Studies On Autophagy Activity In Microspore Mother Cells Of Althaea Rosea(L.)

Autophagy(AP)is a process that the redundant or damaged proteins or organelles in cellsare wrapped by a bilayer membrane and then are transported to a vacuole or lysosome to be degradated and reused in eukaryotes cells.During the organism's normaldevelopment,senescence or sufferring from biological/abiotic stress,the process plays an important role.The early studies on autophagy were mainly concentrated in yeast and mammals.In recent years,the studies in plants have gradually increased,but these studies are mainly confined to the autophagy activity in biological and abiotic stress conditions and plant nutrition growth process.The autophagy on normal growth and development process,especially plant sex reproductive process are rarely studied.Previously,other members in our laboratory found that there were complex autophagosomes/autophagic vacuoles structures in the microspore mother cells(MMCs)of the hollyhock and rapeseed under transmission electron microscopy,but it is unclear whether the structures were true autophagosomes,how they formes and what their functions were.Here,a further qualitative study to the autophagy phenomenon in the MMCs of Hollyhock were performed by the staining of autophagy-specific staining agent MDC,autophagy inhibitor 3-MA and E-64 pretreatment,and sub-microstructure observation.The main results are as follows:(1)Fluorescence microscopy observation after MDC staining showed that the cytoplasm of the normal MMCs in early or meiosis prophase stage displayed green or blue fluorescence,in which there were different numbers of brightly blue spots.These bright spots were mainly distributed in the cell periphery,Sometime some bright spots in the cell periphery gathered into larger bright spots.The number of bright spots in the MMCs of meiosis prophase was significantly more than that in the early MMCs.In the microspore stage bright spots were disappeared.(2)Fluorescence microscopy observation found that after 3-MA pretreatment for24 h and 36 h,the cells also displayed fluorescence,but the numbers of the bright spots increased than the normal conditions slightly relative to that in the normal MMCs,but were significantly reduced compared with DMSO control group.Thenumbers of autophagosomes in the MMCs of DMSO control group were significantly higher than that in the normal MMCs.(3)After E-64 pretreatment for 24 h and 36 h,fluorescence microscopy observation showed that the MMCs displayed blue or green fluorescence,and the numbers of intracellular autophagosomes were significantly increased as compared with the MMCs of normal and DMSO control group.Intracellular autophagosomes numbers in DMSO control group were higher than that in the normal MMCs.(4)Under normal growth conditions,the sizes of microspores were uniform.After 3-MA pretreatment,the microspore volumes of the majority of microspores(70%)were relatively increased and the size becamed nonuniform compared with the MMCs growing in normal condations.After E-64 pretreatment,the volumes of the majority of microspores(80%)were conversely decreasd and the sizes also became nonuniform.(5)RT-PCR analysis demonstrated that the autophagy-related gene ATG8 of hollyhock was expressed in all of the detected tissues,including roots,stems,leaves,MMCs,tetrads and microspores in hollyhock.(6)Sub-microstructure observation revealed that there really were many autophagosomes or autophagic vacole within the hollyhock MMCs.The formation process was that a crescent-like bilayer structure was firsted formed,the membrane continues to extend to form a cup-shaped structure,and then the membrane were fused at both ends to form a phagocytic structure.With cell development some autophagosomes were encapsulated by another membrane to form a structure surrounded by multilayer of membranes,and the inlayer of double membrane of some autophagosomes ruptured and recurlled to form an membrane-bound innclusion.Finally,some of the autophagosomes were fused with plasma membrane and discharged their contents to between plasma membrane and cell wall,and some were fused with vacuole or lysosome to be degradated.All together,these results suggested that autophagic activity really exist in the process of microsporogenesis.