Functional Analysis Of TRAPP? Subunits Trs120 And Trs130 In Arabidopsis Pollen Tube Growth

Male gametophyte(pollen)development is one of the key processes for sexual reproduction in higher plants,and this precisely regulated process is divided into three stages:microsporogenesis,microgametogenesis,germination and growth of pollen tubes,in which vesicle transport plays important roles.In eukaryotic cells,trafficking between organelles within the endomembrane system mainly depends on vesicle transport,which can be divided into four steps:vesicle budding,transport,tethering and fusion.Vesicle tethering is a specific recognition between the vesicle and the target membrane achieved by tethering factors,which is of great significance for the directional transport of intracellular substances.A particular tethering factor resides on subcellular compartment(s)and mediates specific membrane fusion events.Transport Protein Particle II(TRAPPII)is a multi-subunit tethering complex located on TGN(trans-Golgi Network)in plant cell.Previous studies showed that the TRAPPII complex contains two specific subunits(Trs120 and Trs130)in addition to subunits of TRAPPI complex.In Arabidopsis,exocytosis mediated by Trs120 and Trs130 play important roles in plant vascular development and cell growth in shoot and root.Both of them are required for assembly and regulation of cell plate in root cells undergo cytokinesis.But the function of TRAPPII in pollen tube growth is largely unknown.In this study,Arabidopsis functions of Trs130 and Trs120 in pollen tube growth are studied.1.Trs120 and Trs130 genes play important roles in polar growth of pollen tubesAnalysis of maximum-likelihood phylogenetic tree and Real-time PCR showed that Trs120 and Trs130 are conserved in plant kingdom and highly expressed in pollen.The male transmission efficiency of trs120 and trs130 mutation was found significantly reduced.Pollen morphology,nuclear division,and pollen viability of the mutant pollens were examined by SEM,Alexander staining,and DAPI staining respectively.No obvious defects were found in trs120 and trs130 pollen grains.Further in vitro germination experiments showed that mutant pollen grains from trs120/+and trs130/+germinated normally,but the ratio of short and bulged pollen tube were significantly higher than that of wild-type.Therefore,we concluded that absence of Trs120 and Trs130 caused pollen tube growth defects,thereby the male transmission efficiency was reduced.2.Trs120 and Trs130 are involved in subset of secret pathways in pollen tubeWe used immunofluorescence assays to investigate distributions of methylesterified and demethylesterified pectins along the cell wall of trs120 and trs130 pollen tube by antibody JIM7 and JIM5 respectively.The results showed that their distributions in mutant pollen tubes are similar to the wild type ones.G-CaMP5 is a probe for monitoring Ca2+concentration in the cytosol of pollen tube.The pattern of G-CaMP5 indicated that the Ca2+distribution in trs130 pollen tube is normal.On the other hand,GFP-CSLD1 in trs130 pollen tube retained its Golgi localization,but the apical membrane labeling was much decreased.While the labeling of pollen specific Syntaxin SYP124 on the apical membrane increased obviously in trs130 pollen tube.These results suggested that Trs120 and Trs130 are involved in a subset of secret pathways in pollen tube.