Functional Analysis Of ROP Signaling In Pollen Germination In Arabidopsis

Pollen germination is an important step in the fertilization process of flowering plants,which is crucial for seed production and species specificity.Pollen germination is regulated by some protein factors and accompanied by specific cytological activities.However,the major genetic pathways regulating pollen germination remain largely unknown.ROP is a unique class of small GTPase in plants.Active ROPs regulatepolar growth,biotic and abiotic resistance,and plant hormone response by activating its downstream effectors.During pollen germination,a rapid activation of ROPs occured,hinting at the ROP signaling pathway in this process.Howerer,there was no direct genetic evidence supporting this hypothesis.In this study,CRISPR/Cas9 gene editing technology was used to simultaneously knock out pollen expressed ROP1,ROP3,ROP5 and ROP9,and comprehensive cytological,genetic,biochemical and molecular biological methods were used to confirm that the ROP signaling pathway regulates pollen germination.The main research results are as following:（1）Creation of rop mutants using CRISPR/Cas9 gene editing technology.rop1,rop3;9,rop1;3;9 and rop1;3;5;9 mutants were created by CRISPR/Cas9 system.Sequence alignment revealed that one base-pair insertion was introduced in genomes of ROP1and ROP3,one base-pair deletion was introduced in genome of ROP9 and an eleven base-pair deletion was introduced in genomes of ROP5,which caused pre-mature translation termination.（2）The silique of rop1;3;5;9 was sterile.The vegetative growth of all of these mutants were normal at three or five weeks after germination,but the silique of rop1;3;5;9 was sterile.Reciprocal cross assays showed that rop1;3;5;9 as pollen donor produced no seeds,while as female produced full seed set,indicating that the sterility of the rop1;3;5;9 mutant belonged to male sterility.Analysis of progenies from reciprocal crosses or self-crosses of heterozygous mutants and reciprocal crosses experiment demonstrated that the abortion of rop1;3;5;9 could not be transmitted through the male gametophytes.（3）rop mutants exhibited abnormal pollen germination in vitro.To determine what have caused the zero male transmission of rop1;3;5;9,we first analyzed pollen development by using Alexander staining for cytoplasmic viability,4,6-diamino-phenylindole（DAPI）staining for nuclear structure,and scanning electron microscopy（SEM）for pollen coat structure.No abnormality was observed for pollen grains from the rop1;3;5;9 plants by all three approaches.Adhesion and hydration of wild type and rop1;3;5;9 pollen grains on wild type stigmas were comparable.However,pollen grains from rop1;3;5;9 mutant did not germinate at all when incubated in pollen germination medium.Morever,the germination speeds of rop1 and rop3;9 pollen grains were lower than that of wild-type pollen grains,and the pollen germination ratio of rop1;3;9 triple-mutant decreased greatly.Additionally,introducing Lat52:YFP-ROP3 into rop1;3;5;9 quadruple mutant rescued its silique fertility.These results provided direct evidence supporting the regulation of ROP signaling in pollen germination.（4）Screening for effectors of ROP in pollen germination regulation.Inflorescences from transgenic Lat52:mRFP-ROP1 plants were used for immunoprecipitation/mass spectrometry to find out the candidate interacting protein of pollen-expressed ROPs.As a result,a member of BDR（Boundary of ROP）family,namely BDR9 were identified.Arabidopsis transcriptome data suggested that BDR8/9 is relatively highly expressed in mature pollens and pollen tubes,which were confirmed by our q PCR assays as well as GUS siginal of BDR8g:GUS and BDR9g:GUS transgenic plants,indicating that both BDRs are involved in male gametophytic function.（5）ROP1/3/5/9 physically interacted with BDR8/9.Split-LUC experiment verified that all forms of ROP1/3/5/9（wild type,CA form and DN form）interact with BDR8/9.In pull-down assays,BDR8 showed strong affinity with ROP1and ROP1^CA but weak affinity with ROP1^DN,whereas BDR9 showed a comparable affinity with all three forms of ROP1.In addition,FRET assays demonstrated that BDR8/9interacted with ROP1 at the plasma membrane but not in the cytoplasm.In this study,CRISPR/Cas9 gene editing technology was used to create rop mutants to confirm the regulation of pollen-expressed ROPs in in pollen germination.Besides,the possible downstream effectors in this process were identified by immunoprecipitation/mass spectrometry,bioinformatics analysis as well as point-to-point interaction detection.These results are helpful for further study of ROP signaling pathway in pollen germination regulation and provide more information for its application in agricultural biotechnology.