved the chilling resistance of leaves treated with right amount of CaCl₂, while impaired the chilling resistance of leaves treated with three kinds of inhibitors (EGTA, LaCl₃, and TFP), especially EGTA.8. Cerium trichloride precipitation method was used in electromicroscopic-cytochemical assay to study the localization of the Ca²⁺-ATPase in the walnut young leaves in leafing date under chilling stress. The Ca²⁺-ATPase activities were observed on the plasmolemma, cell wall, and in intercellular space, and the strongest on the plasmolemma. It enhanced the Ca²⁺-ATPase activities in the leaves treated with CaCl₂ and LaCl₃ at leafing date. The activities were approximate in the leaves treated with TFP, EGTA, and water. The Ca²⁺-ATPase activities in the leaves treated with CaCl₂ were strong under chilling stress all along.9. The amount of CaM in the leaves treated with TFP was less and increased slowly. The amount of CaM increased remarkably treated with CaCl₂, and increased partially treated with LaCl₃ in leafing date. The amount of CaM in the mesophyll cells increased in initial stage and decreased in late stage of chilling stress treated with every reagent.