Effect Of Calcium And Calmodulin On Neck-bending Of Gerbera Cut-flower

The easy-bending golden breed 'Sondance' of gerbera was selected in this study. The calcium absorption of the scapes during the senescence of gerbera, the chemical location and the ultrastructural changes of calcium in cells of scapes were studied. The study also included the CaM content of scapes, H⁺-ATPase and Ca²⁺-ATPase activity and changes of relative physiological indexs, the impact of calmodulin antagonist over senescence associated enzymes and the effects of exogenous Ca²⁺ . The impacts of Ca²⁺ and CaM over senescence of cut gerbera flower and neck-bending phenomena were discussed, which provided more thorough and reliable theory basis for the mechanism of bending resistance of gerbera and the modulation of exogenous Ca²⁺. The main results were as follows:1. Disposal with 75mg/L CaCl₂ preservative solution, which could effectively reduce neck-bending phenomenon of gerbera and control the increase of series of bent neck. The calcium content was the lowest in the middle part of the stem of cut gerbera flower, i.e. the part which was most likely to bend. A great deal loss of calcium of gerbera due to cutting could be effectively supplemented and the calcium content of the scapes increased rapidly, accumulating massively in the middle part of easy-bending section in the mean time when disposing cut gerbera flower with CaCl₂ preservative solution, which played a crucial role in the improvement of bending resistance ability of gerbera.2. The observation of chemical localization of calcium showed that in the scape cells of gerbera, the precipitates of calcium antimonate mainly appeared in vacuoles and cell walls and there were few distributions of calcium prepcipitates in the middle part of the scapes while more in vacuoles compared with that in the upper and lower part. The calcium in vacuoles gradually came into cytoplasm, which resulted in too high concentration of cytoplasmic calcium in cytoplasm with the vase life time increasing. The calcium in cell walls in the middle part of scapes increased obviously after calcium disposal, which improved the bending resistance ability of cut flower, suppressed the movement of calcium towards cytoplasm and reduced the harms to cut flower.3. The ultrastructural observation of scape cells showed that: the cells in the upper part of gerbera scape arranged densely with large number of chloroplasts averaging to 5-6 per cell, whereas few in the middle and lower part with average number of 3⁴ per cell. In comparison, cells in the middle part of scape arranged much loose and the cell walls were the thinnest. So less material and energy were synthesized in the middle part of scapes with much poor support force.4. Normal chloroplast of scape cells took the spindle form with clear outer membrane, and inner troma lamella arranged regularly, distributing along plasma membrane. The vacuole was big, transparent and no content was seen in membrane. Cells lost water with vase life increasing and cell membrane began to invaginate, causing plasmolysis and confused, irregular cells arrangement when disposing with CK on day 5. Cells began to severely dehydrate and plasmolyzed obviously. Chloroplasts expanded and became round. Chloroplast membrane became obscure and stroma lamella arranged disorderly. Many membraneous inclusions appeared in vacuoles and some cell walls and nuclear membrane disintegrated. In comparison, disposing with calcium maintained good condition and few cells began to plasmolyze until the day 7.5. The condition of CaM content measurement by ELISA was optimized, and the results showed that the optimal coating concentration of the antigen of cut gerbera flower was 10μg/m, the optimal dilution factor for primary antibody was 1:500, and the best dilution multiple was 5. It was of great benefit to the increment of CaM coating efficiency to irradiate with ultraviolet lamp.6. During the whole vase life, in the part of easy-bending section of gerbera, the content of CaM took on the tendency that ascending first, then descending and finally ascending. There existed a peak of CaM content before senescence. The change of Ca²⁺-ATPase activity resembled the changing tendency of CaM content. While the H⁺-ATPase activity increased with the increment of the vase holding days. Before the bent neck appearing,the content of CaM ascend first;bent neck happen on day 4,CaM arrived to a peak,but the content of CaM descend when bent neck increasing. All these showed that the level of CaM is important to modulate neck-bending.7. When disposing cut gerbera flower with 75mg/L CaCl₂ preservative solution, the CaM content, H⁺-ATPase and Ca²⁺-ATPase activity were much higher than disposing without CaCl₂. Disposal with 100μmol/L CPZ could significantly suppress the CaM content and Ca²⁺-ATPase activity. Ca²⁺·CaM system played the role of signal transduction, and the Ca²⁺-ATPase activity was activated and modulated by CaM. Exogenous Ca²⁺ could accelerate the course of signal transduction.8. Study on the cell wall component of gerbera scape showed that the main gradient of pectic substance in scapes was protopectin. The protopectin content decreased and the soluble pectin content increased with the increment of vase holding days. Cellulose content rapidly decreased after 3 days of vase life. While disposal with CaCl₂ could effectively suppress the degradation of cellulose and protopectin, make the relatively slow increment of soluble pectin, and maintain the stability of the cell wall structure. In the meanwhile, disposal with CaCl₂ could accelerate the water and sugar absorption of cut gerbera flower, and benefited maintaining the cell membrane stability.9. During the vase life of gerbera, carboxymethyl cellulose enzyme (Cx) , Polygalacturonase(PG)and pectin methylesterase(PME) activity increased fast. Disposal with CaCl₂ maintained the cell wall hydrolase activity in a low level and disposal with CPZ accelerated the rapid ascending of activity in the late period of vase life, speeding up the process of cut flower senescence. While disposal with exogenous Ca²⁺ could reduce the impact of CPZ over cell wall hydrolase activity. All these showed that Ca²⁺·CaM messenger system may have modulated the cell wall hydrolase to some degree.10. Disposal with Ca²⁺ could rapidly improve the POD,CAT activity of scapes, whereas the disposal with CPZ strongly suppressed the POD,CAT activity and disposal with Ca²⁺+CPZ had the positive modulation effect on this suppression. The whole SOD activity took on the tendency of descending during the cut gerbera flower senescence and the disposal with Ca²⁺ could slow this tendency, while disposal with CPZ aggravated the rapid descending of SOD activity.All these showed that,the following were the main reason of bent neck happened: 1 .On cellular structure,cells in the middle part of scape arranged much loose and the cell walls were the thinnest. 2. The observation of chemical localization of calcium showed that there were few distributions of calcium prepcipitates in the middle part of the scapes while more in vacuoles compared with that in the upper and lower part. 3.The content of calcium: upper part >lower part >lower-middle part >middle part, the calcium content was the lowest in the middle part of the stem of cut gerbera flower, i.e. the part which was most likely to bend,and a great deal of calcium of gerbera lost due to cutting. 4. The content of calmodulin: Before the bent neck appearing,the content of CaM ascending and Ca²⁺·CaM messenger system may have modulated the cut-flower to some degree. When bent neck happened,CaM arrived to a peak,but the content of CaM descend when bent neck increasing. 5. Cell wall component and Cell wall-degrading enzyme:The protopectin content,cellulose content decreased and the soluble pectin content increased with the increment of vase holding days. The rising of cell wall-degrading enzyme activity made the cell wall structure distraction and bent neck happened.Exogenous Ca²⁺ played a crucial role in the improvement of bending resistance ability of gerbera,as the following reason: 1.The loss of calcium of gerbera could be effectively supplemented and accumulating massively in the middle part of easy-bending section when disposing cut gerbera flower with 75mg/L CaCl₂ preservative solution, which played a crucial role in the improvement of bending resistance ability of gerbera. 2. The calcium in cell walls in the middle part of scapes increased obviously after calcium disposal, which improved the bending resistance ability of cut flower. 3. When disposing cut gerbera flower with 75mg/L CaCl₂ preservative solution, the CaM content, H⁺-ATPase and Ca²⁺-ATPase activity were increaming, and mechanism of Ca²⁺·CaM messenger system enhanced. 4. Disposal with Ca²⁺ could delay the degrading of cell wall component and increaming of cell wall-degrading enzyme. 5.Disposal with CaCl₂ could accelerate the water and sugar absorption of cut gerbera flower, and benefited maintaining the cell membrane stability.