Distribution And Function Of F-actin And Myosin In Root Hairs And Pollen Tubes

Root hair and pollen tube are the typical tip-growth plant cells,and have strong cytoplasm streaming,so they are the important model material to be used studying cytoskeleton and organelles movement of plant cell.At present,according to the different research methods,the arrangement form in the tip and sub-apex of root hair and pollen tube is still confusing,and the secretory vesicles of the tip is how to transport to the plasma membrane also remains to be established.In this study,the stably expressed GFP-ABD2-GFP Arabidopsis root hairs and stably expressed ABD2-GFP and GFP-ABD2-GFP Nicotiana tabacum pollens and pollen tubes were observed using confocal microscopes.It was observed that highly dynamic circular F-actin bundles were universally distribution at the sub-apex in growing root hairs and pollen tubes.Using MitoTracker Red CMXRos labeled mitochondria,it was found that mitochondria turnaround along circular F-actin bundles at the sub-apex of root hair and pollen tube;In addition,when the root hair and pollen tube were treated by myosin inhibitor BDM,it was found that mitochondria were almost stop moving.These results demonstrate that circular F-actin bundles provide the track for myosin-driven turnaround and bidirectional movement of mitochondria in the sub-apex of root hairs and pollen tubes.These results can help people to deeply understand the turnaround and bidirectional movement mechanism of organelles in plant tip-growth cells.Secondly,the stably expressed ABD2-GFP and GFP-ABD2-GFP Nicotiana tabacum pollen tubes were observed by confocal microscopes.We found that there was thin F-actin network at the tip of pollen tubes.The F-actin network extended from circular F-actin bundles and branched out to the tip of pollen tubes.Using low concentration Lat B(the depolymerization of microfilament)to treat pollen tubes,it was found that the F-actin network at the tip of pollen tube depolymerized and disappeared rapidly.Lat B treatments also made the thick F-actin bundles extend to the tip,and lead to pollen tube growth stop.Using the same concentration Lat B to treat stably expressed VAMP725-GFP(secretory vesicles marker)Nicotiana tabacum pollen tubes,we also found that gathered distribution of VAMP725-GFP at the tip disappeared rapidly.These results indicate that the F-actin network plays an important role on pollen tube growth,and it may participate in the secretory vesicles transport to the tip.In addition,Atmyosin ?-C and Atmyosin ?-E are found to be abundant expression in mature pollen of Arabidopsis.Therefore we identified homozygote and analysised phenotype of the T-DNA insertion mutants for Atmyosin ?-C and Atmyosin ?-E.In vitro germination,pollen tube length and growth rate between the two T-DNA insertion mutants of Atmyosin ?-C and wild type was no significant difference;But pollen tube length and growth rate of the two T-DNA insertion mutants of Atmyosin ?-E was obviously shorter and slower than that of wild type line.In vivo,pollen tube growth rate of the two T-DNA insertion mutants of Atmyosin ?-E was also slower than that of wild type line.The result indicated that mutation of Atmyosin ?-E affected the pollen tube growth of Arabidopsis.Additionally,we found that the pollen tube of T-DNA insertion mutants of Atmyosin ?-E can completed fertilization,and morphology of silique,seed formation and seed setting rate were no obvious difference compared with wild type line.Thus,we conducted myosin ?-E tail inhibition experiment,i.e.intercepting N-terminal head area,just retaining the tail area including IQ area,and forming fusion protein with mCherry(mCherry-EIQ tail),then transformed pollen of Nicotiana tabacum with gene gun experiment.The results showed that compared with the control,pollen tube growth of Nicotiana tabacum was significantly inhibited after mCherry-EIQ tail expression.Furthermore,through mCherry-E1Q tail of instantaneous transformation in stably expressed VAMP725-GFP Nicotiana tabacum pollen,we found that gathered distribution of VAMP725-GFP at the tip disappeared,and pollen tube growth stopped.In FRAP experiment for VAMP725-GFP,the fluorescence recovery of the tip of pollen tube was slower than control.Conducted double transformation of mCherry-EIQ tail and RLK-GFP(plasma membrane marker),and in FRAP experiment for RLK-GFP,we found that the fluorescence of the tip of pollen tube and plasma membrane could not recover,and the pollen tube growth also stopped.These results illustrate that the expression of mCherry-EIQ tail obviously inhibits the growth of the pollen tube,affects the normal distribution of secretory vesicles,and prevents from transporting vesicles to the tip.Therefore,we speculate that myosin ?-E may be one of the motor proteins,which can transport secretory vesicles to the tip along the F-actin network at the tip of pollen tubes.