MiR164-CUC2 Regulatory Module And FveFDM1 Regulate Organ Development In Woodland Strawberry

Strawberry,a member of the Rosaceae family,possesses unique features during the development of leaves of floral organs.Small RNAs are a class of 18-30 nt noncoding RNA and play important roles in plant growth and development,as well as biotic and abiotic stress responses.Micro RNAs（mi RNAs）act through complement pairing to mediate the cleavage of target m RNAs and inhibition of translation.Si RNAs often affects the establishment of DNA methylation by the RNA directed DNA methylation（Rd DM）pathway.DNA methylation at cytosine is one epigenetic mark regulating genome stability,transposon silencing,and gene expression.So far,little is known about the molecular mechanism of mi RNA and si RNA mediated DNA methylation regulate the growth and development of diploid woodland strawberry.In this study,EMS mutagenized mutants were used as materials to analyze the regulatory mechanism of Fvemi R164a and Rd DM machinery component Fve FDM1 on the development of leaves,flowers and fruits of woodland strawberry.The main results are as follows:1.Conserved and novel roles of mi R164-CUC2 regulatory module in specifying leaf and floral organ morphology in strawberry.des mutants used in this study were obtained in the early stage.Compared with wild-type,both the simple and the three leaflets of des are more deeply serrated and severely wrinkled.However,both the petal and sepal margins become serrated compared with the smooth margins in wild-type.Moreover,the carpels of des are seriously disrupted.In wild-type,each carpel has one upwardly growing style and one ovule inside the ovary.By contrast,some of the des carpels have two or three styles.Consequently,the des flowers are nearly sterile.Gene isolation of the des mutant was performed and this mi R164a is the primary candidate gene for des.The 21-nt mi R164a sequence is located within the first exon,and the SNP（G-A）occurred in its 19^th nucleotide.Overexpressing Fve MIR164A rescued the phenotypes of des/fvemir164a except the petal serrations.A most of target genes are all greatly reduced due to overexpression of Fve MIR164A.Moreover,Fve MIR164A rather than Fve MIR164A^m cleaved the target genes by the vitro GFP fluorescence cleavage assay.In addition to the fvemir164a mutant,we identified two extra EMS mutants,both of them produced leaves with smooth margins.Furthermore,we identified two allelic mutants of Fve CUC2a,one target of Fvemi R164a,which developed leaves with smooth margins and fused leaflets.Phenotypes of the double mutant fvemir164a fvecuc2a indicated that the two genes act linearly in leaf and carpel development,but synergistically in the development of other floral organs and inflorescence architecture.Moreover,Northern blot and small RNA sequencing was performed to examine the presence of mature mi RNAs in the tobacco leaves.Overexpressing Fve MIR164A resulted in the accumulation of 21-nt mature mi R164a,while Fve MIR164A^m overexpression gave rise to a fragment only 20-nt.2.Fve FDM1,an Rd DM pathway component,regulates plant statue and organ size via CHH methylation in woodland strawberryWe screened the EMS-mutagenized M₂ population generated from the F.vesca variety Yellow Wonder（YW）and found one mutant named as ros（reduced organ size）,which was dwarf and developed smaller leaves,flowers,and fruit than wild type.Gene isolation of the ros mutant was performed and this Fve FDM1 is the primary candidate gene for ros.According to the images obtained by differential interference contrast（DIC）,the cell number of the leaf,flower and fruit respectively was significantly decreased in the same area of ros relative to WT.Three lines which are create by the CRISPR/Cas9 technology also developed smaller leaves.The cell number in the same area of leaf epidermis at the adaxial side was significantly reduced in the fvefdm1^CR lines.To assess the functions of Fve FDM1,its coding sequence driven by the constitutive promoter 35S was first stably transformed into the fdm1-1 fdm2-1 double mutant in Arabidopsis.Fve FDM1overexpression restored the DNA methylation levels in fdm1-1 fdm2-1,suggesting that Fve FDM1 shares conserved functions as its Arabidopsis homologs in the DNA methylation regulation via the Rd DM pathway.In addition,two other FDM1 homologs,IDN2 and FDM2 were identified in the F.vesca genome.Protein interaction assays indicate Fve IDN2could interact with itself and Fve FDM1.Whole genome bisulfite sequencing and small RNA-seq indicate that CHH methylation was greatly reduced at any loci of all the chromosomes in fvefdm1 and the downregulated si RNA clusters were more closely associated with CHH hypo-DMRs.Furthermore,RNA-seq was performed in shoot tips,flower buds at stages 1-5,and young fruit at seven days post pollination of fvefdm1 and WT.There are 162 commonly upregulated genes in these tissues,among which the top 2 enriched GO terms are cell cycle and cytoskeleton organization.Some genes regulating organ size,key genes controlling cell cycle and GA signal pathway genes lead to the obstruction of cell division and inhibit the development of organs.In conclusion,This work demonstrates the conserved and novel roles of the mi R164-CUC2 module in leaf and flower development in different plant species,and reveals that the19^th nucleotide of Fvemi R164a is important for its processing;Furthermore,the work also revealed that the Fve FDM1,a Rd DM component regulates the plant organ size via CHH methylation in strawberry.