Reactive Oxygen Species Mediated Tapetal Programmed Cell Death Affects Pollen Development In Tobacco And Tomato

Hybrid vigour is highly valued in agricultural industry,but the cost of artificial emasculation to generate hybrid seed in agriculture was huge.And male sterility is an important agriculture trait,so breeding of new male sterile line is a valuable way to generate hybrid seeds.Pollen development is tightly controlled anther sporophytic tissues which tapetum is the most critical sporophytic tissues through programmed cell death(PCD)to provide material and enzyme for pollen.As previously,the defective of tapetal PCD will lead to pollen abortion.Although the timing and progress of tapetal PCD is tightly controlled by clear and conserved transcriptional network in rice and Arabidopsis,whether intracellular factors involved are evolutionarily conserved is unclear.Recently,there has been demonstrated that NADPH oxidase produce spatiotemporally ROS to regulate tapetal PCD to affect pollen development.To investigate whether the same mechanism that ROS regulate tapetal PCD exists in economically important dicots species or we can use this network to generate new male sterile.We choose two important cash crops such as Nicotiana Benthamiana and Lycopersicon esculentum cv.moneymaker to research their tapetal development.The results and conclusions of this study run as follows:(1)Dynamic ROS levels in tobacco and tomato anthersWe discovered that tobacco and tomato anthers showed a dynamic ROS amplitude which increased at first and then decreased by NBT and H2DCF-DA staining.The ROS accumulated from premeiosis stage,then gradually increased at meiotic and tetrad stage,reached the highest level at microspore stage,finally decreased to basal level at mitosis stage in both tomato and tobacco.(2)Histological and cellular change in tobacco and tomato tapetumWe prepared dissection and transverse anther sections to correlate flower organ size with the specific development stage of tapetum in order to facilitate our work.We prepared plastic sections of these two dicots anthers in order to describe the tapetum and pollen development and events occurred at the cellular level,and we observed tapetal ultrastructure when it underwent PCD by TEM.(3)Tapetal PCD in tobacco and tomatoWe performed TEM and TUNEL assay on tobacco and tomato anthers at different developmental stages to observe tapetal PCD.Indeed,tapetal PCD in tobacco mainly at stage 11,i.e.at mitosis stage,and tomato tapetal PCD earlier and slower than that in tobacco such that it start from stage 10,i.e.at late microspore stage,we also observed TUNEL signaling at stage 11.(4)Pharmacological interference of ROS amplitude impaired pollen developmentTo investigate the relationship between the dynamic amplitude of ROS and tapetal PCD in tobacco anthers,we wondered whether it was the case also for these species.We applied 10?M DPI treat the tobacco anthers to scavenge ROS.The delayed tapetal degeneration by DPI treatment correlates well with a reduced ROS amplitude and significantly higher number of pollen grains were aborted from DPI-treated anthers than that treated with DMSO.These results suggested the mechanism that ROS regulate tapetal PCD also exist in tobacco.(5)Tobacco and tomato RBOH genes expression pattern and function analysisRecently studies have demonstrated the critical roles of the PM-localized NADPH oxidase RBOHE in tapetum ROS signaling in Arabidopsis.It remains unclear how ROS generated in this two dicots tapetum.We performed the BLAST to find the RBOH gene family in tobacco and tomato.We performed qRT-PCR to detect the expression of these RBOH genes in different tissues,thus we found some RBOH gene specifically expressed in anther.Then we performed qRT-PCR to discover the expression of these genes whether were dynamic amplitude in different stage of anther development.We found NbRBOHE1,NbRBOHE2 and LeRBOHE,LeRBOH temporally expressed in tobacco or tomato anthers respectively that means these genes possibly play a role in tobacco and tomato anther in ROS generation.We further silenced the NbRBOHE1 by VIGS,but the gene silencing has no defect in tobacco tapetum and pollen.So we proposed that there must be more RBOH gene expressed in tobacco anthers to regulate tapetal PCD.In the next,we can knockout or overexpress more RBOH gene in tobacco and tomato anthers to observe the development of tapetum and pollen.