Chromatin Remodeling Complex SWR1 Regulates Megasporogenesis In Arabidopsis

In higher plants,the formation of female gametophyte is critical for the successful sexual reproduction and the specialization of germ cells highlights the initiation of sexual reproduction.In Arabidosis,the formation of megaspore mother cell from one of somatic cells in ovule marks the beginning of female germline development.There may be a certain lateral inhibition mechanism that prevents the differentiation of adjacent somatic cells,to ensure only one of somatic cells in ovule to form megaspore mother cell.Intercellular signaling between somatic cells in the ovule is critical for the control of single germline cell formation,but the underlying mechanism is far from clear.Our previous studies had show that both ARP6,one component of SWR1 complex and cytochrome P450 protein KLU participate the process of meiosis during the development of megaspore.In this study,we found that the arp6 klu double mutant produces multiple enlarged megaspore mother cell(MMC)-like cells in ovules.Comparative transcriptome analysis identified that WRKY28 is one of candidate genes in the downstream of SWR1 complex and KLU.Through our cytological assays in combination with genetics and molecular biology evidences we further showed that the SWR1 complex and KLU cooperatively controlled the expression of WRKY28,which is critical for the cell fate decision during the formation of megaspore mother cells.Firstly,through comparative transcriptome analysis,we identified KLU,a gene encoding for a putative cytochrome P450 monooxygenase,was specifically expressed in the inner integument of ovule primordia.Chromosome spreading and FISH assays revealed that the KLU mutation resulted in defect of homologous chromosome pairing during female meiosis suggestting that KLU is involved in the interaction of homologous chromosome during female meiosis.Secondly,we showed that the SWR1 complex genetically interacts with KLU.The double mutant of arp6 klu showed a low seed set rate and reciprocal crossing assays revealed that the low seed set was caused by the defects of female gametophyte.We further show the double mutant of arp6 klu produced multiple enlarged MMC-like cells.We further show that these enlarged cells had gained the morphological and molecular properties of MMC in ovules,suggesting that SWR1 complexes and KLU cooperatively inhibit the formation of multiple megaspore mother cells.Thirdly,by comparing the transcriptome data of the wild-type,arp6,klu single mutant and arp6 klu double mutant,it was found that the expression of WRKY28 was specifically down-regulated in the ovule primordium of the arp6 klu double mutant but not in the single mutants in comparison with wild-type.Quantitate RT-PCR assays show that this gene was highly expressed in the ovules in the stage of MMC specification compared to other tissues including roots,stems,leaves and mature ovules,anthers.Fourthly,we determined the temporal specificity of WRKY28 during megasporogenesis by immune-histochemistry,in situ hybridization and tissue localization analysis.We showed that WRKY28 was specifically expressed in hypodermal somatic cells surrounding the megaspore mother cell and its expression was ended when the functional megaspore was formed.Fifthly,we genetically proved that WRKY28 was critical to inhibit the production of multiple megaspore mother cells.When the function of WRKY28 was disrupted by reverse genetics methods including gene editing by CRISPR-CAS9 method and gene silencing through the expression of SRDX chimeric suppressor,the plants showed abnormal ovule development and multiple enlarged MMC-like cells formation in ovules.Furthermore,we showed that the over-expression of WRKY28 in the arp6 klu double mutant partially restored both the defects of MMC differentiation and the seed setting rate in double mutant implying that WRKY28 is a key downstream gene of SWR1 complex and KLU in the control of megaspore mother cell specification and ovule fertility.Sixthly,CHIP-PCR assays with H2A.Z antibody revealed that H2A.Z deposited at the +1 nucleosome of TSS at WRKY28 locus in the inflorescence of Arabidopsis,while its deposition at WRKY28 locus was significantly reduced in the arp6 mutant.Furthermore,we found that in the mutant of klu,both the enrichment of ARP6 and the deposition of H2A.Z at WRKY28 was significantly reduced suggesting that the KLU may regulate the deposition of H2AZ by recruiting the SWR1 complex to control the expression of WRKY28.Taken together with evidences from phenotyping of arp6,klu,and arp6 klu double mutant,expression profiling of WRKY28,reverse genetics study by gene editing and gene silencing and CHIP assay,our study show that SWR1 complex cooperate with KLU to regulate MMC specification by activating the expression of WRKY28,which is specifically expressed in somatic cells surrounding the MMC in ovules.In conclusion,the female germline specification is a key step in female gametophyte development and our study here revealed the underlying molecular mechanism that ensures only one of somatic cells in ovule to develop into megaspore mother cell.Our study provides insights for the study of how cell fate is decided in the process of plant sexual reproduction.