Regulation Of Early Flower Development By CLV Signaling Pathway In Arabidopsis

Dynamic balance for the maintenance and differentiation of floral meristem is the key for proper flower development.During early flower development of Arabidopsis,the CLAVATA(CLV)-WUSHEL(WUS)signaling pathway maintains the floral meristem homeostasis and ensures the production of normal floral organs.While at certain stage of flower development,floral meristem is terminated by AGAMOUS(AG)-KNUCKLES(KNU)-WUSHEL(WUS)mediated regulatory pathway to ensure the proper formation of carpels.In addition,the chromatin of AG,KNU and WUS genes are all modified by H3K27me3,a repressive histone mark,which is deposited and maintained by Polycomb Group Complex(Pc G).However,it is unclear whether CLV-WUS signaling pathway crosstalks with AG-KNU-WUS regulatory pathway to regulate early flower development,also whether Pc G-mediated epigenetic regulation is involved in it is unknown.For this purpose,we have carried the research as below.1.First,we analyzed the expression of AG,KNU and WUS in the background of clv3-2,which is the null mutant of the stem cell marker gene CLV3.The results showed that AG,KNU and WUS expression are all ectopic in clv3-2,indicating that CLV signaling pathway may crosstalk with AG-KNU-WUS regulatory pathway.2.We used clf-28 loss-of-function mutant,tfl2-1 loss-of-function mutant and35S:GFP-FIE co-suppression line for further analysis,and CURLY LEAF(CLF)、TERMINAL FLOWER2(TFL2)and FERTILIZATION INDEPENDENT ENDOSPERM(FIE)are key factors of Pc G.We analyzed KNU-GUS expression in ag-1 clv3-2 p KNU:KNU-GUS and ag-1 tfl2-1 p KNU:KNU-GUS.KNU expression were all ectopic in the two lines.Through chromatin immunocoprecipitation(Ch IP)analysis,we detected lower H3K27me3 enrichment on KNU locus in clv3-2background compared to wild type.It hints that CLV3 can regulate KNU expression through Pc G-mediated epigenetic pathway.3.We noticed that compared to wild type seedlings,clf-28,tfl2-1 and35S:GFP-FIE has reduced sensitivity to exogenous CLV3 peptide treatments.Further,in clf-28 35S:CLV3 plants,clf-28 mutation could rescue the terminated SAM phenotype of 35S:CLV3.This indicates that CLV signaling pathway requires Pc G activity.4.The phosphorylation level of glycogen synthase kinase-3(GSK3)-like kinases,SHAGGY-like kinases 11/12(SK11/SK12)can respond to CLV signaling pathway according to our Phospho-proteomics data.Further,through Y2 H and Bi FC assays,we found SK11/SK12 can physically interact with CLF protein.In addition,sk11 sk12 double mutant showed a higher sensitivity to CLV3 peptide treatment than wild type seedlings.Meanwhile,the H3K27me3 level on WUS locus increased in sk11 sk12 double mutant compared to wild type.Thus we propose that the interaction between SK11/SK12 and CLF may regulate the function of CLF protein which is necessary for the repression of WUS by CLV signaling cascades.5.Our RNA-seq results show that CLV signaling pathway has a broad effect on the expression of many floral specific genes.And these genes are largely modified by the repressive mark H3K27me3.In conclusion,we propose that CLV signaling pathway maintains stem cell proliferation in early stage flower development by repressing floral specific genes through Pc G-mediated epigenetic machinery,thus ensuring proper flower development.