The Molecular Mechanism Of AtSNAP30 And AtSYP131 In Regulating Pollen Tube Tip Growth

Pollen tube is a typical tip-growth cell.Its growth is regulated by many factors such as cytoskeleton,small GTPases,and Ca2+.The key of pollen tube tip-growth is the fusion of secretory vesicles with the plasma membrane at the apex.The soluble NSF attachment protein receptors(SNAREs)are important regulators of exocytosis and their main role is to mediate the fusion between the vesicles and their target membranes.Usually,the fusion between the vesicles and their target membranes requires Qa-,Qb-,Qc-,and R-SNARE to assemble into tetrameric SNAREs complex(intimal type)or Qa-,Qbc-,and R-SNARE to assemble into trimeric SNAREs complex(plasma membrane type).Studies have shown that pollen-specific Qa-SNARE proteins AtSYP124,AtSYP125,and AtSYP131 are jointly involved in the regulation of Arabidopsis pollen tubes tip-growth.Previous studies in our laboratory have found that R-SNARE protein AtVAMP725 acts on pollen tube tip-growth by regulating the fusion between vesicles and plasma membrane.However,the composition and function of SNARE complex involved in the fusion of apical vesicles of the pollen tube and plasma membrane are not yet well studied.Microarray data showed that there were three genes encoding Qbc-SNARE protein in tihe Arabidopsis genome,namely AtSNAP29,AtSNAP30,and AtSNAP33.In addition,only AtSNAP30 highly expressed in mature pollen,suggesting that it might play an important role in pollen tube tip-growth.In the present study,AtSNAP30 was found to specifically express in Arabidopsis pollen and pollen tube by GUS histochemical activity assay.By observing the phenotype of the pollen tube of Atsnap30 mutant in vitro and in vivo,it was found that the loss of AtSNAP30 could inhibit partially Arabidopsis pollen tube tip-growth.Observation of the pod morphology and seed setting rate of Atsnap30 mutant revealed that loss of AtSNAP30 did not affect fertilization and the formation of seeds.The FRAP experiment of AtPRK1-GFP at the tip of the pollen tube of the Atsnap30 mutant revealed that loss of AtSNAP30 delayed the secretion of the apical vesicles in the pollen tube,which indicated that AtSNAP30 might play an important role in the fusion between the apical vesicles and plasma membrane in pollen tubes tip.In addition,AtSYP131 was found to specifically express in pollen and pollen tube of Arabidopsis by GUS histochemical activity assay.By the experiment of plasmolysis and the co-distribution with the plasma membrane marker protein,AtSYP131 was further determined to locate on the plasma membrane of pollen tube.The FRAP experiment of AtSYP131-GFP at the tip of pollen tube revealed that the site of fusion between AtSYP131 and plasma membrane was mainly at the pollen tube tip.By observing the phenotype of the pollen tube of Atsyp131 mutant in vitro and in vivo,it was found that the loss of AtSYP131 could inhibit partially Arabidopsis pollen tube tip-growth in vitro.Overexpression of AtSYP131C2(transmembrane domain and SNARE domain included)truncated fragments affected the trafficking and/or secretion of the apical vesicles of pollen tube.By observing the phenotype of the pollen tube of Atsnap30 Atsyp131 double mutant in vitro,it was found that AtSNAP30 and AtSYP131 might jointly regulate the tip growth of pollen tube.Furthermore,in vitro and in vivo protein interaction analysis revealed that AtSNAP30,AtSYP131,and AtVAMP725 proteins interacted with each other.In addition,AtSNAP30 and AtSYP131 could bind directly to each other.These results suggest that AtSNAP30,AtSYP131,and AtVAMP725 may form a trimeric SNARE complex in pollen tube of Arabidopsis,which regulates the tip growth of pollen tube by mediating the fusion between the apical vesicles and plasma membrane.The research has important scientific significance for revealing the tip growth mechanism of pollen tube.