Researches On Characters And Mechanism Of Programmed Cell Death (PCD) During The Petal Senescence In Osmanthus Fragrans

Osmanthus fragrans Lour.is an excellent evergreen flower shrub or small woody tree,which belongs to Oleaceae.It originated in China and had a long cultivation history. It is honored as one of top ten most traditional famous flowers in China.Because of its good ornamental value and pleasant fragrance,it is generally applied as avenue tree and shade tree in landscape.And it is selected as City Flower in many cities.Moreover,the petal tissues emit thick scent.Therefore,its petals are often used in the production of tea, food,wine and spice etc.However,the longevity of individual flower can only last for 2-3 days,which reduces its ornamental and economic values.Thus,the studies for prolonging the life span of flower or whole cyme will contribute to extending blooming period of whole trees,vase life of cut flowers and scent emission time.Therefore,our studies focus on the PCD start-up time,status and schedule of different PCD events.We also want to know whether the PCD features occur during Osmanthus petal senescence.Furthermore, the roles of some key factors,such as ethylene,Ca²⁺ and ractive oxygen species(ROS) will be investigated in petal PCD.In the results,the senescent symptoms were observed.The standards for stage division during flowering to senescence were set up.The method for exogenous treatments has been designed.In each stage during flower opening to senescence,the petals attached to trees were sampled for the detection in a series of parameters,like petal water,membrane lipids oxidation(MDA,O₂^-,SOD,POD etc.),protein level,SDS-PAGE, and DNA fragmentation.In addition,the anatomic features of petals have been studied. The relations of key factors and Osmanthus petal PCD are discussed.The main results were shown as follows:1.The structure of Osmanthus petal remained integrated when the flower life is terminated.The vascular bundle still can be distinguished,whereas,the epidermal cells showed obvious hollows.The condensation of nuclear could be observed and camptothecin can enhance his feature.However,only slight smear could be detected by gel electrophoresis,but no 'DNA ladders' appeared.2.The PCD program has been started when flower were fully opening.Membrane lipids proxidation induced by endogenous ROS burst and new protein synthesis might be the former events.Protein degradation and nuclear DNA fragmentation might be latter events or results.SOD and APX may be relative important enzymes in the protective enzymes.In addition,the levels of protein degradation and DNA fragmentation are relative low when flowers attached to trees abscised.But the high level of DNA cleavage could be detected in wilting petal.And the cells with DNA fragments spread randomly in petal tissues.3.The endogenous ethylene could not be detected during petal senescence in different cultivars of O.franrans,and most of cultivars are insensitive to exogenous ethylene. Ethylene could accelerate the petal water loss,ROS production and membrane lipids proxidation,whereas plays little role on antioxidative enzymes.Moreover,ethylene could promote the expression of protein and induced a 63.4 KD protein band,which may be related with new gene expression.In addition,DNA fragmentation can be enhanced by ethylene.4.Ca²⁺ has double roles in PCD during Osmanthus petal senescence:Ca²⁺ with low concentration could help delaying petal senescence,but this role could not persist. However,Ca²⁺ with high concentration could initiate the PCD program.The whole content of Ca²⁺ increased along with petal senescence.Whole Ca²⁺ content in petal tissue would increase after the exogenous Ca²⁺ treatments.It is hypothesized that the Ca²⁺ out of cytoplasm could protect membrane structure whereas the increase of Ca²⁺ in cytoplasm could start up petal PCD.Ca(2+) might play an role in protein degradation, DNA cleavage and other PCD-events by activate the protease and DNase.But above hypothesis needs more evidences.5.Exogenous reactive oxygen species(H₂O₂) could accelerate the Osmanthus petal senescence,while the ROS inhibitors ascorbic acid could delay the petal senescence. H₂O₂ may have little effect on activity of SOD and APX.Ca²⁺ with low concentration can protect the membrane from harm of H₂O₂.H₂O₂ treatment induced new protein bands and 36.8KD,23.9KD and 23.7KD may be specific.And Ca²⁺ can inhibit above new bands appearing in former stage whereas Ca²⁺ promote degradation of most protein.In addition,exogenous ROS could accelerate the cleavage of DNA,but this effect is not rapid,which can be inhibited by Ca²⁺.In conclusion,PCD has been initiated when the flower was fully opening.The membrane lipids peroxidation and new protein synthesis may be the former events.While most protein degradation and nuclear DNA fragmentation may be the latter events or results.The condensation of nuclear and chromatin can be observed.However,the "DNA ladder" can not be detected.Previous researches focus on petal senescence of the commercial cut flowers while few studies are found on petal senescence of the big flowering shrub.So far,our results are consisted to Prunus yedoensis with little typical PCD features.The two species have some common characters.Therefore,the reasonable hypothesis is that there is little biochemical feature in petal PCD in big tree species, which is different from that in herbaceous flowers.It is suggested that the PCD mechanism may be different between above both groups.In addition,ROS may be the most important regulator or signal in Osmanthus petal PCD while Ca²⁺ might have double roles in it.Whereas,ethylene has no effect on most of Osmanthus cultivars.Our comprehensive researches on the regulation mechanism of three key factors in petal PCD had not been reported before.