Summary HUA ENHANCER 3 reveals a role for a cyclin-dependent protein kinase in the specification of floral organ identity in Arabidopsis

The shoot apical meristem (SAM) serves as the ultimate source of the above-ground biomass in plants. Cells in the central zone of the SAM are stem cells that maintain the undifferentiated state, while cells in the peripheral zone participate in organ formation and eventually acquire various identities. Cell division in the meristem must be closely integrated with cell fate specification and organ formation. The SAM generates floral meristems after floral transition. A floral meristem, like the SAM, gives rise to lateral organs (sepals, petals and stamens), but unlike the SAM, terminates in a gynoecium. In Arabidopsis, the termination of stem cells in the floral meristem requires AGAMOUS(AG), a MADS-domain transcription factor (Bowman et al., 1989; Yanofsky et al., 1990). AG represses the expression of WUSCHEL (WUS), a stem cell maintenance gene, in a temporal manner in the floral meristem (Laux et al., 1996; Lenhard et al., 2001; Lohmann et al., 2001). AGalso acts in floral organ identity specification. As a class C floral homeotic gene, AG specifies stamen identity together with the class B and SEPALLATA(SEP) genes and carpel identity together with the SEP genes (Coen and Meyerowitz, 1991; Meyerowitz et al., 1991; Pelaz et al., 2000). In addition to AG, several genes that act in the control of stamen and carpel identities and floral determinacy have been identified through sensitized genetic screens. Recessive mutations in HUA1 and HUA2, hua1-1 and hua2-1, respectively, were isolated as enhancers of the weak ag-4 allele (Chen and Meyerowitz, 1999). hua1-1 hua2-1double mutant flowers show partial carpel-to-sepal and occasional stamen-topetal transformation, phenotypes that are indicative of partial loss of class C activity (Chen et al., 2002; Chen and Meyerowitz, 1999; Western et al., 2002). In a genetic screen in the hua1-1 hua2-1background, recessive mutations in HUA ENHANCER1(HEN1), HEN2, HEN4 and PAUSED (PSD), were found to enhance the weak floral homeotic phenotypes of hua1-1 hua2-1flowers such that stamens are transformed to petals and carpels are partially transformed to sepals (Chen et al., 2002; Cheng et al., 2003; Li and Chen, 2003; Western et al., 2002). The HUA and HENgenes all appear to directly or indirectly promote the expression of AG – in hua1-1 hua2-1 henmutants, AG RNA or protein is of lower abundance than in wild type or in hua1-1 hua2-1(Cheng et al., 2003; Li and Chen, 2003). HUA1 and HEN4 physically interact in the nucleus, and together with HUA2 and HEN2 promote AG expression by preventing the production of alternative transcripts containing intron sequences (Cheng et al., 2003). HEN1 is required for microRNA accumulation and may modulate AG expression or activity through regulation of APETALA2, a class A gene (Park et al., 2002; Chen, 2004)). In this study, we show that HEN3, which encodes an E-type CDK, also acts in the AG pathway. CDKs are a family of serine/threonine protein kinases that control cell cycle progression, and/or coordinate cell cycle progression with transcription regulation. For a fully active state, they require both the association with regulatory subunits, cyclins and the phosphorylation of a conserved threonine residue by CDK-activating kinases (CAKs) (reviewed by Kobor and Greenblatt, 2002; Morgan, 1997). Among the nine known CDKs (CDK1-CDK9) in vertebrates, Cdc2 (CDK1) and CDK2 carry out central cell cycle functions (reviewed by Morgan, 1997). CDK4 and CDK6 are thought to integrate developmental signals and environmental cues into In plants, organs are generated post-embryonically from highly organized structures known as meristems. Cell division in the meristem is closely integrated with cell fate specification and organ formation. The presence of multiple cyclin-dependent kinases (CDKs) and their partner cyclins in plants and other multicellular organisms probably reflects the complexity of cell cycle regulation within developmental contexts. The Arabidopsis genome encodes at least eight CDKs and 30 cyclins. However, no mutants in any CDKs have been reported, and the function of the great majority of these genes in plant development is unknown. We show that HUA ENHANCER3 (HEN3), which encodes CDKE, a homolog of mammalian CDK8, is required for the specification of stamen and carpel identities and for the proper termination of stem cells in the floral meristem. Therefore, CDK8 plays a role in cell differentiation in a multicellular organism.