Mechanism Of PbrTTS1 Regulating Pear Pollen Tube Growth

TTS protein(Transmitting tract-specific glycoprotein,TTS)was a class of proline-rich and highly glycosylated proteins and involve in regulation of the growth of pollen tubes.TTS was subfamily member of AGPs(Arabinogalactan proteins,AGPs).It might act as a signaling molecule to regulate the growth of pollen tubes or as a nutrient to support the growth of pollen tubes,however,the specific mechanism of action of TTS is still unclear.In this study,the pollen and style of 'Dangshan Suli' were used as mainly materials to study the molecular mechanism of how PbrTTSl which was subfamily of AGPs regulate pollen tube growth in‘Dangshan Suli'.The key results are as follows:1.Seven hundred and eighty-two Hydroxyproline-rich glycoproteins(HRGPs)genes were identified from pear genome and the amino acid sequences of HRGPs were rich in proline,serine,alanine and threonine.HRGPs were divided into 3 families:AGPs,moderately glycosylated extensins(EXTs)and lightly glycosylated proline-rich proteins(PRPs).WGD were the major force driving HRGPs expansion.Multiple amino acid sites selection pressure was more than 1,and hydroxyproline(Hyp)-rich motifs could effect the gene evolution,which was mainly reason for the diverse genetic structure of the HRGPs.The genes pairs from WGD event always present highly expression divergence.HRGPs genes were mainly expressed in style,and the expression in pollen and fruit were relatively rare.In conclusion,the highly variable Hyp-rich motif might be the main reason of HRGPs expansion,evolution and founctional divergence.2.PbrTTS1 was a member of the AGPs family.To explore its specific effects on pollen tube growth,prokaryotic expression and purification was used to express and purify PbrTTS1 proteins.PbrTTS1 proteins were used to treatment the pollen tube,the results showed that PbrTTS1 could promote pollen tube growth in vitro,and it is indicated that the glycosylation modification does not affect the biological function of PbrTTS1 to promote pollen tube growth.To explore how the PbrTTS1 promotes pollen tube growth?A cDNA library of pollen that was germinated for 3 h were constructed.PbrTTS1 were regared as a bait to screen candidate proteins and 13 candidate proteins were screened,including F-box protein,defensin-like protein,C2 Domian protein,pectinesterase,protein RALF-like and E3 ubiquitin-protein ligase.Pollen C2 domain-containing protein(PCCP)and rapid alkalinization factor(RALF)were the subject of the study.3.According to results of cDNA,and the function of NaPCCP that has been reported in tobacco.PbrPCCP1 was isolated from pear by bioinformatics analysis,tissue localization analysis,expression pattern analysis.PbrPCCP1 mainly expressed in pollen and localized in the plasma membrane.Y2H,BiFC and LCI analysis showed that PbrPCCPl can interact with PbrTTS1,and the Pollen ole e I conserved domain of PbrTTSl was responsible for interaction The growth of pollen tube was inhibited when the expression level of PbrPCCP1 in the pollen tube were knockdown using antisense oligonucleotide(As-ODN).After inhibiting the expression of PbrPCCPl in pollen using ODN,the promoting effect of PbrTTSl on pollen tube growth was attenuated.4.The RALF genes are cysteine-rich peptide and involve in multiple processes of plant growth.Twenty-four PbrRALF were identified from pear,and PbrRALF2 can inhibit the growth of pollen tubes by altering the phosphorylation level of receptor-like kinase(RLK)PbrCrRLK1L13.Therefore,combined the results of cDNA,the interaction between PbrTTS1 and 24 PbrRALF were further tested by Y2H,and the results showed that PbrRALF5/17/19 could interact with PbrTTS1.RT-PCR and RT-qPCR analysis indicated that the expression level of PbrRALF 19 was up-regulated in style after pollination.Moreover,Y2H analysis showed that PbrTTSl interacts with PbrCrRLK1L13-ECD.The affinity constant between PbrTTSl and PbrCrRLK1L13-ECD was calculated by BLI,KD?3.07E-06M.Y3H results showed that PbrTTS1 could inhibit the interaction between PbrCrRLK1L13 and PbrRALF2.PbrTTS1 could promote pollen tube growth after knockdown the expression of PbrCrRLK1L13 using as-ODN.In summary,PbrTTS1 competes with PbrRALF2 for interaction with the extracellular domain of PbrCrRLK1L13 to regulate the growth of pollen tubes.Additionally,PbrRALF19 interacts with PbrTTSl,and thereby attenuating the inhibitory effect of PbrTTS1 on PbrRALF2 and PbrCrRLK1L13.