| Rice is an important food.To a great extent,most of studies on rice were focused on the molecular mechanisms of panicle and tiller development,which greatly contribute to the yield.Root,as an important organ,plays important roles in anchoring plants,absorbing and transporting water,nutrients.Although a few genes have been identified and characterized during root system development,the initiation and growth mechanism of root remains elusive in rice.Rice root system includes primary root(PR),lateral roots(LR)and crown roots(CR).These three type roots display similar structure and function at mature stage,while they initiate from different type of cells,respectively.The molecular mechanism and gene expression reprogramming during the three type roots initiation are not known.In this study,we elaborated the characters of transcriptome and cytosine methylome dynamics of root initiation stages by RNA-seq and Bisulfite-seq.We also investigated the molecular mechanism of WOXI1 and OsPIW1 in regulating crown root development.The major results are shown as follow:1.By utilizing RNA-seq and BS-seq technologies coupled with laser microdissection,we analyzed the transcriptomes and DNA methylomes of different stages of three distinct root types(24 samples in total).The sample generated from different cells show notably diversity in transcriptome reprogramming.Convergence tendency was observed during crown and lateral root maturing,except for embryonic root.In addition,plenty of genes were up-regulated during crown and lateral root initiation,while the number of expressed genes in embryonic root decreased.Cluster analysis indicated that hormone responsive genes(PIN,IAA,RR)and transcription factor genes(AP2,MADS,MYB)were spatiotemporally expressed,which suggesting functionally differentiation of these genes during roots initiation process.Methylomes revealed drastic CHH(H=A,T,C)dynamic during three root types initiation.In addition,CHH methylation decreased during crown and lateral roots initiation,but elevated in embryonic root.Compared with the gradually decrease in crown root,a drastic dynamic of genomic CHH methylation was observed in lateral root initials.Further analysis indicated that CHH DMRs were mainly located at flanking regions of gene body.Although no general correlation was observed between dynamics of gene expression and DNA methylation in the genome-wide,the negative correlation of gene expression and CHH methylation were observed in some functional genes.2.In this study,we dissected the molecular mechanisms of WOX11 and OsPIW1 by utilizing RNA-seq and methods of molecular biology,cytobiology,and genetics.According to our RNA-seq data,664(230 down regulated and 434 up regulated)mis-regulated genes were identified in root tips of wox11 mutant,and these differential expressed genes were involved in root development,cytokinin pathway,stress response,and redox metabolism pathway.Among these genes,41%(273/664)contained WOX11 binding motif,which was mainly located in promoter and intron regions.The result implied that these genes were the potential targets of WOX11.Quantitative RT-PCR and in-situ hybridization were used to verify the repeatability of differentially expressed genes in our data.EMSA and Chromatin immunoprecipitation(ChIP)indicated that WOX11 could bind directly to some of the downstream target genes.The work built a hierarchical regulatory model of WOX11 in rice crown root development.OsPIW1 encodes a protein characterized by a transmembrane domain at the N-terminal and C3H2C3 zinc finger domain at the C-terminal.Quantitative RT-PCR and in-situ hybridizations indicated that OsPIW1 were highly expressed in the meristem zone of root tip and crown root initials,and could be induced by exogeneous cytokinin treatment.Root Impaired phenotype including short length,less number,decreased initiation speed,smaller meristem zone of crown root was observed in OsPIW1 RNAi and knock out lines.Edu staining indicated a decline in cell division in OsPIW1 RNAi lines.Transcriptomes of OsPIW1 RNAi lines showed that genes involved in cytokinin,cell wall metabolism,and redox were influenced by OsPIWl.Collectively,these results suggested that OsPIWl might play a role in maintaining meristem cell division ability through cytokinin pathway.These studies could help to understand transcriptome and methylome reprogramming during root initiation and regulation network of crown root development.It provides theoretical and molecular basis for the subsequent studies on plant organ development... |
