DG1 Regulates The Pollen Development And The Pollen Tube Growth In The Pistil Of Arabidopsis

The interaction between pollen tube and stigma is the key step for pollination recognition,which determines the germination of pollen and the growth of pollen tube on stigma.The study on the interaction mechanism between pollen tube and stigma is the basis for understanding the sexual reproduction and pollination recognition in angiosperm.Endocytosis is important regulation for the growth of pollen tube and the interaction between pollen tube and pistil during pollination;however,the underlying molecular mechanism remains to be investigated.Arabidopsis Defective Pollen Tube Growth1(DG1)encodes an adaptor protein like(AP-like),DG1 was expressed in pollen grain(after the Stage 11)and the tip of pollen tube,and localized in the plasmalemma of pollen grain and flank region of the tip of pollen tube,which indicate DG1functions in the pollen development and the growth of pollen tube.In order to investigate the roles of DG1 in the development of pollen,we created a mutant of DG1,dg1-3/+,by CRISPR/Cas9 system,in which an adenine base was inserted in the coding region of DG1 leading to premature translation termination.Half of the dg1-3/+pollen showed an abnormal morphology and could not germinate on medium.Alexander staining revealed that the mutant pollen lost their viability.Additionally,the mutant pollen from stage 12 stage showed abnormal accumulation of callose and abnormal distribution of pectin and cellulose.To investigate the roles of DG1 in pollen tube growth,we obtained the transgenetic Arabidopsis lines expressing the estradiol-induced artificial microRNA construct pER8::amiRNA-DG1.The pollen tubes of the transgenetic lines showed normal growth on the stigma surface of the pistil treated with estradiol,but could not grow into the transmitting tissue.These results indicate that DG1 plays a role for the growth of pollen tube in pistil tissue.To identify the cargo proteins of DG1,a yeast two-hybrid analysis was performed by search against the cDNA library of Arabidopsis pollen using DG1.And,a Pectin methylesterase PME and a Shaker family potassium ion(K~+)channel SPIK were identified.We confirmed their interactions through DUALmembrane Yeast two-hybrid system and Pull-down analyses.Furthermore,we found that DG1 interacts with SPIK through the Y-I-L-V sequence of SPIK.PME-RFP was localized in the plasma membrane and cell wall in growing pollen tube in the pPME::PME-RFP transgenic lines.We isolated the T-DNA inseration mutant of PME,pme,which showed similar pollen phenotypes with the dg1-3.Furthermore,we had constructed the pER8::amiRNA-PME and investigate the growth of pollen tube in pistil treated with estradiol,which would help us to understand the roles of PME for the growth of pollen tube in pistil.