Molecular Mechanisms Of Arabidopsis T-SNARE SYP31/SYP32 And COG5 Subunit Of COG Tethering Complex In The Regulation Of Male Gametophyte Development

Male gametophyte development is one of the key processes for sexual reproduction of angiosperms,and it involves important events such as the formation of pollen intine,the formation of cell plates during the first mitosis of pollen,and the growth of pollen tubes.The synthesis and transport of cell wall material plays a key role in these developmental events.In plants,the molecular mechanisms and physiological role of intra-Golgi traffiking and Golgi interity maintenance are unclear.In this study,two kinds of Golgi-localized protein T-SNARE SYP31/SYP32 and tethering factor COG5 were analyzed by biochemistry,cell biology and genetics methods.This thesis are divided into two parts.Part I:Function of SYP31/SYP32 in the Development of Male GametophytesIn yeast and mammalian cells,SED5/Syntaxin5 are believed to be involved in the retrograde transport in Golgi.Consistently,their homologs in arabidopsis SYP31 and SYP32 are localized in Golgi and strong evidences support that SYP31 is confined at cis face of Golgi.Over expression of SYP31 in protoplast inhibits the trafficking between ER and Golgi.It's also reported that SYP31 is localized at cell plate in dividing plant cells.which implies a role in cytokinesis.However,the physiological function of SYP31 and SYP32 during plant growth and development has not been explored.The first part of this thesis is the functional study of SYP31/SYP32 in the development of male gametophytes.The main results are as follows:1.Role of the SYP3 family in male gametophyte developmentThe results of multiple sequence alignment showed that SYP31 and SYP32 had conserved domain.The RNA-seq data.GUS staining and real-time PCR analysis showed that SYP31 and SYP32 were both abundantly expressed in pollen,suggesting that there were functional redundancy between them.Consistent with this.backcross of the single mutants or double mutants indicated a fully penetrant,synthetic transmission defect of syp31 and syp32 through the male gametes.Consistent with a defect in male gametophyte development.the results of Alexander staining showed that?22%pollen from syp31/+:syp32/+anther was inviable,by contrast,<1%of the pollen was inviable in either wild-type or single heterozygous null syp3 alleles.Together,these results demonstrate that SYP3 family have necessary and redundant function during pollen development.2.syp31 syp32 double mutant pollen displayed altered Golgi morphology,which led to absence of cellulose in pollen intine,ectopic callose deposition and defective cell plates.Analysis by DAPI staining indicates that the arrest of double mutant pollen occurs between the unicellular and the bicellular stage.By TEM(transmission electron microscope)analysis,the Golgi in the unicellular pollen from the double mutant anther displayed short and swelling cisternae;the integrity of Golgi in the bicellular pollen of the mutant was disrupted with shorter and reduced number of cisternae.The results of aniline blue staining indicate that the formation of cell plate during PMI is defective and ectopic callose deposition was found in bicellular pollen.Calcoflour white staining of semi-thin sections revealed the defective deposition of cellulose in the intine of double mutant pollen.Together.These results indicate that SYP31 and SYP32 are involved in maintenance of Golgi integrity,therefore play a role in the synthesis of cellulose in pollen intine and the formation of pollen cell plates.3.SYP31/SYP32 modulate retention of Golgi protein and pectin secretionEMP12,a Golgi resident protein,was mislocated into vacuole in syp31 syp32 mutant pollen,suggesting a defect in COPI trafficking.In the GS-TAP experiment,a large number of COPI coat complex components were purified with SYP31 and SYP32,which further supported that SYP31/SYP32 modulated COPI trafficking.The highly methylated pectin is synthesized on Golgi apparatus.and then transported to the plasma membrane,which is involved in the formation of the inner wall.The results of JIM7 immunofluorescence showed that highly methylated pec tin accumulated inside the syp31 syp32 double mutant pollen,which indicated that the speed and accuracy of pectin secretion was affected.4.Interaction of SYP31/SYP32 with COG tethered complex and COPI coat complexGS-SYP31 and GS-SYP32 were expressed in PSBD cells of Arabidopsis thaliana.Using tandem affinity purification(TAP)and mass spectrometry analysis we identified the co-purified proteins of SYP31 and SYP32.including COG tethered complex and COPI coat complex.The interaction of COG3 with SYP31 and SYP32 was further verified by yeast two-hybrid,in vivo immunoprecipitation and luciferase complementary imaging and GST pull-down experiments.SYP31/SYP32 colocalize with COG3 in tabacco leaves,and in syp31 syp32 double mutant,the Golgi localization of COG3 was disrupted,impling that SYP31/SYP32 may act as the recruiter of COG3.Previously reported abnormal morphology of Golgi bodies in Arabidopsis COG complex subunits cog3 and cog8 mutant,showing a short and swollen phenotype,similar to the Golgi phenotype observed in syp31 syp32-1 double mutant pollen.Similar to the pollen of syp31 syp32,?-COP in cog3 and cog8 mutants lost their close association with Golgi(Tan et al.,2016),suggesting that SYP31/SYP32 and COG3 jointly participate in COPI-mediated retrograde transport in Golgi.Part ?:Function of COG5 in Pollen Tube Growth1.Mutation in COG5 caused defective pollen tube growthGUS staining demonstrates high expression of COG5 in pollen,and back cross of cog5/+mutant suggest a transmission defect through male gametophyte.DAPI staining,Alexander staining and SEM analysis of cog5/+ mutant and wild-type didn't show any differences,which demonstrated the normal microsporogenesis and pollen development.In vitro germination and pollen tube growth of cog51/+was examined and many ruptured and short pollen tubes were observed in the mutants and the ratio of these abnormal pollen tubes was significantly higher than that of wild-type showed by the statistical analysis.Immunofluorescence and histochemical analysis showed that the deposition of cell wall components in cog5 pollen tube was abnormal.2.Golgi apparatus morphology was changed and Golgi marker protein were mislocalized in cog5 mutantThe dot-like fluorescence signal of EMP12 in cog5 mutant pollen suggested the change of Golgi morphology and the TEM analysis showed that the lengths of the mini-stacks cisternae were reduced significantly and the distance between cisternae was also larger than that of the wild-type Golgi.Besides,the aberrant Golgi in mutant pollen was surrounded by a number of vesicles.The fluorescence of ERD2-GFP in cog5 mutant pollen was detected as a network in contrast with the fluorescent disks and bars in wild-type.Together.these results suggested that mutation in COG5 caused change of Golgi apparatus morphology and mislocalization of Golgi marker protein.3.COG functions in a protein network together with RabD2B GTPase to mediate COPI-dependent vesicle traffickingBy yeast two-hybrid screening,we identified the interacting proteins P-COP and RabD2B with COG5.The interaction between COG5 and ?-COP was further validated by immunoprecipitation,bimolecular fluorescence complementarity test and GST-pull down test in vitro.The results of bimolecular fluorescence complementarity test showed that the interaction site between COG5 and ?-COP was in Golgi.The fluorescence signal of P-COP in cog5 mutant pollen is weak and fuzzy,and the background fluorescence signal around it is enhanced and there is a distribution on tiny vesicle-like structure,which may be caused by the failure of COPI vesicles to tether and fuse to Golgi.Activated and inactivated RabD2B yeast expression vectors were constructed by point mutation.Yeast two-hybrid;luciferase complementary imaging and GST-pull down experiments in vitro showed that COG5 interacted with activated RabD2B,suggesting that it might participate in COPI-mediated protein transport in Golgi as a downstream effector of RabD2B.In this study,we found that the Golgi bodies in cog5 mutant pollen were shorter under transmission electron microscope,which were similar to those reported previously in cog3 and cog8,and in the Golgi phenotype observed in syp31 syp32-1 double mutant pollen.In these different mutants,gamma-COP lost the close relationship with the Golgi body;COG5 and beta-COP were directly related to COG3 and SYP31/SYP32.Interaction.These results suggest that SYP31/SYP32 and COG complex are involved in the regulation of COP I vesicle-mediated reverse transport in Golgi and the maintenance of Golgi morphology and structure,and play a role in the development of male gametophytes in Arabidopsis.