Arabidopsis KETCH1 Mediated Gametogenesis Via Nuclear Accumulation Of Ribosomal Protein RPL27a

Double-fertilization of both egg and central cell with sperm cells is a major distinguishing feature of flowering plants reproduction.Development of gametophytes is critical for plant reproduction.Male and female gametophytes are generated from micro-or megaspore mother cells through consecutive meiotic and mitotic cell divisions.Researches show that mutations affecting mitotic cell division during gametogenesis often cause lethality,hinting at a key role of mitotic regulation during gametogenesis.Gametophytic lethality due to defective mitosis is also observed in mutants that are compromised in ribosome biogenesis.Although large amount of ribosomal proteins(Ribosomal Proteins,RPs)have been identified in plants based on homology with their yeast and metazoan counterparts,how these RPs are regulated,such as dynamic subcellular targeting,is little known.We report here that an Arabidopsis importin ?,KETCH1(karyopherin enabling the transport of the cytoplasmic HYL1),is critical for male and female gametogenesis via nuclear accumulation of ribosomal protein L27a(RPL27a).The main results and conclusions presented in this thesis are as follows:(1)Functional loss of KETCH1 compromises male and female transmissionIt was reported previously that mutations of KETCH1 resulted in embryo lethality and thus no homozygous mutants of KETCH1 could be obtained(Zhang et al.,2017).However,the heterozygous mutant of KETCH1,ketch1-2/+,contained around 47% tiny and wrinkled ovules,much higher than 25% as expected for embryo lethality.In addition,the small and wrinkled ovules in ketch1-2/+ were unfertilized,containing irregularly distributed nuclei.By analyzing the progenies of recriprocal crosses beween wild type and ketch1-2/+,we determined that both male and female gametophytic transmissions of ketch1-2 were severely reduced,suggesting that KETCH1 is critical for male and female gametophytic functionality.(2)Functional loss of KETCH1 results in the arrest of gametogenesisTo determine the cause of reduced male transmission,we performed Alexander staining of cytoplasmic viability,DAPI staining of nuclear structure,and scanning electron micrographs(SEMs)of pollen coat structure.Compared to wild-type pollen grains,around 32% pollen from ketch1-2/+ were abnormal.We analyzed anthers at different developmental stages by plastic embedding and semi-thin transverse sections.Pollen development seems comparable between wild type and ketch1-2/+ before stage 10.However,microspores containing only one nucleus and few large vacuoles at stage 11 in ketch1-2/+ anthers in contrast to those of wild type,indicate that ketch1-2 microspores are defective during PMI.To determine the defects of ketch1-2 female gametophytes,we examined ketch1-2/+ ovules at different developmental stages by optical sections through CLSM.Although all stage 3-I ovules in ketch1-2/+ contained a FM,FM in 30% ovules failed to go through mitosis,result in the embryo sac contained irregular numbers of nuclei,ranging from one to six.These results show that KETCH1 is critical for the mitotic cell division of unicellular microspores and functional megaspore.(3)KETCH1 is constitutively expressed and high in reproductive tissuesKETCH1 is constitutively expressed and high in reproductive tissues such as inflorescence,ovules,and pollen by quantitative RT-PCRs and the KETCH1 promoter-driven GUS reporter lines.Analysis of the KETCH1 promoter-driven nuclear-localized YFP lines show that KETCH1 is expressed in generative cell during gametogenesis.(4)The role of KETCH1 during gametogenesis is independent of HYL1KETCH1 was recently reported to mediate the nucleocytoplasmic transport of HYPONASTIC LEAVES 1(HYL1),a miRNA regulator.Although the null mutant of HYL1,i.e.hyl1-2,was reported to show reduced fertility,both male and female gametophytic transmission of hyl1-2 were comparable to those of wild type,suggesting that compromised nucleocytoplasmic transport of HYL1 was not responsible for KETCH1 function during gametogenesis.(5)Nuclear accumulation of ribosomal protein RPL27 aC requires KETCH1Phylogenetic analysis suggested that Arabidopsis KETCH1 is closely related to mammalian importin 5(IPO5),which mediates the nuclear import of several ribosomal proteins in mammals.We first tested the interaction between KETCH1 and RPL27 aC by bimolecular fluorescence complementation(BiFC)and by in vitro pull-down assays.And our results also suggested a pair-wise specificity between importins and their cargos.Furthermore,the nuclear accumulation of RPL27 aC was significantly reduced in pollen grains and pollen tubes of ketch1-2 as well as in leaf protoplasts by down-regulating KETCH1 through RNAi,suggesting RPL27 aC as another cargo of KETCH1.Indeed,RPL27 a is critical for male and female gametophytic transmission,consistent with its role in KETCH1-regulated gametogenesis.Thus,a likely scenario is that the reduced nuclear accumulation of RPL27 a in ketch1-2 compromised ribosomal biogenesis,leading to the arrest of gametogenesis.Karyopherins are molecular chaperons mediating nucleocytoplasmic protein transport.Our report show that functional loss of KETCH1 caused the arrest of male and female gametophytic development after meiosis.The role of KETCH1 during gametogenesis is independent of HYL1,a previously reported KETCH1 cargo.Instead,KETCH1 is critical for the nuclear accumulation of RPL27 a,whose mutations caused similar gametophytic defects.Reduced nuclear accumulation of ribosomal proteins may cause reduced translational capacity and trigger the arrest of mitotic cell cycle progression during gametogenesis.