The Study Of The Molecular Mechanism Of Arabidopsis Rab-H1b Regulates Hypocotyl Growth

Arabidopsis hypocotyl is widely used as a model system to study the regulation of cell elongation.A strong yet flexible cell wall allows directional cell growth driven by vacuolar turgor pressure.Cellulose is an important component and the major load-bearing element in the cell wall,which is believed to be synthetized by cellulose synthase(CESA)complexes(CSCs)in the plasma membrane(PM).The proper distribution and function of PM-localized CSCs are precisely regulated by both endocytosis and exocytosis.However,the mechanism underlying the trafficking of CSCs between endomembrane compartments and the PM is poorly understood.Many regulators are involved in vesicle trafficking.For example,Rab GTPases are involved in membrane identity,vesicular budding from donor membranes,vesicle motility,docking,tethering,and fusion with acceptor membranes.In A.thaliana,57 Rab GTPases family members have been identified,which are grouped into 8 clades(named as Rab-A to Rab-H)based on their putative function in various trafficking pathways.To further study the mechanism during hypocotyl growth,a pool of T-DNA insertion lines was screened for mutant that exhibited short etiolated hypocotyls.rab-h1b displayed short etiolated hypocotyls.From a genetic screen for mutants having defects in hypocotyl elongation,a mutant displaying short and fragile etiolated hypocotyls was identified.Further analysis revealed that the defect in hypocotyl growth was attributed to a loss of function of RabHlb.Using scanning electron microscope,a thinner cell wall was detected in rab-h1b mutant cells.Additonally,a reduction in cellulose content was revealed as well.Stably expression of FP-tagged CESA6 indicated an over accumulation of CSCs in the PM of rab-hlb mutant cells,in addition to a reduced velocity of PM-localized CSCs.Expression of FP-fused RabHlb indicated a Golgi localization pattern of Rab-Hlb.Besides,some Rab-Hlb siganls could be detected in small endomembrane compartments that partially overlapped with exocytosis marker GFP-VAMP721,and travelled close to trans-Golgi network/ealy endosome(TGN/EE).Using transmission electron microscope,Rab-Hlb loss of function was found to result in abnormal Golgi apparatus had an increased number of cisternae,and surrounded by overaccumulated vesicles,suggesting a key role of Rab-H1b in cisternal homeostasis.Fluorescence recovery after photo bleaching(FRAP)analysis revealed that the delivery of CESA6 to the PM was impaired in rab-h1b.Additionaly,the endocytosis of PM indicated by FM4-64 was also reduced.This study demonstrates that Rab-H1b is involved in cellulose biosynthesis by participating in the traffick of CESA6 to and from the PM,and links Rab GTPases to cellulose biosynthesis,uncovering a novel mechanism in regulation of cell elongation and hypocotyl growth.Furthermore,this study also demonstrates the importance of Rab-Hlb mediated Golgi morphology and cisternal homeostasis in exocytosis and endocytosis of CESA6,which provides new insights in vesicle trafficking regulation in plant cells.