Functional Analysis Of Arabidopsis ASNAP In Sexual Reproduction

Sexual reproduction is essential for plant survival and reproduction.Gametophytic development is the pre-requisite for plant reproduction and seed yield,whose study is not only informative to understand fundamental biological questions but also potentially applicable to agricultural biotech.Vesicular trafficking mediates the transport of proteins and lipids among endomembrane systems in eukaryotic cells.The fusion between vesicles and target membrane is controlled by SNARE protein complexes,which consists of one RSNARE and three Q-SNAREs.The SNARE complexes depolymerize after membrane fusion to recycle.In yeasts and animals,N-ethylmaleimide-sensitive fusion protein(NSF),together with soluble NSF attachment protein(?-SNAP),promotes the disassembly of SNARE complexs.Functional loss of ?-SNAP seriously compromises the reproduction of animals.Plant genomes encode homologs of ?-SNAP.However,their function is yet to be characterized.In this study,we identified the homologs of ?-SNAP in Arabidopsis thaliana(designated as ASNAP),and examined its function by reverse genetics.The main results and conclusions of this thesis are as follows:(1)ASNAP was constitutively expressed.ASNAP is constitutively expressed based on quantitative real-time PCRs and genomicGUS reporter analyses.(2)Functional loss of ASNAP gametophytic lethality.Because there were no T-DNA insertion lines within the ASNAP genomic locus,we generated asnap mutants by using the CRISPR/Cas9 system.Our studies of the mutants showed that ASNAP is essential for both male and female gametogenesis since no asnap mutant alleles can be transmitted at all.Thus,ASNAP is the first plant gene essential for both male and female gametogenesis,whose study is only made possible by genome-editing such as CRISPR/Cas9.(3)Functional loss of ASNAP caused pollen abortion and the arrest of embryo sac development.To identify the cause of male and female gametophytic lethality by ASNAP loss-offunction,we conducted detailed analyses.By Alexander staining,DAPI staining,scanning electron micrographs(SEMs),optical sections,semi-thin transverse sections and ultrastructure studies,we demonstrated that the development of asnap microspores was arrested at pollen mitosis I.By optical sections,whole-mount ovule clearing,pollination assays with ProLAT52: GUS etc,we demonstrated that the development of asnap embryo sacs was arrested right after the establishment of functional megaspore(FM).(4)asnap-pc,a weak ASNAP allelic mutant,showed abnormal sporophyte development.Partial complementation of asnap by GFP-ASNAP resulted in a weak allele,asnap-pc(for partial complementation).The mutant plants showed short roots and abnormal cotyledon development,indicating that ASNAP is also critical for the development of sporophytic cells.(5)ASNAP is targeted to the cytoplasm and endomembrane compartments.We generated GFP-fused ASNAP transgenic plants.By using RFP-fused endomembrane markers in confocal laser scanning microscopy(CLSM),we demonstrated that ANSAP associates with almost all endomembrane compartments,in addition to the cytoplasm,consisting with its roles in dissembling SNARE complexes.(6)ASNAP interacts with NSF who also participates in the regulation of fertility.In yeast and animals,?-SNAP interacts with NSF to dissemble SNARE complexes.We demonstrated that such interaction also occurs in Arabidopsis by bimolecular fluorescence complementation(BiFC).Downregulating NSF by RNA interference resulted in a significant decrease in plant fertility,suggesting that ASNAP and NSF also function as complexes in plants.In this paper,we used the CRISPR/Cas9 technology to creat asnap mutant.We revealed the key roles of ASNAP in the development of male and female gametophytes and preliminarily discussed its mechanism.These results provide more clues for analyzing the dynamics of vesicle transport and the mechanism of vesicle transport regulating growth and development in plants.