Study On Transcriptional Regulation Of Transcription Factor TDF1 On Tapetum And Pollen Wall Development Of Arabidopsis Thaliana

The life cycle of high plants can be subdivided into sporophytic vegetative development and gametophytic reproductive development. During the male gametophytes process, it’s well known that sporophytic tapetum plays important roles in pollen and pollen wall development, such as providing the necessary nutrients for microsporocytes development, releasing the microspores from tetrads after callose degradation, storing and transporting the sporopollenin precursors for sexine formation and activating PCD. Defective in tapetal development and function 1(TDF1) encodes an R2R3 MYB transcription factor, which is highly expressed in tapetum and microspores during the early stage of anther development, playing a critical role in regulating tapetum function and pollen development. However, the transcriptional regulation network of TDF1 and other downstream genes remains unknown and the biological pathways of TDF1 required for normal tapetum and pollen development are unclear. Here, we construct the transgenic plants with GFP/MYC/FLAG/HA, showing TDF1 can bind to the promoter of AMS with AACCT binding site in vivo and in vitro by Ch IP and EMSA system. Furthermore, TDF1 directly regulates AMS verified by the transient expression assay in tobacco. However, obtaining the Pro DYT1:AMS transgenic lines with tdf1 background, we find AMS cannot rescue the phenotype of tdf1 even if AMS is normally expressed. Therefore, we show that TDF1 and AMS can interact with each other by Co-Ch IP assay in tobacco, suggesting that this interaction may function in the activation of downstream target genes of AMS. Analyze the microarray data, TDF1 is required for normal expression of pathways including miscellaneous enzymes familes, cell wall and development, revealing that TDF1 has a close relationship with cell differentiation, cell wall modification and cell development. Combined with the phenotype of tapetum in tdf1 with abundant cell division and vacuolation, we find that TDF1 can directly regulate SKS11, SKS13, SKS18 and EXPB5 to inhibit the cell division and accelerate cell wall expansion meeting with the transformation of secretory tapetum. SKS18 is specifically expressed in tapetum at stage 6~7. SKS18-GFP is exclusively located on the cell wall/cell plasma membrane of tapetum at stage 6~7, then SKS18-GFP is gradually migrated onto the plasma membrane of microspore and anther locule. On the other hand, we find that AMS directly regulates the expression of TEK and MS188 in tapetum for the nexine and sexine formation in vivo and in vitro, respectively. Additionally, we observe the Pro AMS:TEK:GFP and Pro AMS:MS188:GFP transgenic lines with wild-type background, finding that premature expression of TEK and MS188 lead to male sterile. This futher indicates the importance of sequential pollen wall development. In conclusion, our results demonstrate the important roles of TDF1 in regulating anther transcriptome and function; show TDF1 function in tapetum differentiation and pollen wall development based on its transcriptional regulatory networks.