Author Summary Stem cells have the capacity to self renew while producing daughter cells that undergo differentiation. While some stem cells remain as stem cells throughout the life of an organism, others are programmed to terminate within developmental contexts. It is presumed that stem cell termination is simply the differentiation of stem cells into a specific cell type(s). Using floral stem cells as a model, we show that the temporally regulated termination of floral stem cells is genetically separable from stem cell differentiation, and thus we reveal the presence of a temporal program of stem cell regulation. We show that two microRNAs, miR172 and miR165/166, and two argonaute family proteins, ARGONAUTE1 (AGO1) and AGO10, regulate the termination of floral stem cells. We establish the homeodomain-leucine zipper (DH-Zip) genes, targets of miR165/166, as crucial factors in floral stem cell termination. While AGO1 is the major miRNA effector, the molecular function of AGO10 has been elusive. Here we demonstrate that AGO10 is also a miRNA effector in that AGO10 is associated with miRNAs in vivo and exhibits ‘‘slicer’’ activity in vitro. Despite the similar biochemical activities, AGO1 and AGO10 promote floral stem cell termination by exerting opposite effects on miR165/166. (OC) located underneath the stem cells [2]. The OC signals to the overlying stem cells to maintain their ‘‘stemness’’. In loss-of- function wus mutants, the stem cells in both the SAM and the floral meristems terminate precociously. For example, flowers of the wus-1 mutant lack a full complement of floral organs and terminate in a central stamen. WUS expression commences as soon as the floral meristem is formed (at stage 1; stages according to [3]). By stage 6, when the carpel primordia are formed, WUS expression is shut off [4], and this temporally precise repression of WUS expression underlies the proper termination of floral stem cells. The temporal regulation of WUS expression requires at least two transcription factors with opposing activities, AGAMOUS (AG) and APETALA2 (AP2), which are best known for their roles in the specification of reproductive and perianth organ identities, respectively, in flower development (reviewed in [5]). The MADS- domain protein AG promotes floral meristem termination by repressing WUS expression at stage 6 [6,7]. In an ag mutant, such as ag-1, WUS expression persists throughout flower development [6,7] and the floral meristems lose determinacy to result in a flowers-within-flower phenotype [8]. Flowers of the ag-1 wus-1 double mutant resemble wus-1 flowers, indicating that AG causes floral stem cell termination by repressing WUS expression [2]. While AG acts to terminate floral stem cells, AP2, an AP2- domain containing transcription factor gene [9] regulated by the microRNA (miRNA) miR172 [10,11], promotes stem cell maintenance. In plants that express the miR172-resistant AP2 cDNA AP2m3 [11] from the AP2 promoter, flowers have numerous stamens that are continuously produced by indetermi- nate floral meristems [12]. WUS expression is prolonged in AP2p::AP2m3 flowers, and the prolonged WUS expression underlies the indeterminate phenotype because wus-1 is completely epistatic to AP2m3 [12]. AP2 is known to repress the transcription of AG in the outer two floral whorls [13]. De-repressed AP2 in AP2m3 plants causes a moderate reduction in AG mRNA levels in the center of the floral meristem and this partially underlies the stem cell termination defects of AP2m3 flowers [12]. De-repressed AP2 also compromises floral stem cell termination in an AG- AGO10 and Floral Stem Cell Termination independent manner, which is reflected by the stronger pheno- types of AP2m3 ag-1 relative to ag-1 [12]. Small RNAs of 21–24 nucleotides (nt) are sequence-specific guides in RNA silencing at the transcriptional and posttranscrip- tional levels in plants (reviewed in [14]). ARGONAUTE (AGO) proteins associate with small RNAs and serve as effectors in RNA silencing (reviewed in [15]). Different AGO proteins associate with, and mediate the functions of, distinct types of small RNAs. Among the ten AGO proteins in Arabidopsis, AGO1, 2, 4, 5, 6, 7, and 9 have been shown to bind specific sets of endogenous small RNAs [16–19]. AGO1, the founding member of the family, binds most miRNAs, trans-acting siRNAs (ta-siRNAs), and transgene siRNAs and exhibits ‘‘slicer’’ activity, an endonucleolytic activity that precisely cleaves target mRNAs [20]. ZWILLE (ZLL)/ PINHEAD (PNH), which will be hereafter referred to as AGO10 (unless specific alleles or constructs are referred to), is the most related to AGO1 among the ten Arabidopsis AGO proteins [21]. While AGO1 is ubiquitously expressed [22], AGO10 expression is restricted to meristems, the vasculature, and adaxial sides of lateral organ primordia [23,24]. AGO10 was initially identified as a factor required for SAM maintenance [23–25]. Null mutations in AGO10 lead to the absence of a SAM with incomplete penetrance. Examination of several miRNA target genes at the mRNA and protein levels in ago10 mutants implicated AGO10 in miRNA- mediated translational repression [26,27]. However, the lack of knowledge of the small RNA binding and enzymatic properties of AGO10 has hindered the understanding of the molecular mechanisms of action of AGO10 in development and in RNA silencing, especially in relationship to AGO1. In our previous studies, we found that four genes with potential roles in RNA metabolism, HUA1, HUA2, HUA ENHANCER2 (HEN2), and HEN4, promote AG expression [28]. Loss-of-function mutations in any one gene do not sufficiently compromise AG expression or flower development, but double or triple mutants have reduced AG expression and phenotypes indicative of partial loss-of-function of AG. In a genetic screen in the hua1 hua2 background and in two separate genetic screens in backgrounds compromised for AG expression or function, we isolated ago10 mutations with defects in floral stem cell termination. We also establish a role of AGO1 in floral stem cell termination. Genetic evidence suggests that AGO10 acts to terminate floral stem cells in part by facilitating miR172-mediated repression of AP2. We demonstrate that the HD-Zip genes PHABULOSA (PHB) and PHAVOLUTA (PHV), targets of miR165/166 [29,30], play a previously unknown yet crucial role in the proper termination of floral stem cells. Like AGO1, AGO10 associates with miR172 and miR165/166 in vivo and exhibits slicing activity in vitro. Intriguingly, AGO1 and AGO10 exert opposite effects on miR165/166 in vivo, yet both contribute to the termination of floral stem cells. Results AGO10 promotes floral determinacy To identify genes with functions similar to those of AG in promoting stamen/carpel identities and terminating floral stem cells, we performed ethyl methanesulfonate (EMS) mutagenesis to screen for enhancers of the hua1-1 hua2-1 double mutant, which will be hereafter referred to as hua1 hua2. hua1 hua2 flowers are largely normal except that the gynoecia are shorter and more enlarged at the apical end than wild type (Figure 1A). The floral phenotypes of hua1 hua2 are sensitive to the dosage of functional AG. Combination of ag-1/+ or ag-4 with hua1 hua2 drastically enhances the homeotic and floral determinacy defects of hua1 hua2 PLoS Genetics | www.plosgenetics.org 2 March 2011 | Volume 7 | Issue 3 | e1001358