Seminars

1) In aerial part of plants, continuous organ initiation and outgrowth relies on the proliferation and differentiation of stem cells maintained by the CLAVATA(CLV)-WUSCHEL (WUS) negative feedback loop. In meristems of initial stage flower buds, CLV-WUS feedback loop continues to function spatially to maintain stem cells which are to be consumed in floral organ formation. At floral stage 6, the floral homeotic gene AGAMOUS (AG) induces the repressive zinc finger protein KNUCKLES (KNU), which subsequently terminates WUS and stem cell activity to ensure the proper reproductive development. However, in early floral stages, how the spatial maintenance and timed termination of stem cells are properly coordinated remains a mystery.
Here, we show that the key genes in the temporal regulation of floral stem cells, as AG and KNU, are both ectopically expressed in clv3-2, a loss-of-function mutant. Further, through RNA-seq analysis, we notice that CLV3 represses a set of floral specific genes that are expressed in differentiating cells. These genes include AG, KNU, and B-class genes APELTALA3 (AP3) and PISTILLATA (PI), all of which could also be epigenetically regulated by Polycomb-group (PcG) proteins. By ChIP analysis, we find that the H3K27me3 level on AG and KNU chromatin both decreases in clv3-2 mutant flowers. Therefore, our data suggest that CLV3-mediated repression may prevent premature differentiation of stem cells through PcG-mediated epigenetic mechanism, laying a new layer of regulatory network for meristem maintenance.
In addition, we find that mutants of PcG components are resistant to over-expressed CLV3 and fertile stamens and carpels are still observed. This indicates functional CLV signaling requires PcG activity. To further analyze the underling mechanism, we are working on the phosphorylation of PcG proteins via different approaches.

2) DNA methylation performs important functions in gene silencing, genome stability, genomic imprinting, and other developmental aspects. DNA methylation patterns are found to be established by the combined as well as concerted actions of DNA methylation and demethylation. In model plant Arabidopsis, DNA glycosylase/ lyases, like ROS1, DME, DML2 and DML3, are proven to be DNA demethylases which are involved in active DNA demethylation and anti-silencing processes. It has been discovered that DNA glycosylase/ lyases, ZDP and APE1L are involved in removing methylated cytonsine bases, and thus generate abasic sites on DNA strands. Several enzymes, presumably DNA polymerases and ligases, may act downstream of such DNA glycosylase/ lyases to add an unmethylated cytosine to the abasic site and repair the lesion. In addition, a few proteins, such as IDM1, IDM2, MBD7, etc., are implicated with binding on methylated DNA and acetylating histone H3 in order to create a favorable chromatin environment for ROS1 actions. However, unlike RNA-directed DNA methylation (RdDM) which has been well studied, ROS1-mediated DNA demethylation process is as yet poorly understood, and the counterpart proteins which play similar roles as the protein factors in RdDM are largely not identified. To address this question, we performed extensive genetic screening for novel proteins which probably take part in DNA demethylation. Our screening has led to the discovery of two proteins which were thought to be associated with chromatin decondesation and dsRNA binding. Massive efforts to unravel the molecular details of both proteins’ involvement in the ROS1-mediated DNA demethylation process are currently underway.