Molecular Mechanism Of PbrPME44 Involved In The Self-incompatibility Of Pyrus

Pear is one of the most significant temperate fruit species.China ranks first in the world of pear cultivation area and total production,accounting for about 70%of the world.Pear belongs to Rosaceae and shows a typical S-RNase based gametophytic self-incompatibility.Self-incompatibility results in the failure of fructification,which greatly influencs the sustainable development of pear production.Thus,it makes a significant concern on detecting the molecular mechanism on self-incompatibility,creating comnpatible germplasm and guaranteeing the yield and quality of pear.In this study,the pollen of ’Dangshansuli’ was used as the material to isolate and clone three genes(PbrPPA5,PbrbZIP1 and PbrPME44),and the regulatory network among the three genes were explored.The results are as follows:1.Thirty-nine PPA genes were identified from five Rosaceae species including pear(Pyrus bretschneideri),apple(Malus domestica),peach(Prunus persica),strawberry(Fragaria vesca),and Japanese apricot/mei(Prunus mume).Based on the structural characteristics and phylogenetic analysis,the members of PPA gene family were classified into two main subfamilies(A and B).Pear contained 12 members of PPA gene family.Whole-genome duplication(WGD)and dispersed gene duplication were the main force,which drove the expansion of PPA gene family in pear and other Rosaceae species.Purifying selection played a dominant role in the evolution of PPA genes.Subcellular localization analyses demonstrated that PbrPPA genes located in the cytosol and nucleus.PbrPPA genes showed multiple expression patterns in different pear tissues.Six members of pear PPA gene family presented particularly higher expression level in pollen tube of pear,and PbrPPA5 showed tissue specific expression in pollen tube.To further analyze the function of PbrPPA5,we performed gene cloning,protein purification,and ODN to declare the important roles of PbrPPA 5 in pollen tube cell wall development.2.To further detect the important roles of PbrPPA5 in pollen tube cell wall development,we used PbrPPA5 as bait to screen a yeast cDNA library prepared by pear pollen tube.In the putative 14 candidates,we found PbrbZIP1,a member of bZIP transcription factor gene family.Expression analysis showed PbrbZIP1 had high expression level in pollen tube.Phylogenetic analysis revealed that there were 80 members of bZIP gene family in pear,and pear bZIP gene family was divided into 10 subfamilies.Sequence analysis showed that PbrbZIP1,AtbZIP34 and AtbZIP61 belonged to subfamily E and they were highly homologous.Knocking down the expression of PbrbZIP1 gene in pear pollen using antisense oligonucleotides induced the defect in the development of pollen tube cell wall,and the pollent tubes showed the same phenotype as the results of knocking down the expression of PbrPPA5 gene.Yeast two-hybrid assay,BiFC and LCI assay showed that PbrPPA5 and PbrbZIP1 interacted in the nucleus.Both PbrPPA5 and PbrbZIP1 participated in the development of pollen tube cell wall,and maybe the biological function of PbrPPA5 in regulating the development of pollen tube cell wall were worked throught PbrbZIP1.3.Pectin is an important component of pollen tube cell wall,and its methylesterified/demethylesterified level dynamically regulates the structure of pollen tube cell wall.Pectin methylesterase(PME)is an important factor for controlling pectin methylesterification/demethylesterification.The swelling in pollen tube tip was induced by excessive extracellular Ca2+concentration.LM20 immunohistochemical analysis showed that this expansion accompanied with the accumulation of methylesterified pectin.qPCR analysis revealed that the expression level of PbrPME44,a pectin methylesterase,was down-regulated.There are 81 members in pear PME gene family.Knocking down the expression of PbrPME44 gene in pear pollen using antisense oligonucleotides also induced the defect in the development of pollen tube cell wall.Pollen tubes treated with PbrPME44 recombinant protein showed changes in content and distributions of methylesterified pectin in pollen tube tip.Yeast one-hybrid assay and DLR assay revealed that PbrbZIP1 could bind to the promoter region of PbrPME44,and PbrbZIP1 could act as a transcription repressor of PbrPME44,and its repressor activity is enhanced by PbrPPA5.Furthermore,the BLI assay showed that Ca2+could enhance the interaction between PbrbZIPl and PbrPPA5.Taken together,our results suggested that Ca2+could regulate the interaction between PbrbZIPl and PbrPPA5 to control the expression level of PbrPME44.4 S-RNase is the pistil determinant factor in self-incompatibility of pear.It can recognize the self-pollen tube and play a cytotoxic role,and triggers the programmed cell death in the self-pollen tube.In this study,the S-RNase recombinant protein was expressed and purified from E.coli.The RNase activity of S-RNase recombinant protein was affected by pH.After treating the self-pollen tube with S-RNase recombinant protein,the pollen tubes expanded/swelled and methylesterified pectin were accumulated in the pollen tube cell wall.Yeast two-hybrid assay revealed that S7/34-RNase could not interact with PbrPPA5,PbrbZIP1 or PbrPME44.However,qPCR analysis showed that PbrPME44 was down-regulated in self-pollen tube.S-RNase could induce the increase of Ca2+concentration in self-pollen tube at the later stage of self-incompatibility.In this study,the increase of Ca2+ concentration in self-pollen tube inhibited the activity of PbrPPA5,and it influenced the interaction between PbrPPA5 and PbrbZIP1,which further resulted in disordered expression level of PbrPME44.Finally,the balance between methylesterified pectin and demethylesterified pectin in pollen tube cell wall was broken.The inner and outer turgor pressure of pollen tube was changed,and the pollen tube cell wall at the tip of the pollen tube is unstable,which would cause the expansion or rapture in pollen tube tip.In conclusion,the identification and characterization of PPA,bZIP and PME gene family in pear were investigated.PbrbZIP1 could bind to the ABRE-like element in the promoter region of PbrPME44,and acted as a transcription repressor of PbrPME44.PbrPPA5 could act as a co-repressor with PbrbZIP1 to regulate the expression level of PbrPME44.The swelling and accumulation of methylesterified pectin in the tip of self-pollen tube were induced by the down-regulated expression level of PbrPME44.These results provide a set of evidence for the understanding of the development of pollen tube cell wall and self-incompatibility in pear.