| Pollen germination and pollen tube polar growth is essential for sexual reproduction of flowering plants.It is well known that many factors are involved in the regulation of tube growth,for example,hydration,transport,the cytoskeleton arrangement,signal transduction and cell wall biosynthesis etc.Pollen germination and pollen tube growth depends on the process of vesicle trafficking system.The exocyst complex is comprised of eight subunits and required for tethering and fusion of the vesicles and plasma membrane at the sites of polarized exocytosis.Mutations in plant exocyst subunits that are encoded by one or two genes(SEC5,SEC6,SEC8,SEC 15)cause defects in pollen germination and tube growth.Here we obtained a T-DNA insertion mutant by utilizing reverse genetics method.Our finding suggested that SEC3A,a subunit of the exocyst complex,which was essential for the pollen germination.1.sec3a insertion mutant specifically block male gametophytic transmission by affecting pollen germination.In the process of propagation of sec3a mutant plants,the homozygous mutant lines were not found,and only the wild type and heterozygous plants could be obtained.The progeny of self-fertilized sec3a/+ showed segregation between mutants and wild type at a ratio close to 1:1.To investigate if sec3a affected male or female gametophytic function,the sec3a/+ plant was used as male or female to cross with wild-type.Apparently,genetic transmission of sec3a mutation through the male was affected in the mutant,while female gametophytic transmission was normal.To characterize the mutant phenotypes,sec3a were introgressed into the quartet1(qrtl)mutant background.Pollen morphology,nuclear division,and pollen viability of the mutant plants were examined by SEM,DAPI staining,and Alexander staining,respectively.These results indicated that sec3a mutation did not affect pollen grain development.We then examined the germination of sec3a/+ pollen grains in vitro.We can see that 41%of the pollen grains from sec3a/+ plants were able to germinate,compared to that 78%of wild-type pollen grains could germinate under the same conditions.sec3a allowed tetrad analysis of pollen germination ability in vitro.Under the same conditions,31.7%of qrtl/-plants had three or four pollen grains germinated,while no sec3a/+;qrtl/-quartet was found to have three or four pollen grains germinated.The result of pollen germination in vivo was also consistent with the above observations.2.Phenotypes of sec3a are rescued by wild-type SEC3A genesTo demonstrate that the mutant phenotype is caused by the disruption of SEC3A gene,complementation experiments were performed with cDNA of SEC3A.The ratio of plants with T-DNA insertion increased from about 1:1 to 2:1 in progeny of selfed complemented plants and we can obtain homozygous line(PRsec3a).The in vitro germination rates of the pollen grains from the two independent complemented lines selected were significantly recovered and close to that of wild-type pollen grains.RT-PCR analysis of PRsec3a indicating that a truncated mRNA were expressed,but the transcripts of spanning the respective T-DNA insertion sites were not expressed.We also constructed two plasmids pLAT52:SEC3A-GFP and pSEC3A:gSEC3A that could complement the phenotypes of sec3a/+.3.SEC3A gene is strongly expressed in pollen and SEC3A is located at the pollen germination poreThe time and location of gene expression often implies its function.The GUS staining wasn’t observed until binucleate pollen stage and increased gradually at trinucleate pollen stage.SEC3A-GFP was localized in the cytoplasm,and the signal was observed from bicellular pollen,as the same result as the GUS activity analysis.About 30 min after mature pollen was incubated in germination medium,SEC3A-GFP signal was strongly shifted to a cortical region,which predicted the germination pore.Therefore,SEC3A may play an important role in the process of pollen germination.4.The cell wall components cannot be gathered in the germination pore in sec3a insertion mutantsec3a/+ mutation blocks male gametophytic transmission by affecting pollen germination.The hydration was the initial signal of the pollen germination.On the medium,the imbibition showed no significant difference for the wild-type pollen grains compared with the mutant ones.During the pollen germination,the cell wall materials gathered at the germination pore,which provided the material accumulation for the pollen tube growth.In the mutant pollen grain the cell wall components were unable to deposit at the germination plaque.We speculate that SEC3A is used as the target site on the receptor membrane in the process of vesicle material transport during pollen germination.5.The reduction of PI(4,5)P2 does not affect the polarity of SEC3A in the germination holeIn yeast Sec3p directly interacts with PI(4,5)P2 in the plasma membrane.So we want to test whether SEC3A can directly bind PIPs in vitro.We purified recombinant HIS-SEC3 A fusion proteins and used them in PIP strip overlay binding assays.SEC3 A can selectively bound to various phosphoinositides.PIP5K4 produce PI(4,5)P2 and have been implicated in vesicle trafficking and cytoskeletal rearrangements.We found that the reduction of PI(4,5)P2 does not affect the polarity of SEC3A in the germination pore by using a pip5k4 mutant.6.SEC3A located at the apex of the pollen tube and its polar localization depends on the cytoskeletonSEC3A-GFP was associated with small vesicles that underwent cytoplasmic streaming at the top of growing pollen tubes and formed a dynamic cone-shaped area in the clear zone of the growing pollen.The polar localization of the SEC3A was disturbed when we used Lat B to disrupt the actin organization in pollen tube.These data suggest that the polar localization of SEC3-GFP is dependent on actin filaments.7.SEC3A gene is required for the auxin accumulation and root gravitropic responseIn GUS staining experiments,we found that the SEC3A gene is not only strongly expressing in pollen,but also has strong expression in the root tip and embryo.Because LAT52 is a pollen specific promoter,we can further explore whether the deletion of SEC3A gene cause severe sporophytic defects in Arabidopsis.The development of the embryos were normal in PRsec3a,but the columella cells identity was lost.And the PRsec3a displayed significant gravitropism defects.Therefore SEC3A not only plays an important role in male gametophytic transmission but also play a certain role in columella cells identity.Taken together,we conclude that SEC3 A is located at the pollen germination pore and sec3a insertion mutant specifically block male gametophytic transmission by affecting pollen germination. |
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