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In plants, leaves and flowers originate from the shoot apical meristem. In an indeterminate shoot apical meristem, stem cells persist for the life of the plant. In a determinate meristem, a certain number of organs are produced before the meristem is terminated; this characterizes the floral meristem derived from the shoot apical meristem. In Arabidopsis, stem cell identity is sustained by expression of the gene WUSCHEL. Expression of WUSCHEL can be terminated by the zinc finger protein KNUCKLES (KNU), with the result that stem cell identity is inactivated. KNU expression is induced by the floral homeotic protein AGAMOUS (AG), but that induction process requires ~2 days and invokes modification of histones resident at the KNU locus. Here, we show that the 2-day time lag is a consequence of a regulated molecular mechanism and that this mechanism can be embedded in a synthetic regulatory system to invoke a similar time lag.
For transgenic Arabidopsis plants, we accelerated or inhibited cell cycles with pharmacological agents and studied the resulting KNU expression in response to AG induction. We used chromatin immunoprecipitation to study the presence of Polycomb proteins on the KNU locus at specific times during flower development. We used insertional mutagenesis to alter the function of the Polycomb response element (PRE) and analyzed the response from a heterologous promoter in Arabidopsis cell cultures. We constructed a synthetic mimic in Arabidopsis floral buds of the AG function by using the DNA binding domain of the lactose operon repressor (lacI) with its cognate binding sites. To test the logic that the delay in downstream gene induction was caused by the need to evict Polycomb group (PcG) proteins from their residence, we simulated the competition between PcG proteins and DNA binding proteins by using lacI, designed transcription activator–like effector DNA binding proteins, and synthesized promoters in Arabidopsis cell lines.
AG induces KNU with a time delay regulated by epigenetic modification. In wild-type plants, KNU expression begins in the center of the floral meristem and follows cell cycle progression. The binding sites for AG in the KNU upstream region are located within the PRE sequences required for the repressive histone modification. Binding of AG displaces PcG proteins, leading to the failure to maintain the repressive histone methylation. The combination of lacI operator sequences with a chimeric protein that contained the lacI DNA binding domain but lacked the activation domain was able to mimic the AG activity in Arabidopsis floral buds. We also reconstituted the cell division–dependent delayed-induction circuit in cell lines.
Our results indicate that flower development in Arabidopsis employs cell division to provide stem cells with a window of opportunity to change fate. The competition we observed between repressive PcG proteins and an activating transcription factor may reflect a general mechanism. The logic of the molecular circuit we have uncovered here may impose timing control on diverse growth and differentiation pathways in plants and animals.
A Matter of Timing
Plants flower only when their developmental programs give the go-ahead; otherwise floral genes remain repressed. Sun et al. (10.1126/science.1248559; see the Perspective by Zhang) analyzed the regulatory program that controls expression of the transcription factor KNUCKLES (KNU), which is required in the control of floral genes. KNU expression was silenced by the presence of Polycomb group (PcG) proteins. The floral homeotic protein AGAMOUS competed for control of KNU and activated its expression, but with a 2-day lag time. Thus, eviction of PcG by activating DNA binding proteins can insert a lag time before a switch in gene expression takes place.
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.