PI3P Binds To ADF1 To Modulate Actin Dynamic And Plays A Role In Hypocotyl Elongation In Dark

Phosphatidylinositol is an important phospholipid that exists widely in eukaryotic cells.It plays a very important role in cell morphology,metabolic regulation,signal transduction response to stress,and various physiological activities in cells.Phosphatidylinositol-3-phosphate(PI3P)is the main component of the phosphatidylinositol signal,and is catalyzed by phosphatidylinositol-3 kinase(PI3K).PI3 P plays an important role in the process of membrane dynamics and transport regulation.It mainly participates in the regulation of root hair growth,polar apical growth,pollen development,pollen tube elongation,vesicle transport,stomatal switch movement,vacuole morphological changes,and actin dynamics regulation.However,little is known about the detailed mechanism of PI3 P in regulating plant growth and development,and further research is needed.The acitn cytoskeleton is a filamentous network that plays a vitol role in the regulation of various biological processes including cytoplasmic circulation,hypocotyl elongation,pollen tube germination,stomata movement,vesicle transport,the maintenance of cell architecture,and cell division.Actin cytoskeleton generally exists in two forms,spherical monomers and filamentous polymers.Actin cytoskeleton undergoes constant dynamic rearrangement,through the rapid turnover between polymerizing and depolymerizing process.This rapid rearrangements of actin cytoskeleton is strictly regulated in time and space by actin binding proteins.Actin depolymerizing factor(ADF)is an important actin binding protein.In plants,ADF has actin cleavage activities to play roles in the depolymerization of filamentous polymers,and participates in the regulation of plant growth and development,as well as the control of stomatal movement.Therefore,ADF is an important actin binding protein for actin severing and depolymerizing regulaiton.The actin activity of ADF protein is strictly regulated,which mainly includes phosphorylation-mediated ADF inactivation,the regulation of ADF protein by PH,and phosphatidylinositol,PI(4,5)P2-mediated inhibition of ADF.PI(4,5)P2 binds to ADF protein,reduces the actin binding activity of ADF,and inhibits actin depolymerization.However,little is known about whether there are other types of phosphatidylinositol regulating ADF actin activity.The research and results of this paper mainly include the following aspects:1.PI3 K specific inhibitors: Wortmannin and LY294002,inhibit hypocotyl elongation in darkIn this experiment,the Arabidopsis seedlings were treated with the specific inhibitors of PI3 K,Wortmannin and LY294002.The phenotype of hypocotyl elongation was observed after growing in the dark for 4-5 days.The results show that Wortmannin and LY294002 inhibits the elongation of hypocotyl in the dark.2.The transgenic plants overexpressing YFP-2×FYVE exhbit impaired hypocotyl elongation phenotype in the darkThe PI3P-binding domain FYVE was overexpressed in Arabidopsis transgenic lines to mimic the environment where the PI3 P content was reduced.After 4-5 days of growth in the dark,the hypocotyl elongation phenotype was observed.When overexpressing PI3P-binding domain FYVE,the hypocotyl elongation is inhibited.Thus,it shows that PI3 P participates in the process of hypocotyl elongation in the dark.3.Dynamic changes of actin filaments in hypocotyl cells is altered after treatment with WortmanninArabidopsis seedlings growon in the dark for 4-5 days were treated with Wortmannin,and the actin stability in hypocotyl cells were observed with two-photon confocal microscopy.The result of this study shows that the depolymerization of actin filaments is more obvious after Wortmannin treatment.It proves that PI3 P is involved in the dynamic regulation of actin filaments in hypocotyls.4.PI3 P binds to ADF1It is known that PI3 P is involved in the process of hypocotyl elongation in the dark and functions in actin dynamic regulation.In this process,which proteins bind to PI3 P are involved in regulating cell elongation and actin dynamic regulation? In order to further make sure the mechanism by which PI3 P participates in the regulation of hypocotyl elongation and the dynamic regulation of actin filaments,we constructed an ADF1 protein expression vector,purified the ADF1 protein,and performed the in vitro binding assay including spot membrane experiments and microthermokinetic analysis(MST).It suggests that PI3 P binds to ADF1.5.ADF1 phosphorylation level is reduced after Wortmannin treatmentTransgenic Arabidopsis seedlings exprocessing Flag-ADF1 were used in this exprement.The Flag-ADF1 protein was immuneprecipitated with flag beads.Phosphorylation assay was performed using anti-phospho antibody.After Wortmannin treatment,ADF1 phosphorylation level is reduced obviously.The phosphorylation plays a key role in the regulation of ADF1-mediated actin filament depolymerization.ADF1 participates in the regulation of the hypocotyls.The innovations of this experiment are:This experiment demonstrates that PI3 P binds to ADF1 to regulate the phosphorylation level of ADF.PI3 P is involved in regulating acitn dynamic and functions in the regulation of hypocotyl elongation.These results have innovative significance for further expanding the biological function of PI3 P,and have important value for further discovering and exploring the regulatory mechanism of ADF protein.