Molecular Mechanisms Of ARR10/12/18 In Regulating Arabidopsis Female Gametophyte Development

Mature female gametophyte,also known as embryo sac,is the place to complete the double fertilization process and produce seeds.Therefore,clarifying the regulatory mechanisms of embryo sac development has important scientific significance and agricultural value.However,because the embryo sac is deeply buried in the ovule,the female reproductive organ in angiosperms,there are certain challenges for embryo sac research.The study of regulatory mechanisms of embryo sac development has always been one of the highlights in plant reproductive biology.The elements of twocomponent signaling system in higher plants include histidine kinases on cell membrane,intracellular histidine phosphate transfer proteins,and response regulators in the nucleus.It has been reported that the histidine kinase CYTOKININ INDEPENDENT 1(CKI1)regulates cell fate decision in the embryo sac via an ARABIDOPSIS HISTIDINE PHOSPHATE TRANSFER PROTEIN(AHP)-dependent pathway.However,how the CKI1-AHP signaling pathway regulates embryo sac development remains unclear.In order to further explore the regulation of cytokinin signaling pathway on female gametophyte development,transgenic plants expressing NLS-YFP driven by the promoters of Arabidopsis cytokinin response regulators(ARR)were created.Three ARRs expressing in the mature embryo sac,namely ARR10/12/18,were identified.The T-DNA insertion mutants of ARR10/12/18 were further analyzed,while no homozygous triple mutant arr10 12 18 could be isolated by genetic cross.Consistently,in the developing siliques of arr10-5/+ arr12-1 arr18-2,arr10-5 arr12-1/+ arr18-2,and arr10-5 12-1 18-2/+,severe seed abortion was found.The promoters and coding regions of ARR10/12/18 that were fused with the YFP sequence could completely rescue seed abortion defects of the arr10 12 18 mutants,suggesting that ARR10/12/18 redundantly regulate reproductive development in Arabidopsis.The expression patternsand protein localization of these three ARRs during embryo sac development were analyzed in detail,and the results showed that all three ARRs were expressed in the embryo sac from FG1 to FG7.These three ARR proteins were mainly located in the chalazal nuclei after the FG4 stage.The results of reciprocal cross and gamete transmission efficiency analysis showed that the defects of female gametophyte caused seed abortion of the mutant.The development process of embryo sac in wild type and mutant was observed by differential interference contrast microscopy and confocal laser scanning microscopy.The results showed that about half of mature embryo sacs at the FG6/7 stage in the mutants collapsed and degraded,which was very similar to that reported in cki1 and ahp2 3 5 mutants,suggesting that ARR10/12/18 may function in the CKI1-AHP pathway to regulate embryo sac development.The expression patterns of embryo sac cell markers were analyzed in wild type and arr10-5 12-1 18-2/+ mutants.The results showed that the chalazal cell properties were lost and the micropylar cell marker extended to the chalazal end in the embryo sac of arr10-5 12-1 18-2,which was similar to that in cki1-9 and ahp2 3 5,further suggesting that ARR10/12/18 may play a role in the CKI1-AHP2/3/5 pathway to regulate embryo sac cell fate specification.In addition,the quadruple mutant arr10-5 12-1 18-2/+ cki1-9/+ and the sextuple mutant arr10-512-1 18-2/+ ahp2-2 3 5-2/+ showed more severe seed abortion and a higher proportion of embryo sac with altered cell properties like arr10-5 12-1 18-2/+,demonstrating that ARR10/12/18 function together with CKI1 and AHP to regulate embryo sac cell fate determination.Furthermore,ectopic expression of activated ARR10/12/18 in the embryo sac could generate additional central cell-like cells in micropylar end,which was consistent with that of ectopic expression of CKI1.The expression of activated ARR 10/12/18 driven by the promoter of ARR18 or CKI1 could rescue the cki1-9embryo sac defects and obtain fertile homozygous cki1-9 plants,indicating that ARR10/12/18 located downstream of CKI1-AHP to regulate cell fate in the embryo sac.To further explore the molecular elements downstream of the CKI1-AHP2/3/5-ARR10/12/18 pathway,the CRISPR/Cas9 technology was used to obtain homozygous mutants of arr10-6 12-3 18 cr and cki1 cr,both of which showed similar complete seed abortion phenotype.Ovaries of wild type,arr10-6 12-3 18 cr,and cki1 cr at the 12 th flower stage were harvested for RNA-seq analysis to identify the differentially expressed genes(DEGs)in the ovaries of both mutants by comparing to wild type.It was found that a large number of genes were down-regulated in both arr10-6 12-3 18 cr and cki1 cr,which further demonstrated that CKI1 and ARR10/12/18 were in the same signaling pathway to regulate female gametophyte development.GO terms analysis of the down-regulated genes showed that a large number of genes encode secreted polypeptides and proteins involved in cell wall metabolism.The expression levels of some down-regulated genes were further analyzed by RT-q PCR and expression pattern analysis,and the results were consistent with those of the RNA-seq.Taken together,this study revealed that ARR10/12/18 function downstream of CKI1-AHP2/3/5 to regulate cell fate specification during embryo sac development.