Functional Analysis Of Rice Homeobox Gene WOX11 In Shoot Apical Meristem And Root Apical Meristem Development

Embryogenesis in higher plants gives rise to two major meristems: shoot apical meristem(SAM)and root apical meristem(RAM).All aerial organs of a plant such as leaves,stem,axillary buds,and seeds derived from SAM occupy a vast majority of the whole plant and are associated with morphogenesis,photosynthesis and biological /nonbiological threats responses.While the root system derived from RAM plays an important role in plant anchoring and water and nutrients absorbing.Proper establishment and maintenance of SAM and RAM define the development of a whole plant.Thus,it is of vital importance to study the mechanisms involved in SAM and RAM development in most important crops such as rice.The homeobox gene WOX11,previously reported to activate crown root growth.In this thesis,we studied the molecular mechanism of WOX11 in controlling crown root development,and found that WOX11 functioned also as a regulator of SAM development.Histological section assay showed that mutation of WOX11 led to a smaller SAM in size and overexpression of WOX11 resulted in a flatter SAM.Both mutation and overexpression of WOX11 affected shoot development from the juvenile stage to the heading stage and also altered plant sensitivity to brassinosteroids.To identify the mechanisms involved in WOX11 regulation of downstream genes,yeast two hybridization assay was performed and a histone H3K27me2/me3 demethylase JMJ705 were identified and further proved to interact with WOX11 in vitro and in vivo.To identify common target genes that could be activated by WOX11 and JMJ705,genome-wide RNA-sequencing(RNA-seq)was performed in wox11 and jmj705 SAM and their overexpression plants.Data analysis reveals that WOX11 and JMJ705 commonly activate the expression of genes that are enriched for H3K27me3 and are involved in primary metabolism,light-induced plastid biogenesis and hormone signaling in the SAM.We validated up-regulated genes involved in hormone synthesis and signaling,gibberellin biosynthesis and signaling/defense response from the ox WOX11 and ox JMJ705 RNA-seq data with quantitative RT-PCR and found that those genes were activated in ox WOX11 and ox JMJ705 and particularly repressed in mutant SAM.Ch IP assay further showed that WOX11 and JMJ705 protein were enriched on those loci and JMJ705-mediated demethylation of H3K27me3 activated their expression.Deletion of WOX11 reduced JMJ705 binding to those loci with only WOX11 binding site.For those genes with both WOX11 and JMJ705-binding sites,the WOX11 mutation significantly reduced JMJ705 association to the WOX11 binding sites,and in some cases altered JMJ705 enrichment for the JMJ705 binding sites.On the other side,JMJ705 mutation also caused a significant reduction of WOX11 enrichment on some loci with WOX11 binding site alone.To further investigate the mechanism involved in WOX11 regulation of crown root development,we identified a WOX11-interacting partner——ERF3,an auxin-and cytokinin-responsive AP2 transcription factor,which was expressed in crown root initials and during crown root growth.Further studies shows that ERF3 promotes crown root initiation and primary root elongation in rice.In crown root initials,ERF3 directly binds to RR2 and upregulates its expression,resulting in promotion of crown root initiation.In emerging crown roots,WOX11 expression was turned on and its binding to RR2 was enhanced by interaction with ERF3,leading to inhibition of ERF3 function or direct repression of RR2 and promotes crown root elongation.RNA-seq analysis of wox11 mutant found that differentially expressed genes in the mutant were enrichment in stimulus response,implying that WOX11 may be involved in stress processes which were perceived through root system due to the root defects in the mutant.Further studies showed that wox11 mutant displayed a quicker water loss rate than wild type and it was hypersensitive to drought in the field at the heading stage,which may be associated with root hair defects in wox11.Mutation of WOX11 also reduced the response of JA-induced gene expression during the induce process of JA.Besides,overexpression of WOX11 leads to seed abnormality and analysis showed that the expression of AFL family genes were activated in ox WOX11 endosperm at 7 DAP,implying that WOX11 may regulated protein accumulation in seeds through AFL pathway.Both mutation and overexpression of WOX11 affected rice development in multiaspects,suggesting that it is necessary to define the expression of homeobox gene expression in rice.We tried to understand the epigenetic mechanisms involved in WOX11 regulation and found that the suppression of WOX11 in mature organs may be a consequence of hyper H3K27me3 and hyper DNA methylation on the promoter region of WOX11.By searching mutants in which WOX11 expression was activated in mature organs,the DNA methyltransferase DMT706 and the chromatin remodeling factor DDM1 were identified.Genome-wide bisulfite sequencing showed that DNA methylation level,especially CHG and CHH type DNA methylation level in the promoter region of WOX11 locus which contained a MITE transposon and a retrotransposon were reduced in dmt706 and ddm1,implying that DMT706 and DDM1 were responsible for the methylation of those sites and deletion of this region may activate WOX11 expression.