Microfilament Cytoskeleton Affects Stem Cells Specification Through Mediating Auxin Polar Transport Duirng In Vitro Shoot Regeneration In Arabidopsis

Microfilament cytoskeleton has been proved to play an important role in regulating plantgrowth and development. Using the in vitro shoot regeneration system, we focused onstudying the mechanism of microfilament cytoskeleton regulated-stem cells specificationduring shoot meristem initiation. Firstly, we detected the dynamic distribution of themicrofilament cytoskeleton during shoot regeneration, by combining pWUS::DsRED-N7with35S::GFP-ABD2-GFP to study the microfilament cytoskeleton in organizing center cells.Secondly, we studied the influence of changing dynamic distribution of microfilamentcytoskeleton on stem cell specification and in vitro shoot regeneration. Finally, we analyzedthe function of actin depolymerizing factor (ADF) during shoot regeneration. The resultsdemonstrate that stem cell specification is associated with the process of microfilamentdepolymerization during in vitro shoot regeneration. Inhibiting microfilamentdepolymerization affects auxin polar transport and distribution, thus inhibits stem cellspecification in in vitro shoot regeneration. ADFs, the actin depolymerization factors, areshown to play an important role in this process. The main results are as follows:1) Stem cell specification is associated with the process of microfilamentdepolymerization during in vitro shoot regeneration.Polymerizedly filamentous distribution of microfilament cytoskeleton was observed incells of the callus. After induction of shoot, partial depolymerizing of microfilaments occurredin organizing center cells of de novo formed shoot promeristem. Later, actin filaments mainlydecorated as randomly distributed punctiform, granular, short rod-like bundles in theorganizing center of the shoot meristem. However, callus cells around shoot primordial stillharboured polymerizedly filamentous microfilament cytoskeleton. These results indicate thatstem cell specification is associated with the depolymerization of microfilament cytoskeletonduring in vitro shoot regeneration.2) Inhibition of microfilament depolymerization affects stem cell specification and shootregeneration.Application of phalloidin, the inhibitor which restraint the microfilament depolymerization in shoot-induced medium resulted in the inhibition of stem cellspecification and shoot regeneration. WUS could not be detected in callus during shootregeneration under treatment of phalloidin. Furthermore, PIN1polar localization in the calluswas obviously disrupted，too. This indicates that inhibiting microfilament depolymerizationaffects stem cell specification and shoot regeneration.3) Down-regulation of expression of Actin depolymerizing factors (ADFs) disrupts auxinpolar transport and distribution during shoot induction.qRT-PCR analysis showed that expression of most ADFs was up-regulated during shootinduction. While the expression of polymerizing factor ADF9was significantlydown-regulated. The adf single mutant showed no obvious phenotype in shoot regeneration.Whereas, simultaneously suppressing the expression of ADF1, ADF2, ADF3and ADF4inshoot induction could reduce the rate and frequency of shoot regeneration. Furthermore, auxinpolar transport and distribution was disrupted in these tissues. All these results suggest thatADFs function redundantly in regulating distribution of microfilament cytoskeleton, resultingin auxin polar transport and distribution changed required for in vitro shoot regeneration.