Molecular Mechanism Of PbrCalS5 Regulating Pollen Tube Growth In Pear

Pear,belonging to the Rosaceae family,are cultivated all over the world.The cultivation area and total production of pear rank among the top three in our country.The successful pollination and fertilization is the foundation of fruit setting.The transport of sperm cells to ovule by the pollen tube is essential for the successful pollination and fertilization.As one of the main components of the pollen tube cell wall,callose can form into the callose plugs,which ensure the pollen tube growth.In this study,‘Dangshansuli’was used to as the material to investigate the molecular mechanism of PbrCalS5 regulating the callose synthesis in pear pollen tube growth.The main research results are shown as follow:1.Callose synthase(CalS)is a key factor in regulating callose deposition.Sixty-nine CalS genes were identified from seven Rosaceae species,including strawberry,apple,sweet cherry,pear,Chinese plum,peach and black raspberry,and were divided into six subfamilies using maximum-likelihood method.Each subgroup contained at least one CalS gene from each species.The syntenic analysis suggested that the CalS genes in seven Rosaceae species were randomly distributed across different chromosomes and had evolutionary trajectory.In addition,the highest number of syntenic CalS gene pairs was found in pear.Dispersed duplication was the main force for the expansion of CalS gene family in seven Rosaceae species.Purifying selection played the key role in the evolution of the CalS genes.In Chinese white pear and apple,the whole-genome duplication also played the predominant role in the evolution of CalS gene.The CalS proteins consisted of three conserved domains.Among 13 pear CalS genes,three PbrCalS were expressed in pear pollen tubes.The result of q RT-PCR analysis showed that PbrCalS5 plays an important role in pear pollen development,germination and pollen tube growth.Low temperature stress significantly increased the expression of PbrCalS5 and accumulated the callose deposition in pear pollen tube wall.2.Aniline blue staining showed that callose was deposited in both the wall and callose plugs in pear pollen tube.In addition,the callose is distributed in the shank of the pollen tueb.Callose plugs are observed in pear pollen tubes after culturing 3 h,and the number of callose plugs increase to 3 after 6 h of culturing.The position of the first callose plug determines the length of pear pollen tube.The closer callose plug near the pollen grain,the longer the pollen tube grows.In the subcellular localization assay,the PbrCalS5 is a membrane-and cell wall-localized protein that plays key role in the callose synthesis of pear pollen tubes.The 2-Deoxy-D-glucose significantly inhibited the pollen tube growth,and resulted in the decreased of cell wall thickness and the suppression of the callose content,which indicated that the reduced callose content inhibits pollen tube growth.Using antisense oligonucleotide,we found that PbrCalS5 affected the pollen tube growth by regulating both callose deposition in the pollen tube cell wall and the formation of callose plugs.Taken together,these results suggested that the knockdown of PbrCalS5 expression resulted in the decrease of callose content in the pollen tube and subsequently shorten the length of the pollen tube.3.In order to further explore the molecular mechanism of PbrCalS5 affecting pollen tube growth in pear,we found a transcription factor PbrbZIP30 by transcriptome data.PbrbZIP30 had a high expression level in pollen tube.The expression trend of PbrbZIP30 and PbrCalS5 was consistent during pollen tube growth.Ssubcellular localization analysis showed that PbrbZIP30 protein was localized in nucleus,cell membrane and cytoplasm.The results of yeast one-hybrid and EMSA demonstrated that PbrbZIP30 could bind to the CAGCT sequence in the PbrCalS5 promoter,and the dual luciferase reporter demonstrated that PbrbZIP30 could activate the expression of PbrCalS5.Using antisense oligonucleotide,PbrbZIP30 inhibited the pollen tube growth,the callose deposition in the pollen tube cell wall and the formation of callose plugs,and also inhibited the expression level of PbrCalS5.Taken together,these results revealed that potential function of PbrbZIP30 to regulate the development of pollen tube were dependent on activating the expression of PbrCalS5.In conclusion,callose regulates the pear pollen tube growth;PbrCalS5 is an important factor for the synthesis of callose and can promote the growth of pollen tubes.The transcription factor PbrbZIP30 binds to the CAGCT motif of the PbrCalS5 promoter,to activate the expression of PbrCalS5 and then regulate the callose deposition in pear pollen tube.