Study On Autophagy Processes In Laticifers Of Euphorbia Kansui L.

Euphorbia kansui Liou which belongs to Euphorbia in Euphorbiaceae is a perennial herbaceous species and an endemic species in China.It possesses high medicinal value.E.kansui contains a kind of highly specialized cell,named laticifers.The laticifers of E.kansui is a nonarticulated type,which longitudinal extend to the ends with plant growth and contain white latex.In recent years,a number of reports studied on its chemical constituents and pharmacological activities,etc.However,the degradation mechanism of laticifer cytoplasm has less hitherto been detailed reported.In the present study,our group has used the electronic microscopy to observe the characteristics of E.kansui laticifers development: At the early development,laticifers possessed dense cytoplasm with various and many organelles.Along with development,there were lots of vacuoles originated from endoplasmic reticulum contain latex particles.Furthermore,these vacuoles fused with each other.Ultimately,they fused with the central vacuole.Until laticifers reached maturity,the cell was nearly filled with a central vacuole and a thin layer cytoplasm remained.Therefore,the degradation of part of cytoplasm and organelles might happen during laticifer development.As a result,the questions of how part of protoplasm in E.kansui nonarticulated laticifers degrade,whether the autophagy involves in the protoplasm degeneration has attracted our attention.Based on the above issues,this study researched the forming and degradation process of autophagosomes in laticifers,as well as discussed the relationship between autophagy and development of laticifers using techniques of western blot,immunofluorescence combined with confocal laser scanning electron microscopy,colloidal gold immune electron microscopy and transmission electron microscopy.All of these studies would provide more theoretical knowledge for development regulation of nonarticulated laticifers,and also provide fundamental materials to deeply understand the issues about autophagy participating in plant growth.The main results were as follows: Firstly,autophagy related protein ATG8 s has been used as a marker to identify autophagy related structure for resent years.Cysteine protease(CP)is a main lysosome hydrolase,which can be used to identify the lysosome like structure.Therefore,firstly,the protein of E.kansui latex was extracted,and the expression of ATG8 a and CP in laticifers were analyzed with western blot.Anti-ATG8 a antibodies identified a single band of estimated 14 kDa and Anti-CP antibodies identified another single band of estimated 40 kDa,corresponding to the molecular sizes of the two kinds of protein,which illustrate that the availability of Anti-ATG8 a antibodies and Anti-CP antibodies as well as the existence of two kinds of protein as endogenous proteins in E.kansui laticifers.Secondly,the colocalization of Anti-ATG8 a antibody-Alexa Fluor 488 with Lyso Tracker Red(LTR),and colocalization of anti-ATG8 a antibody-Alexa Fluor 488,LTR and monodansylcadaverine(MDC)were performed respectively for immunofluorescence analysis.The Anti-ATG8 a antibody-Alexa Fluor 488 can label the autophagosomes and autolysosomes,indicated by green fluorescence.The LTR and MDC have been reported as indicators to primarily detect lysosomes and autolysosomes,indicated by red fluorescence and purple fluorescence,respectively.The results showed that with the development of the stem,laticifer cells also gradually mature,all three fluorescence signals showed regular changes: from weak to strong to weak.Similar results also could be got from quantitative analysis.These regular changes were consistent with the development characteristics of laticifers.It also indicated that autophagy participates in the protoplasm degradation of E.kansui laticifers.In addition,LTRstaining red fluorescence and MDC-staining purple fluorescence were perfectly overlapped with ATG8a-labeled green fluorescence,suggesting that lysosome-like structures and autolysosomes present in laticifers.Thirdly,through transmission electron microscope,the complete autophagy process in E.kansui laticifers had been observed.At the beginning of autophagosome formation,we found that many isolation membranes(IMs),derived from endoplasmic reticulum,swelled and spread out into the regions possessing thick cytoplasm or abundant organelles.In the process of wrapping,the IMs firstly formed the cup-shaped structure.Finally,with the IMs gradual elongation,the completed double-membrane structure termed mature autophagosome formed.Additionally,lots of small electron-dense vesicles were found to originate from the dictyosomes in the cytoplasm of laticifer cells,which were written as lysosome-like vesicles by us.These vesicles were often distributed around autophagosomes or near degradable organelles,and could fuse with autophagosomes.In addition,the dynamic degradation process of cytoplasm components in autolysosome was observed in E.kansui laticifers as well.Fourthly,the subcellular localization of ATG8 a and CP in E.kansui laticifers by using colloidal gold immunoelectron microscopy indicated that immuno-gold particles with Anti-ATG8 a antibodies were distributed in the swollen endoplasmic reticulum,the membrane structures of autophagosomes and the cavity of autophagosomes.The immuno-gold particles with Anti-CP antibodies were mainly distributed in the electron-dense lysosome-like vesicles,as well as autophagy related structures around the lysosome-like vesicles.In conclusion,autophagy is involved in part of cytoplasm degradation during development of E.kansui laticifers,with a way that endoplasmic reticulum can wrap some organelles and cytoplasm to form autophagosomes,and then fuse with lysosome-like vesicles to form autolysosomes.