Studies On Autophagy During The Course Of Microspore Embryogenesis In Nicotiana Tabacum L

Autophagy is an important biological event,which can help plants to resist abiotic stress and play a very important role for the growth and development of plants by degrading or recycling the large molecular substances,toxic proteins,organelles and other substances in thecell.Early autophagy-related studies were primarily focused on animal,yeast,and eukaryotic organisms,less than few research on plant,particularly on plant embryogenesis.Microspore embryogenesis is a very complicated process that microspore can change its developmental fate from gametophytic pathway to sporophytic pathway when exposed to stress treatments in culture.In this process,the cytoplasm of the microspore with the embryogenic potential is reconstructed,the cytoskeleton is rearranged,and the normal cell cycle enters the new cycle after the temporary termination.While the cells without embryogenic potential will died in the process of stress treatments.However,the mechanism of embryogenesis is still not clear.During the course of microspore embryogenesis,does the presence of autophagy determines the microspore transformed from the gametophytic pathway to the embryogenesis pathway,the generation of autophagy is to help the cells resistant to external stress or to cause death of cells.Microspore embryogenesis will be effect if autophagy is inhibit?The present study investigated the autophagy during the course of microspore embryogenesis from cell morphology,molecular biology,and bioinformatics,and compared it with early gametes of the gametophyte pathway.The main experimental results are as follows:(1)The system of microspore embryogenesis was established,and the time and pattern of cell division in the process of embryogenesis were clarified,through morphological observation,we have compare the differences between the two developmental pathways of the early stage,which provided a reference for the follow-up study.(2)The cells were stained and fluorescently observed during the day,three days,five days,and the prophase of gametophyte development using autophagy specific dye mondansulfonamide(MDC).It was found that in that process of embryogenesis,autophagy begins at the first day of the stress treatment,the intensity reach a maximum at the third day,decay is started at the 5th day,and no autophagy signals are detected in the gametophyte pathway.It is speculated that the attenuation of the number and intensity of autophagy may be related to the change of cytoplasmic concentration.(3)The ultrastructure of the autophagy was observed by slicing,and the ultrastructure of the autophagy was observed on both the first and third days of stress treatment,and the autophagy-encapsulant mitochondria were relatively common,However,autophagy was not found in the binucleate pollen and mature pollen,which was consistent with the MDC test results.There is a lot of starch grains in mature pollen inside the cell accumulation,but in the microspore of equal division for the first time there are high density of mitochondria,which suggests that there is a clear difference in the occurrence of autophagy between the two developmental pathways of microspores.(4)When autophagy is inhibited,the activity of microspores rises,while the embryo's capacity declines,which show that during the embryogenesis,autophagy may play a role in screening microspores which capable the potential of embryogenesis.(5)RNA-seq data showed that there are multiple expression patterns of autophagy-related genes during embryogenesis.RT-qPCR was used to detect the relative expression of 8 autophagy-related genes,The results showed that in addition to NtATGlb,the expression levels of the other seven autophagy-related genes showed a clear upward trend,and the up-regulation of NtVPS15 and NtVTI12b was particularly evident during the induction.NtVPS15,NtATG4,and NtVTI12b were not expressed in the gametophytic pathway,and the expression level of NtVPS15,NtATG4,and NtVTI12b gradually increased during stress induction.It is indicated that the specific expression of these three genes also plays a role in promoting the embryogenesis of microspores.NtATG8e and NtVTI12b down-regulated expression in gametophyte,and up-regulated expression in the stress of induced,speculated that the two genes for changing the microspore developmental pathway plays a very important role.