Arabidopsis ALA3 Regulates Pollen Tube Growth And Guidance By Affecting The Anionic Phospholipid-mediated Tip Localization Of PRKs

Pollen tube guidance regulates the rapid growth and ovule targeting of pollen tubes in pistils,which is crucial for flowering plants to complete sexual reproduction.Arabidopsis pollen-specific receptor kinases（PRKs）family members PRK3 and PRK6are specifically tip-localized and essential in pollen tube growth and guidance.However,the mechanisms controlling PRK polar localization at the pollen tube tip are vague.The Arabidopsis P4-ATPase（Arabidopsis aminophospholipid ATPase3,ALA3）helps establish the polar localization of apical phosphatidylserine（PS）in pollen tubes.Here,we discovered that loss of ALA3 function caused pollen tube defects in growth,ovule targeting and sensitivity to the attractive peptide At LURE1.2 and significantly affected the polar localization pattern of PRK3 and PRK6.We revealed that PRK3 and PRK6contained two polybasic clusters in the juxtamembrane domain and bound PS in vitro.PRK3 and PRK6 with polybasic cluster mutations reduced or abolished their binding to PS,altered their polar localization pattern and failed to effectively complement the pollen tube guidance defective phenotypes of the prk mutants.These results suggest that ALA3 influences the precise localization of PRK3,PRK6 and other PRKs by regulating the distribution of PS,which plays a key role in regulating pollen tube growth and guidance.In this paper,we mainly used cell biology,molecular biology,genetics and biochemistry to study the function of ALA3 in pollen tube growth and guidance.And the mechanism of ALA3 regulates pollen tube growth and guidance by affecting the anionic phospholipid-mediated tip localization of PRKs,the following results were obtained:1.Loss of ALA3 function induces defects in fertility.The ala3 mutant showed slow growth in pollen tube,abnormal ovules targeting and aborted,and was insensitive to the attractor peptide At LURE1.2 secreted by the female gametophyte,indicating that the loss of ala3 function resulted in defects in pollen tube growth and pollen tube guidance,and eventually aborted.Those results suggest that ALA3 affects the key modules involved in the At LURE1-mediated pollen tube guidance signaling pathway.2.The loss of ALA3 function resulted in the change of the distribution pattern of PRKs at the tip of pollen tube.The polar localization pattern of PRK1,PRK3,PRK6and PRK8 in ala3 mutant pollen tubes was significantly changed,which may be the main reason of slow pollen tube growth,abnormal guidance and abortion in the ala3mutant.3.PS-mediated Rab vesicle transport pathway indirectly regulates the polarity distribution of PRK6 at the tip of pollen tube.The loss of ALA3 function leads to the inability of PRK6 to be effectively transported to the pollen tube tip.Meanwhile,the loss of ALA3 function affects the PS-mediated polar distribution pattern of the pollen tube tip of Rab A4d,but the change of PRK6-m Ruby2 distribution in raba4d mutant pollen tube is different from ala3 mutant.raba4d mutant pollen tubes responded to At LURE1.2.Therefore,ALA3 establishes and maintains the polarity distribution of PS at the tip of the pollen tube,which indirectly affects the polar transport of PRK6 to the tip of the pollen tube.4.Some members of PRK family have polybasic clusters,which can bind anionic phospholipids.We primarily screened polybasic clusters of the PRK family members,except PRK5 and PRK7,contained polybasic cluster in the juxtamembrane domain.And the lipid overlay experiment demonstrated that PRK family members with polybasic clusters bound anionic phospholipids,but the binding patterns of phospholipids were distinct.5.Genetic studies have proved that ALA3 was genetically related to PRK3 and PRK6.The ala3 prk3 prk6 mutants showed slow pollen tube growth,abnormal guidance and abortion,but these phenotypes exhibited by the ala3 prk3 prk6 triple mutants were not aggravated compared with those of the ala3 mutant and prk3 prk6double mutants.Thus,the abortion and pollen tube-related phenotypes of ala3 mutants were partially caused by the effects of PRK3 and PRK6.They might be genetically in the same pathway.6.The binding of PRK3 and PRK6 to phospholipids is critical for their subcellular localization.We created the transgenic rescued plants of polybasic clusters mutations in PRK6 and PRK3,and found that the mutations in different polybasic clusters affected their localization patterns at the tip of the pollen tube to varying degrees.These results indicate that anionic phospholipids,such as PS and PA,may be directly involved in regulating the precise localization of PRK3 and PRK6.7.Mutant PRK3 and PRK6 could not completely complement the phenotype of prk mutants.PRK3^8Q-GFP and PRK3^9Q-GFP could not completely compensate for slow pollen tube growth and the abortive of prk3 prk6 double mutant,nor could PRK6^6Q-GFP and PRK6^8Q-GFP completely compensate for the damaged pollen tube ovules and At LURE1.2 insensitive phenotypes of prk6.Therefore,the ability of PRK3 and PRK6 to bind anionic phospholipids,such as PS and PA,is essential for their polar localization in the pollen tube and for the regulation of pollen tube growth,guidance and abortion.To summarize,this study investigated the physiological functions of ALA3,a member of the Arabidopsis P4-ATPase family,in the process of pollen tube growth and guidance to elucidate the molecular mechanism of Arabidopsis P4-ATPase regulating pollen tube guidance and the fine regulation mechanism of maintaining the polarity localization of PRKs pollen tube tip.This provides a critical theoretical foundation for optimizing pollen tube polar growth and comprehending the chemical mechanism of pollen tube guidance.