The Study On The Mechanism Of Arabidopsis ADF5 Regulating The Seed Development And Bundling Actin Filaments

Higher-order structures of the actin cytoskeleton are essential for massive physiological processes.However,the mechanisms underlying the formation and maintenance of actin bundles in plant cell remain largely unclear.Actin Depolymerizing Factors(ADFs)family is an important class of actin binding proteins,which exists in all eukaryotes,and typically contributes to actin turnover by severing and/or depolymerizing actin filaments.Recently,ADF9 is shown actin-bundling and actin-stabilizing activities in vitro.We also identified another atypical ADF isovariant,ADF5,which also exhibits pH-sensitive actin bundling and stabilizing activities in vitro.Up to now,little is known about the physiological and biochemical functions of ADF5.Thus,our research goal is to understand the mechanism of ADF5 and ADF9 bundling actin filaments,and regulating the dynamic of the actin cytoskeleton during the seed development.The main obtained results are as follows.1.We found some physiological traits in ADF5 T-DNA inserted mutant(adf5)were significantly distinct from those in wild type,including the size,length,weight of seed,the silique width and product per single plant as well as the cotyledon cell area but not cell number.All above phenotypes can be rescued by exogenous ADF5,which indicated ADF5 participating the seed development.2.The embryo development of adf5 lagged behind those in WT after we observed the whole embryo developmental process in Arabidopsis.Comparing with WT,the seed development of mutant will take more 2-3 days from fertilization to seed maturation.3.Loss of function of ADF5 can obviously reduce actin and actin filament bundling in cell,moreover,the cytoskeleton in mutant cell is extremely sensitive to actin depolymerized drug LatB,which indicated that ADF5 might be an actin bundling protein to take part in forming and maintaining actin bundles.4.By means of high and low speed co-sedimentation assays,it was found that ADF5 could bundle and maintain actin filament in pH-dependent manner,conflicting with other conserved ADF5.In addition,the N-terminal of ADF5 can bind actin.If this domain is lost,ADF5 would be induced to deficient in bundling actin.And then,when each N-terminal part of ADF5 and ADF1 are exchanged,the recombinant protein ADF5N-ADF1 C can also bundle actin,these results were indicated that the N-terminal of ADF5 possessed functional site to bind F-actin,which is vital for determining ADF5 biochemical traits.Moreover,we found that ADF5 does not form polymers in vitro.After the conserved site binding actin of C-terminal of ADF5 was site-specific mutated,the function of ADF5 bundling actin would be abated.It was confirmed that the natural function of actin bundling of ADF5 was dependent on sites binding to F-actin within both N-and C-terminal.5.Exogenous ADF5 derivatives including N-terminal,lack of N-terminal and site-mutated Cterminal of ADF5 cannot rescue the seed development defects in adf5.It was demonstrated that ADF5 played a primary role on bundling and maintaining actin to regulate seed development.In this paper,we first reported the third subfamily member of ADF named ADF5 in Arabidopsis,which functioned for bundling and maintaining actin in vivo and vitro as well as participated in forming and stabilizing higher-ordered cellular structure via two actin binding sites existing in N-and C-terminal of ADF5.Furthermore,it was proven that actin cytoskeleton played an important role on plant seed development process.