Arabidopsis SYP121 Takes Action In ROP2 Signaling Pathway To Regulate Root Hair Tip Growth

Root hairs are fast-growing tubular protruisons on root epidermal cells that play important roles in water and nutrient uptake in plants.Root hair cell is typically tip growing cell,and its growth is regulated by many factors,including Ca2+,organization and dynamics of the cytoskeleton,vesicle trafficking,small GTPase.Previous studies showed that the tip-focused polarized growth of root hairs is accomplished by the secretion of newly synthesized cell wall and membrane materials to the tip via polarized membrane trafficking.However little is known about the mechanism of secretory vesicle fusion with the plasma membrane(PM),which needs further study.In eukaryotic cells,the membrane fusions are mediated by a family of SNARE(Soluble N-ethyl-maleimide-sensitive factor attachment protein receptor)proteins.In this study,we investigated the function of a Qa-SNARE protein,SYP121,in root hair tip growth and the underlying regulatory mechanism.The phenotype analysis demonstrated that the syp121 T-DNA insertion mutants had shorter root hairs and the slower growth rate compared to wild type.Two complemented lines displayed normal root hair length and growth rate,which confirmed the role of SYP121 in root hair growth.The data from GeneChip arrays in the Genevestigator database(www.genevestigator.etha.ch)indicated that SYP121 expressed in root hairs.GUS activity staining aslo revealed that SYP121 expressed in root and root hairs.Sub-cellular localization assay showed that SYP121 mainly localized at the apical PM and the inverted cone region at the tip in the growing root hairs,while the signal at the inverted cone region at the tip was disappeared in the root hairs that ceased growing.Further investigation revealed that this polar localization was oscillates during root hair tip growth and the oscillation was very important for root hair tip growth.The localization of SYP121 and the root hair phenotype of syp121 mutant are similar to previously reported ROP2 localization in root hairs and the phenotype of rop2-1 mutant,suggesting that SYP121 may coordinate with ROP2 to regulate root hair tip growth.The root hairs of rop2-1 syp121-1 double mutant were similar to rop2-1 root hairs,and overexpressing SYP121 in rop2-1 mutant partially recovered the phenotype of rop2-1 root hairs,indicating that ROP2 and SYP121 may regulate root hair tip growth in the same pathway.Further research showed that GFP-ROP2 and mCherry-SYP121 co-localized at the apical PM of root hairs and their oscillation mode had certain correlation.SYP121 was found preferably interact with active ROP2 in vitro and in vivo,suggesting that SYP121 is likely a downstream effector of ROP2.It was also revealed that subcellular localization pattern of SYP121 was changed in root hairs from both rop2-1 mutant and CA-rop2(Constitutively Active GTP-bound rop2)transgenic plants.Biochemical analysis and BiFC(Bimolecular fluorescence complementation)assay demonstrated that active ROP2 interacted with the N terminal and H domain of SYP121 both in vitro and in vivo,which facilated SYP121 interacting with other SNARE proteins to form a SNARE complex for functioning in membrane fusion.The present demonstrated that ROP2 signaling pathway was able to regulate root hair growth through signaling to SNARE protein SYP121 to modulate vesicle fusion with the apical PM.This study reveals a key protein that links the ROP signal pathway and vesicle fusion process,providing a new insight in understanding the regulatory mechanism of ROP2 in root hair tip growth control.