The Molecular Mechanism Of PbrSEC1 Regulating Pollen Tube Growth

Being extensively cultivated in the world,pear has crucial values both in economy and nutrition.Among all countries,China has the largest cultivation area of pear.The pollination and fertilization are the majority of sexual reproduction and the basis for guaranteeing the fruit yield in pear.Consequently,exploring the potential proteins that are involved in the interaction between style and pollen and pear pollen tube growth is important for advancing the research of pear.In this study,we performed the transcriptome and proteomic sequencing using different combinations of pear pollen and style to screen the potential genes and proteins that have regulatory functions in the process of pear pollen tube growth.Base on the result of sequencing analysis,the protein of Pbr028556.1 has the significant trend in protein expression accompany the growing of pollen tube in style.Therefore,we explore the potential regulatory functions in pear pollen tube growth and related molecular mechanism of Pbr028556.1 in the following study.1.In the day of bud stage,we employed the‘Dangshansuli’pollens and the‘Cuiguan’pollens to pollinate the styles of‘Dangshansuli’,respectively.In addition,we set a group of‘Dangshansuli’styles that were unpollinated.The three groups of styles were respectively collected after 24 h,48 h,72 h and 120 h of the bud stage,and then were used for transcriptome and proteomic sequencing.By carrying out the co-expression analysis,the genes and proteins that had similar expression profiles were allocated to the Clusters.Subsequently,we predicted the potential functions of Clusters.In the Cluster 5 of protein,Pbr028556.1 was identified to exhibit the significant expression tendency in the process of pear pollen tube growth.Consequently,we selected it as the protein of interest in the study.2.We identified a total of 23 OGTs in eight Rosaceae species.The phylogenetic analysis was applied to classify them into two genomic branches,including SECRET AGENT（SEC）and SPINDLY（SPY）.The result of the evolution profile analysis illustrated the foremost roles that WGD/segmental and dispersed gene duplication play in the evolutionary processes of SECs and SPYs.The PbrSEC1（Pbr028556.1）was characterized to have the predominant expression in the major pear tissues including root,stem,leaf,fruit,pollen and pollen tubes.Additionally,it exhibited the significant trend of gene expression in the process of pear pollen tube growth.The subcellular localization assay confirmed that the PbrSEC1 was localized both in nucleus and cytoplasm.By employing the stable overexpression experiment,we explored the potential functions of PbrSEC1 in the process of plant growth and development.Based on the results,PbrSEC1 promoted the growth and development of plant by interacting with RGA1 and thus inhibiting its correlations with target genes including PIF1,IAA19 and IAA29.By performing the as-ODN assay,we explored the potential function of PbrSEC1 in regulating the pear pollen tube growth.By conducting CM-H₂DCFDA and S4B stainings,we found that PbrSEC1 up-regulated the apical ROS content and cellulose content in pollen tube wall.3.Inhibiting the expression of PbrSEC1 promoted the resistance of F-actin against the actin depolymerization induced by LatB.By overexpressing the PbrSEC1 into the sec mutants,we ascertained the role of PbrSEC1 inhibiting the growth of pollen tubes.The Y2H screening and co-expression analysis were conducted to screen the potential interaction proteins of PbrSEC1.As a result,the actin cross-linking protein PbrCROLIN1was found to directly interact with PbrSEC1,and has the significant function of maintaining the stability of F-actin under the LatB treatment.By performing diverse functional experiments,we explored the signal pathway of PbrSEC1-PbrCROLIN1 that influence the F-actin stability and pollen tube growth.4.By applying the Fluo-4-AM staining,the PbrSEC1 was found to significantly inhibit the[Ca²⁺]_cytconcentration in pear pollen tubes.Applying PbrSEC1 as the bait to carry out the Y2H screening,we found that PbrCNR8（Pbr034562.1）,which belong to the PLAC8-containing protein family,can interact with PbrSEC1 and has the potential regulatory function in pear pollen tube growth.The direct interaction relationship between PbrSEC1and PbrCNR8 was ascertained via Y2H,GST Pull-down and LCI assays.In addition,we truncated the protein of PbrCNR8 and subsequently utilized the Bi FC and Y2H experiments to verify that the PbrSEC1 interacted with the intracellular domain of PbrCNR8 in cytoplasm.The as-ODN assay and stable overexpression in Arabidopsis was performed to explore the function of PbrCNR8 in pear pollen tube growth.Based on the result,PbrCNR8inhibited the elongation of pollen tubes.By conducting the Fluo-4-AM staining and patch clamp assay,we characterized that the PbrCNR8 regulated the pear pollen tube growth by affecting the[Ca²⁺]_cytconcentration and Ca²⁺current in tube cell.The signal pathway of PbrSEC1-PbrCNR8 regulating pear pollen tube growth was also explored.The calmodulin-binding protein PbrIQD1 was identified to directly interact with PbrCNR8-IND.Therefore,we put forward the molecular mechanism that PbrSEC1-PbrCNR8 regulated the[Ca²⁺]_cytconcentration and pollen tube growth by mediating PbrIQD1.In summary,we utilized the transcriptome and proteome sequencing to screen out the O-GlcNAc transferase PbrSEC1 which played the significant role in the process of pear pollen tube growth.Various experiments and assasys were perfomed to explore the potential functions of PbrSEC1 regulating the pollen tube growth,and subsequently uncovered the mechanisms of PbrSEC1-PbrCROLIN1 regulating the stability of F-actin and PbrSEC1-PbrCNR8 regulating the[Ca²⁺]_cytconcentration and growth rate of pear pollen tubes.