Developmental regulation of the floral meristem ensures that plants of the same species have similarly sized flowers with a fixed number of floral organs. The maintenance of stem cells in the floral meristem is terminated after the production of a fixed number of floral organ primordia. Precise repression of the Arabidopsis thaliana homeobox gene WUSCHEL (WUS) by the floral homeotic protein AGAMOUS (AG) plays a major part in this process. Here we show that KNUCKLES (KNU) mediates the repression of WUS in floral meristem determinacy control. AG directly induces the transcription of KNU, which encodes a C2H2-type zinc finger protein with a conserved transcriptional repression motif. In turn, KNU represses WUS transcription to abolish stem cell activity. We also show that the timing of KNU induction is key in balancing proliferation and differentiation in flower development. Delayed KNU expression results in an indeterminate meristem, whereas ectopic KNU expression prematurely terminates the floral meristem. Furthermore, KNU induction by AG is preceded by changes in repressive histone modification at the KNU locus, which occurs in an AG-dependent manner. This study provides a mechanistic link between transcriptional feedback and epigenetic regulation in plant stem cell proliferation.[Keywords: Arabidopsis thaliana; flower development; homeotic control; MADS protein; AGAMOUS; WUSCHEL; stem cell] Supplemental material is available at http://www.genesdev.org.

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Plant floral stem cells divide a limited number of times before they stop and terminally differentiate, but the mechanisms that control this timing remain unclear. The precise temporal induction of the Arabidopsis zinc finger repressor KNUCKLES (KNU) is essential for the coordinated growth and differentiation of floral stem cells. We identify an epigenetic mechanism in which the floral homeotic protein AGAMOUS (AG) induces KNU at ~2 days of delay. AG binding sites colocalize with a Polycomb response element in the KNU upstream region. AG binding to the KNU promoter causes the eviction of the Polycomb group proteins from the locus, leading to cell division-dependent induction. These analyses demonstrate that floral stem cells measure developmental timing by a division-dependent epigenetic timer triggered by Polycomb eviction.

SummaryOrchids are members of Orchidaceae, one of the largest families in the flowering plants. Among the angiosperms, orchids are unique in their floral patterning, particularly in floral structures and organ identity. The ABCDE model was proposed as a general model to explain flower development in diverse plant groups, however the extent to which this model is applicable to orchids is still unknown. To investigate the regulatory mechanisms underlying orchid flower development, we isolated candidates for A, B, C, D and E function genes from Dendrobium crumenatum. These include AP2-, PI/GLO-, AP3/DEF-, AG-and SEP-like genes. The expression profiles of these genes exhibited different patterns from their Arabidopsis orthologs in floral patterning. Functional studies showed that DcOPI and DcOAG1 could replace the functions of PI and AG in Arabidopsis, respectively. By using chimeric repressor silencing technology, DcOAP3A was found to be another putative B function gene. Yeast two-hybrid analysis demonstrated that DcOAP3A/B and DcOPI could form heterodimers. These heterodimers could further interact with DcOSEP to form higher protein complexes, similar to their orthologs in eudicots. Our findings suggested that there is partial conservation in the B and C function genes between Arabidopsis and orchid. However, gene duplication might have led to the divergence in gene expression and regulation, possibly followed by functional divergence, resulting in the unique floral ontogeny in orchids.