Mechanism Analysis Of PbrBAK1 And PbrANXUR Receptor-Like Kinases Regulated Pollen Tube Growth In Pear

Receptor-like kinases(RLKs)are important signaling molecules in plant cells and play crucial roles in the process of plant growth and responding physiological stresses.RLKs were divided into subfamilies according to different binding domains of the extracellular ligand.The BRI1-associated receptor kinase 1(BAK1)in the leucine-rich repeats receptor-like protein kinase(LRR-RLK)subfamily and ANXUR in the Catharanthus roseus receptor-like kinase(CrRLK1-L)subfamily were two important protein kinases regulation the plant cell growth.BAK1 is the co-receptor of BRI1(the receptor of Brassinosteroids(BR)and participates in mediating the signaling transduction pathway of BR regulating plant cell growth.ANXUR receptor kinases regulate the cell wall integrity by regulating Nicotinamide adenine dinucleotide phosphate(NADPH)oxidase at the pollen tube tip and further affect the pollen tube growth.However,details of the underlying mechanism of ANXUR regulating pollen tube growth and whether it participated in the BAK1 mediated Brassinolide(BL)signaling pathway are unclear.To resolve the above question,this study conducted a series of research and the following were the the main results:1.This study analyzed the LRR-RLK subfamily in five main Rosaceae species and screen out the possible BAK1 gene in pear.LRR-RLK is the largest gene subfamily of RLKs and actively participates in regulating growth and development in plants.However,the patterns of LRR-RLK gene family evolution in the five main Rosaceae species for which genome sequences are available have not yet been reported.In this study,we performed a comprehensive analysis of LRR-RLK genes for five Rosaceae species:Fragaria vesca(strawberry),Malus domestica(apple),Pyrus bretschneideri(pear),Prunus mume(mei),and Prunus persica(peach),which contained 201,244,427,267,and 258 LRR-RLK genes,respectively.All LRR-RLK genes were further grouped into 23 subfamilies based on their sequence similarity with previously defined hidden Markov models for each subfamily that developed from 4 model plant species.RLK-Pelle_LRR-?-1(?-1),RLK-Pelle_LRR-?-1(?-1),and RLK-Pelle_LRR-?(?)were the three largest subfamilies.Synteny analysis indicated that there were 236 tandem duplicated genes in the five Rosaceae species,among which subfamilies ?-1(82 genes)and ?-1(80 genes)comprised 68.6%.The results indicate that tandem duplication made a large contribution to expansion of the subfamilies.The gene expression,tissue-specific expression,and subcellular localization data revealed that LRR-RLK genes were differentially expressed in various organs and tissues,and the largest subfamily ?-1 was highly expressed in all five Rosaceae species.In pear,we identified five PbrBRI1 genes and five PbrBAK1 genes that belong to the Xb-1 and ? subfamilies,respectively.Taken together,the results provided an overview of the LRR-RLK family in Rosaceae genomes and the basis for further functional studies.2.In this study,it was found that exogenous Epibrassinolide(epiBL)could promote the pear pollen tube growth.This study treated pear pollens by exogenous epiBL with in vitro culture and in vivo pollination.Firstly,the pollens of 'Dangshansuli' that cultured in the liquid medium were found promoting pollen tube growth under the low concentration epiBL,while inhibited under the high concentration epiBL.And the best concentration that promoting the pollen tube growth was 0.01 ?M epiBL.Propiconazol(PPZ),the inhibitor of exogenous Brassinosteroids(BR),could inhibit the pollen growth of pear,and the growth of pollen tubes were resumed when adding exogenous epiBL.At the same time,exogenous BR can promote depolymerizing the pollen tube microfilament skeleton and the ROS accumulation at the pollen tube tip and activate the Ca2+ channel in pollen tube tips and sub-tips.In addition,the effects of exogenous epiBL,glutamic acid(Glu)and spermidine(Spd)on the growth and germination of living pollen tubes were analyzed by liquid pollination.It was found that exogenous epiBL and Spd treatment can increase the pollen tube growth and fruit setting rate.This indicated that a suitable concentration of BR could promote the pollen tube growth and development and reduce the adverse effects of low temperature and other abiotic stress on pollination and fertilization of pear trees,and also provided a basic theory for clarifying the mechanism of BR promoting pollen tube growth.3.This study found that ANXURs(ANX1 and ANX2)could not able to regulate NADPH oxidase active directly,while ANXURs were regulated by exogenous epiBL but not directly through BAK1.BAK1 as an important receptor-like kinase involved in multiple signaling pathways and as a co-receptor involved in regulating pollen tube growth and immune response and other processes.ANXUR as another important receptor kinase is specifically expressed in pollen tubes and regulate the pollen tube growth by regulating NADPH oxidase activity.PbrBAK1d,PbrANXl and PbrANX2 were expressed in pollen and located in plasma membrane.The expression of PbrANXl and PbrANX2 was regulated by BR.It was found that through Antisense Oligodeoxynucleotide(A-ODN)inhibit the PbrBAK1d expression the pollen tube growth was inhibited but inhibit the PbrANXl and PbrANX2 could promote the growth,which indicated that BAK1 and ANXURs were not in a same singaling pathway in regulating pollen tube growth.Using yeast two-hybrid and Bimolecular Fluorescence Complementation(BiFC)experiments,this study found that BAK1 and ANXURs genes did not directly interact with each other in pear and Arabidopsis.It was also found that ANXURs and Respiratory burst oxidase homologue H/J(Rboh H/J)did not directly interact with each other.This result suggested that ANXUR receptor kinase regulates pollen tube growth without direct phosphorylation of Rboh H or RbohJ.ANXURs may mediate BL to control the pollen tube growth by relating to other proteins under the BL signaling pathway.4.This study indicated that Mitogen activated protein kinase(MAPK/MPK)downstream of ANXURs to receive signals and activate NADPH oxidase to regulate pollen tube growth.ANX1 and ANX2 are CrRLK1-L subfamily important proteins,which have typical transmembrane receptor kinase structure and involved in pollen tube growth.The receptor protein kinase with transmembrane structure have most recognized by MAPK.There is no report that MAPK directly regulate the pollen tube growth and development.We found that ANXUR was not directly intreact with Rboh H/J to regulate the pollen tube growth.In this study,the intracellular domain of PbrANX1(PbrANX1-ID)was used as a bait,two sequences similarity to AtMPK3 and AtMPK6 were screened in the 'Dangshansuli'pollen nucleoprotein yeast library.Four pear MAPK genes were screened by comparison,named PbrMPK5?PbrMPK7?PbrMPK15 and PbrMPK18,respectively.PbrMPK18 interacted with PbrANX1 by yeast two-hybrid and Agrobacterium-mediated transformation.Gene AtANX1 and AtANX2 interacted with AtMPK3 and AtMPK6 in Arabidopsis.This study found that only the complete intracellular domain of ANXURs existed can interact with MPK.Besides,this study found PbrMPK18 and PbrRboh H/J also exist interactions between each other.Similarly,Arabidopsis gene AtMPK3/6 and AtRboh H/J also interact with each other.In this study,MAPK may be downstream of ANXUR,and received the signal from ANXUR extracellular domain than delivered into the cell.MAPK also contact with Rboh H/J to induce the ROS producing to regulate the pollen tube growth.In a word,this thesis on the basis of the project group long-term research and the woody fruit tree of pear pollen as the main object.We applied some technical means and research methods including physical measurement,gene cloning,tissue and cell positioning,function analysis,and build a signal transduction to in-depth analyses the promotion or inhibition that same receptor kinase gene in regulation of pollen tube growth in pear.Through the research strategy of pharmacology and genetics,we discussed BAK1 and ANXUR regulation the signal pathway of pear pollen tube growth and development on molecular level.Pollination and fertilization was the key processes of pear production.The results of this study provided a theoretical guidance for the pollen tube growth regulation.