The Role Of Actin Depolymerizing Factor ADF7 In Root Hair Growth And Development In Arabidopsis Thaliana

Root hair plays an essential role in the absorption of water and nutrients,and root hair growth and development is an important mechanism of plant response to abiotic stress.This is regulated by many factors,for example,microfilament skeleton is essential for the growth and development of root hairs.However,the molecular mechanism of microfilament dynamics in the growth and development of root hairs under abiotic stress has been rarely reported.Osmotic stress is one of the most important abiotic stresses,which can cause cell water loss and inhibit plant growth and development.This study revealed that,under osmotic stress,the Actin Depolymerization Factor 7(ADF7)inhibits the expression and function of the actin bunding protein VILLIN1(VLN1)in regulating the dynamics of microfilaments in the root hair growth and development,and this is directly regulated by the transcription factor CAPRICE(CPC).(1)Genetic analysis showed that the loss of ADF7 function resulted in a decrease in the number of root hairs.It’s because of the decrease in the percentage of root hair growing in root hair cells(H cells)in root epidermal cells and the density of H cells.In addition,the loss of ADF7 function also resulted in significantly shorter root hair length.However,ADF7 OE seedlings had more root hair number and longer root hais.Furthermore,the morphology of microfilaments and the dynamics of single microfilaments in root and hair cells were observed and analyzed.These results illustrate that ADF7 plays a positive role in root hair growth and development through depolymerize and sever single F-actins,which leads to a significant decrease of thick bundles and increase of F-actin turnover.(2)RT-q PCR and Western Blot analysis showed that VLN1 expression was significantly increased in adf7 mutants,but inhibited in ADF7 OE seedlings.While ADF7 expression in roots wasn’t significantly changed in vln1 and VLN1 genotype seedlings.The root hair phenotype of adf7 vln1 seedlings were similar to single vln1 mutants.The morphology of microfilaments in root cells and hair cells and the dynamics of single microfilaments were observed.We found that ADF7 inhibited VLN1-mediated thick bundle formations,leading to the increase of depolymerization and F-actin turnover in root epidermal cells and root hairs.These results indicate that ADF7 upstream inhibits VLN1 expression and VLN1-mediated microfilament binding(Hereinafter referred to as the ADF7-VLN1 module).In addition,we found that the number and length of root hairs of all genotype seedlings are reduced under the treatment of reactive oxygen inhibitor DPI.Meanwhile,the reactive oxygen species(ROS)production and expression of ROS-related gene ROOT HAIR DEFECTIVE2(RHD2)were significantly lower in adf7,while the ROS production and expression of RHD2 were higher in adf7 vln1,which was similar to vln1.These results illustrate that ADF7-VLN1 module leads to the depolymerization of microfilaments and the increase of the turnover of single microfilaments,which may affect the expression of RHD2 and lead to the increase of ROS level,thus promoting the formation and growth of root hairs.(3)The detection of gene expression under osmotic stress showed that with the increase of mannitol treatment concentration,VLN1 expression decreased gradually,while the relative expression of ADF7 increased gradually.Phenotypes of related genotype seedlings showed that adf7 vln1,vln1 and ADF7 OE were similar under osmotic stress,with more root hair number,longer root hair length,larger leaf area,dry weight and water content,while adf7,VLN1 OE,and adf7 VLN1 OE seedlings displayed contrasting phenotypes.These results illustrate that the root hair growth and development mediated by ADF7-VLN1 contributes to osmotic stress tolerance.In addition,similar to vln1,the root epidermal cells and root hairs of adf7 vln1 had thin and dense microfilament arrangement in osmotic stress.While the F-actin array of adf7-2seedlings displayed contrasting phenotypes.It indicated that ADF7-VLN1-mediated root hair growth and development respond to osmotic stress by inducing F-actin depolymerization.Compare with adf7-2,the ROS accumulation of root epidermal cells and root hairs in adf7 vln1 seedlings was similar to vln1 with high level under osmotic stress.These results illustrate that ADF7-VLN1 module mediates root hair growth and development by inducing the F-actin depolymerization in root cells and root hairs,increasing the accumulation of ROS and responding to osmotic stress.(4)GUS staining and Western Bloting analysis showed that the expression of CPC in root under the mannitol treatments was significantly increased.In addition,under mannitol treatment,cpc has fewer and shorter root hairs than Col-0,and the microfilaments in root epidermal cells and root hairs of cpc are thicker and sparser.These results suggest that CPC is involved in osmotic stress and may affect the growth and development of root hairs by inhibiting the formation of thick microfilament bundles in cells.Ch IP and EMSA results showed that CPC could directly recognize ADF7 and VLN1.RT-q PCR analysis indicate that CPC promotes ADF7 and inhibits VLN1 expression.We researched the root hair number and length of mutants and hybrid materials under the osmotic stress,compare with adf7-2,cpc has fewer root hairs and similar length of root hairs.cpc adf7 and cpc vln1 are similar to adf7-2 and vln1-2 respectively.It implies that CPC might via directly regulating ADF7-VLN1 module affect the root hair growth and development in response to osmotic stress.In conclusion,the results showed that under osmotic stress,CPC regulated ADF7-VLN1 module,affected ROS accumulation by regulating the F-actin dynamics in cells,and participated in root hair growth and development.This provided a new evidence for the dynamics of microfilaments and their molecular mechanisms in root hair growth and development under abiotic stress.