Functional Analysis Of YKT61 In Arabidopsis Thaliana

In eukaryotic cells,cell compartmentalization is a hallmark.Functional and spatially independent reaction chambers exist inside cells,which mainly refer to membrane-wrapped vesicles.Each reaction chamber is composed of organelle specific biomolecules.The establishment and maintenance of complex eukaryotic compartments require comprehensive transport activities between different locations in the endomembrane system.Vesicles transfer biomacromolecules by shuttling between organelles with cargo and connecting these organelles,ensuring spatio-temporal dynamic distribution of biomacromolecules,which is important for maintaining cell function and responding to various environmental stimuli.Vesicle transport is divided into four basic steps,including vesicle budding,movement,tethering,and fusion.These four steps are strictly controlled to ensure that the vesicles generated from the donor compartment can be delivered to the correct acceptor compartment.Specific vesicle fusion during vesicle transport is mediated by the SNARE（soluble N-ethylmaleimide-sensitive factor attachment protein receptor）protein.SNARE is a superfamily of highly conserved protein.According to sequence analysis,64 genes encoding SNARE protein have been found in the Arabidopsis genome,which have been proved to be located in different subcellular compartments and are commonly expressed at various stages of plant growth and development.In the past few decades,SNARE proteins have been found to be involved in the regulation of basic plant physiological processes,such as cell division,gametophyte development,defense response,stem and root geotropism,osmotic stress,salt stress,and ion channel regulation through comprehensive cell biology and biochemistry studies,as well as forward and reverse genetic screening.YKT61 is a unique SNARE protein.Unlike other SNARE proteins,YKT61 lacks transmembrane domain and is mainly located in the cytoplasm,suggesting that YKT61 may have a different biological function.However,the function of YKT61 in Arabidopsis thaliana has not been reported.In this paper,Arabidopsis YKT61 was the main object of study.The molecular function of YKT61 and its roles in gametophyte cell development and sporophyte cell development were analyzed in detail.The research results and main conclusions of this paper are as follows:（1）YKT61 is essential for the development of male and female gametophytesYKT61 is a constitutive expression gene that is expressed in various tissues and organs of Arabidopsis thaliana.We edited YKT61 using CRISPR/Cas9 gene editing technology to obtain two alleles,ykt61-1/+and ykt61-2/+,which introduced a premature stop codon into the transcript due to base editing.Through multiple analyses,we found that ykt61/+silique is aborted,and the results of cross-pollination showed that ykt61 male and female gametophytes did not generation at all.We analyzed the development of pollen,and found that ykt61 pollen was completely aborted and began to develop abnormally in Pollen Mitosis I.In terms of female gametophyte development,we found that ykt61 female gametophytes result in failed pollen tube guidance and reception by optical section,12HAP GUS and aniline blue staining experiments.These results indicate that YKT61 is essential for the development of female and male gametophytes.（2）YKT61 affects cell division and differentiation by regulating dynamic targeting of BRI1.By constructing YKT61 functional knockdown weak mutants ykt61-pc（ykt61-partially complemented）and RNAi materials,we found that knockout YKT61 would lead to shorter primary roots,smaller rosette leaves,plant dwarfing and reduced fertility.At the cellular level,YKT61 is essential for dynamic biogenesis of vacuoles,morphology maintenance of Golgi,and endocytosis.The above results indicate that YKT61 is involved in regulating the growth and development of sporophyte cells.Using the techniques of genetics,cytology,molecular biology and pharmaceutics,we found that the introduction of bri1-116,a mutant of brassinolide receptor BRI1,could completely inhibit the ectopic excessive expression of ERF115,abnormal proliferation of quiescent center cells and abnormal deposition of starch granules in columella stem cell initials after knockdown YKT61.This indicates that YKT61 is involved in regulating brassinolide signaling pathway,and BRI1 is located in the downstream of YKT61.The results of medicaments treatment showed that YKT61 regulated brassinolide signaling pathway differently in different cell environments.YKT61 positively regulates brassinolide signaling pathway in meristematic cortex cells,but negatively regulates brassinolide signaling pathway in quiescent center steady-state maintenance.YKT61 directly interacts with BRI1,and participates in the brassinolide-mediated division and differentiation of root apical meristem and quiescent center by regulating BRI1 localization and cycling on plasma membrane.（3）YKT61 interacts with multiple SNARE proteins on different membrane components.By Bi FC and pull-down experiments,we found that YKT61 can interact with multiple SNARE proteins in different organelles,suggesting that YKT61 may mediate different vesicle fusion events and play a role in multiple membrane structures.The deletion of eighth amino acid,which is the key site for its function,leads to the abnormal development of gametophytes and embryos,altered localization,and loss of interaction with multiple SNARE proteins.In addition,the 195th position of Cys in YKT61 is also the key site for its function.Palmitoylation of YKT61 at this site is essential for membrane localization of YKT61.YKT61^C195S cannot complement the phenotype of ykt61/+,but can still interact with SNARE proteins.These results indicate that interaction between YKT61 and other SNARE is a necessary but insufficient condition for YKT61 to function.（4）Palmitoylation is essential for the function YKT61.Through homologous protein sequence alignment and protein palmitoylation detection experiments,we found that Arabidopsis YKT61 was palmitoacylated and the modification site is cysteine residue at 195 position.This site mutation resulted in abnormal localization of YKT61,and the mutant could not be rescued,indicating that palmitoylation is crucial for the function of YKT61.However,the mutation of C195 site did not affect the interaction between YKT61 and other SNARE,indicating that YKT61 function is not only involved in the formation of SNARE complex and mediating vesicular fusion,but also has other ways to exert its function.We mutated YKT61 from a R-SNARE to a Q-SNARE,and found that its localization,interaction,and its ability to complement the mutant were not affected,indicating that YKT61plays a unique role in Arabidopsis thaliana and does not function as a classical R-SNARE.In addition to palmitoylation,YKT61 may also function as a palmitoyl transferase.Yeast YKT6 is a palmitoyl acyltransferases,and its palmitoylation substrate is the fusion factor Vac8,which has no homologous protein in Arabidopsis thaliana.We found that the interacting proteins of YKT61,VAMP721,VAMP722 and VMAP727,were palmitoacylated,and the VAMP721,VAMP722 and VMAP727 had altered localization in the mutant of YKT61 knockout.The VAMP7s can be combined with Q-SNARE by replacing YKT61.In combination with the study of YKT61 in Drosophila melanogaster,we hypothesized that cytoplasmic YKT61 was located on the membrane after palmitoylation,and then combined with Q-SNARE to participate in SNARE complex assembly.When R-SNARE VAMP7s reach the fusion site through vesicular transport,palmitoacylated by YKT61,and VAMP7s replace YKT61 to form SNARE complexes with fusion ability,thus completing the vesicular fusion process,which further enriched the research progress of palmitoylation.