The Arabidopsis Armadillo Protein ZAK IXIK Is Involved In Floral Organ Development Through Interaction With The F-box Protein SAP

The flower of flowering plants contains two reproductive organs,stamen and pistil,which produces male and female gametophytes,respectively.During anthesis,the male gametophyte(pollen)produced in anther of stamen is released and transferred to stigma of pistil.It then gets hyfrated,germinates and generates a pollen tube.The pollen tube grows through the transmitting tracts in pistil,and finally enters the female gametophyte(embryo sac)in the ovule and discharges the two sperm cells that further fuse with central cell and egg cell,respectively.The resulting fertilized egg(zygote)develops into an embryo that further grows into a plant.Therefore,flower and embryo developmental processes are important for the generation alternation of plant.They are also the basis of seed production that is closely related to agricultural production.Studies on the genetics of flower and embryo development processes not only enable us to understand the genetic mechanisms that control flower and embryo development,but also could provide a theoretical support to improvement of crop breeding and cultivation technologies.Our previous study identified two embryo-defective mutants kd616 and sgt4712 that were generated by insertion of Ds transposon element.The molecular characterization showed that the Ds elements in the two mutants were inserted in the same gene AT4G00231 that encodes an Armadillo protein.Recently,the AT4G00231 was named as ZAK IXIK(ZIX).Therefore,we renamed kd616 and sgt4712 as zix-1 and zix-4,respectively.Genetic and phenotypic analyses showed that the two mutants did not have impact on male and female gamophyte development,but drastically affected embryo development.The mutant embrogenesis was disrupted at the globar stage,finally resulting in abortion of seed development.No homozygous zix mutant plant was naturally generated available.This study was to further characterize the zix-1 and zix-4 mutants and study the roles and mechanisms of the Arm protein gene ZIX in plant development.The expression pattern analysis showed that ZIX is constitutively expressed in many tissues.Especially,it is more highly expressed in the tissues with active cell division and growth.These results indicated that ZIX may also play roles in other tissues besides embryos.Therefore,an embryo-rescue assay was performed by embyo-specific expression of ZIX in the zix-1 mutant plants driven by the promoter of the embryo-specific gene ABI3 to generate the embryo-rescued homozygous zix-1 mutant(EmR-zix-1)plants.The EmR-zix-1 plants exhibited multiple defects in plant growth and development.The main phenotypes were that the EmR-zix-1 plants grew dwarf and smaller,and could not flower;or they could have a normal growth pattern and could flower,but had drastical defects in floral organ development,including incomplete petals or lack of petals,reduction in stamen number or defective stamen with abnormal pollen chamber or without pollen chambers,unclosed carpels and exposed ovules.The RNA-Seq assays using the EmR-zix-1 inflorescences showed that the mutation in ZIX affected expression of many genes,including those involved in various aspects of cellular signal transduction,transcription regulation,oxidation-reduction,transport,stress response and metabolism.Compared with those in wild-type plants,the expression of the flower homeotic genes and floral organ developmental genes in EmR-zix-1 plants were significantly changed.Furthermore,in some floral homeotic mutants,expression of ZIX gene was also changed.These results indicated that ZIX is not only important for embryo development,but also plays important roles in flower development.The ZIX gene encodes a 54 kD Armadillo protein with 475 amino acids.The protein shares a low homology with the Armadillo proteins from other species,but it has a conserved Ataxin10 homologous domain at its C-terminus.The ZIX protein was predicted to have a typical right-handed super-helical solenoid.Structural analysis also showed that the protein may form homologous polymer,and work through association with the biological macromolecules such as DNA or protein.BiFC,LCI,pull-down and Native PAGE assays demonstrated that the ZIX protein was able to form homo-polymers.A candidate for ZIX-associating protein,namely the F-box protein SAP,was then isolated using the method of tandem affinity immunoprecipitation.The interaction of ZIX with SAP was further confirmed by pull-down,BiFC,LCI and Co-IP assays.The protein subcellular localization assays showed that ZIX protein was located in the cytoplasm and nucleus,and the SAP protein in the nucleus,indicating that they had an overlapped protein subcellular localization pattern.The expression of SAP gene in Arabidopsis thaliana was detected in many tissues,especially in inflorescences and silliques,which overlaped with that of ZIX gene.In addition,phenotypic comparison using the cas9-sap-92 mutant generated by the CRISPR/Cas9 system and sap-flag mutant obtained from Dr.Kaufmann laboratory showed that the homozygous sap mutants had the floral phenotypes similar to those of the embryo-rescued zix mutant(EmR-zix-1).They also exhibited defects in petals,stamen and carpels.However,the sap mutants did not exhibit any defect in embryo development,different from the zix mutants that disrupted embryogenesis and caused seed abortion.These results indicated that ZIX may play important roles in flower development through interaction with SAP.However,whether SAP is also involved in embryo development remains unclear.The transcription activiation assay showed that the ZIX-BD fusion protein has transcriptional activation activity in yeast.Cell free degradation experiments showed that ZIX protein was unstable in vitro,and such instability could be inhibited by proteasome inhibitor MG132,suggesting that the protein may be degraded through the 26S proteasome pathway.The GST-ZIX protein degradation assays using the treatments with the total proteins extracted from SAP-overexpressing plants,sap mutant plants and wild-type plants showed that the total protein extract from sap mutant plants could not promote degradation of the GST-ZIX protein,but the total protein extract from SAP-overexpressing plants could promote degradation of GST-ZIX like that from wild-type plants.The expression levels of ZIX mRNAs in the SAP-overexpressing and sap mutant plants were not significantly different from that in wild-type plants.Furthermore,the amount of ZIX protein in SAP-overexpressing was similar to that in wild-type plants.However,the amount of ZIX protein in sap mutant plants was increased compared to that in wild-type plants.These results indicated that ZIX may be a target of SAP.SAP may regulate expression of the downstream genes through regulating degradation of ZIX.Taken all together,the ZIX gene is not only essential for embryo development,but also plays important roles in flower development through interaction with the F-box gene SAP.These results could provide a new clue for understanding of genetic mechanisms of flower development.They could also provide a new knowledge about the roles of the Armadillo proteins in plant development.