Lorelei-Like Gpi-Anchored Proteins 2/3 Regulate Pollen Tube Growth

In angiosperms,pollen tube growth is crucial for successful fertilization.In flowering plants,when the pollen lands on the stigma of the pistil,after adhesion and hydration,it will germinate into a pollen tube.The pollen tube then growing down along the style carrying two sperm cell until it reaches the embryo sac.The pollen tube bursts and the sperms are released to complete double fertilization.In Arabidopsis thaliana,ANXUR(ANX)/BUPS receptor kinase complex located at the apical plasma membrane of pollen tubes responds to the autocrine rapid alkalinization factor 4/19(RALF4/19)to maintain pollen tube growth;however,the molecular and cellular mechanisms underlying the ANX/BUPS-mediated regulation of pollen tube growth remain unclear.By phylogenetic anlysis and transcriptional level examination,we found that LORELEI-like glycosylatedphosphatidylinositol anchored proteins 2 and 3(LLG2 and LLG3)were highly expressed in pollen and that the pLAT52::LLG2/3-RNAi-23/27/53 lines showed obvious fertility defects.Reciprocal cross by limited pollination was found that the decrease of LLG2/3 expression level led to the defect of male gametophyte,which was mainly manifested in the interrupted growth,increased burst rate of pollen tubes and abnormal deposition of cell wall components.Above results indicate that LLG2 and LLG3 are involved in regulating pollen tube growth.Cell biology analysis showed that the self-fusion protein of LLG3 is located on the apcial plasma membrane of the pollen tube,which may be related to the transport and localization of LLG2/3 in assisting ANX1/2.In the pLAT52::LLG2/3-RNAi transgenic lines,ANX1/2-GFP could not be delivered to the plasma membrane in the apical region of pollen tubes.Further results showed that the reactive oxygen species(ROS)levels in the pollen tubes of pLAT52::LLG2/3-RNAi were significantly reduced,and changing the reactive oxygen species content in the pollen tube could significantly affect the growth of the pollen tube.Moreover,we found that RALF4 induced the production of reactive oxygen species,which stimulated pollen tube growth and reduced pollen burst rate.In summary,our study proposes a point that LLG2/3 assist ANX/BUPS in transporting to the apical plasma membrane of pollen tubes,and might act as coreceptors for ANX/BUPS to sense the RALF4/19 signal,thereby maintaining the pollen tube growth by activating downstream reactive oxygen species signaling pathways.Thus,the signal pathway of RALF-ANX/BUPS-LLG2/3-ROS regulating pollen tube growth was established.This study deepened the understanding of the pollen tube growth regulation signal pathways and broadened the research of reactive oxygen species regulating the pollen tube growth.