Molecular Mechanism For RALFs Regulating Pear Pollen Tube Growth

Polypeptide signals play important roles in plant growth and development.Rapid alkalinization factor is a cysteine-rich polypeptide and involved in many plant growth and development processes.In this study,the pollen of 'Dangshansuli' was used as the material to study the molecular mechanism of RALF which expressed in pear pollen regulating pollen tube growth.The main results are as follows:1.A total of 24 PbrRALFs were selected from pear genome and divided into 4 subfamiliesaccording to the phylogenetic analysis..Duplication mode analysis showed that genome-wide replication(WGD)was the main driving force in the expansion of this family.Semi-quantitative expression analysis of PbrRALF genes showed that 16 PbrRALFs genes expressed in pear pollen.In order to further study the regulation of RALF in pear pollen tube growth,we made the 16 PbrRALFs expressed and purified using multiple techniques,including gene cloning,construction of prokaryotic expression vectors,prokaryotic expression and purification,and obtained 16 recombinant PbrRALF proteins.The systematic analysis of pear RALF family provides an inportant theoretical foundation for the study of its function.2.To study the effect of RALF on pear pollen tube growth,we used 16 recombinant PbrRALF proteins which expressed in pollen to treat pear pollen,and found three PbrRALFs(PbrRALF2,PbrRALF7,and PbrRALF 11)could significantly inhibit pollen tube growth,of which PbrRALF2 showed a dominative inhibitory effect.Therefore,PbrRALF2 was primarily used for subsequent experiments.After knocking down the expression of PbrRALF2 gene in pear pollen using antisense oligonucleotides(as-ODN),we observed a significant promotion in pear pollen tube length.These results suggest that PbrRALF2 was an inhibitor of pear pollen tube growth.Different concentrations of PbrRALF2 were used to treat the pollen of 'Dongshansuli',and we found that PbrRALF2 inhibited the pear pollen tube growth in a dose-dependent manner.PbrRALF2 could significantly increase the production of ROS at the tip of pear pollen tube,promote the depolymerization of pollen tube microfilament skeleton,increase the amount of pectin in pollen tube cell walls and cause cell wall thickening.The regulation of PbrRALF2 on pear pollen tube growth further deepens our understanding of RALF involved in the regulation of cell expansion.3.CrRLK1L receptor kinases often act as plasma membrane surface receptors to participate in multiple signal transduction pathways during plant growth and development.In this study,26 CrRLK1L family members were selected from pear genome.Evolutionary model analysis showed that the genome-wide replication event(WGD)was the main driving force in the expansion of this family.We established a specific signaling pathway for RALF-regulated pear pollen tube growth using a series of techniques such as yeast two-hybrid,pull down,receptor dynamics and antisense oligonucleotides.We found that PbrRALF2 specifically interacted with the extracellular juxtamembrane region of PbrCrRLK1L13.The relative affinity of PbrRALF2 for PbrCrRLK1L13 was at the submicromolar level,consistent with the values of ligand-receptor kinase pairs and the IC50 for PbrRALF2-mediated inhibition of pear pollen tube growth,suggesting that PbrCrRLK1L13 was the receptor of PbrRALF2.Meanwhile,PbrRALF2 increased the phosphorylation level of PbrCrRLK1L13 after binding to its extracellular domain,indicating that the binding of PbrRALF2 with PbrCrRLK1L13 was functional.Furthermore,PbrMPK18 interacted with the intracellular domain of PbrCrRLK1L13.When the expression of PbrCrRLK1L13 and PbrMPK18 were respectively knocked down,the ROS production and pollen tube growth inhibition were also less sensitive to PbrRALF2.The results showed that PbrRALF2 interacted with PbrCrRLK1L13 and mediated PbrMPK18-induced reactive oxygen species(ROS)production at the tip of pollen tubes,while excessive accumulation of ROS caused pear pollen tube growth inhibition.The present results demonstrated auto-regulated signaling could control the integrity growth of pollen tube,suggesting that the RALFs-CrRLK1L pathway might represent a general mechanism for inhibiting cell extension.4.Small GTPase proteins are involved in many processes of plant growth and development In this study,112 small GTPase proteins were screened in pear genome and divided into 4 subfamilies.The duplication pattern analysis showed that genome-wide duplication or segmental duplication was the main driving force in the expansion of this family.Transcriptome data and qRT-PCR analysis showed that PbrROPl and PbrROP2 highly expressed in pollen.After knocking down the expression levels of PbrROP1,PbrROP2 and PbrGEF8 in pollen respectively,the growth rate of pollen tubes decreased significantly.Yeast two-hybrid demonstrated that PbrGEF8 interacted with both PbrROP1 and PbrROP2,indicating that PbrGEF8 could mediate pollen tube growth regulation through PbrROP1 and PbrROP2.At the same time,we found that PbrGEF8 also intreacted with PbrCrRLK1L13.When the expression of PbrGEF8 was knocked down,the pollen tube growth inhibition was less sensitive to PbrRALF2 as well.Therefore,we proposed a new mechanism for ROP-dependent regulation of pollen tube growth:PbrRALF2-PbrCrRLK1L13 complex interacted with PbrGEF8 and may inhibit its activity,and then mediated PbrROP to regulatepollen tube growth.