similar small RNA binding specificities and that they both have ‘‘slicer’’ activity. A previous study also documented that AGO10 antagonizes AGO1 function. ago10 mutations partially suppressed the leaf margin serration phenotype of the hypomorphic ago1-27 allele [27]. Moreover, defects in transgene PTGS and miRNA- mediated gene silencing in the hypomorphic ago1 mutant were also restored by ago10 mutations [27]. The elevated AGO1 protein levels in the double mutants reveal that AGO10 is a negative regulator of AGO1 at the translational level. By showing that AGO10 binds miR168, which targets AGO1, we provide the molecular evidence for the direct regulation of AGO1 by AGO10 through miR168. Our studies support overlapping roles for AGO10 and AGO1 in the temporal regulation of floral stem cells but also reveal that the two proteins do so with both similar and different molecular mechanisms (Figure 6). It is well established that AGO1 mediates the functions of miR172 and miR165/166 in different aspects of plant development. It is likely that AGO1 promotes floral stem cell termination by repressing AP2 and HD-Zip expression. Consistent with this, de-repression of AP2 or HD-Zip genes compromises floral stem cell termination. We show here that AGO10 also associates with these two miRNAs in vivo. Genetic evidence suggests that AGO10 mediates the functions of miR172 in both stamen identity specification and floral determinacy. Therefore, AGO10 reinforces the functions of AGO1 in the regulation of AP2. Perhaps AGO1 alone is not sufficient to mediate the functions of Figure 6. A model of AGO1 and AGO10 in floral stem cell regulation. miR172 promotes floral stem cell termination by repressing the expression of AP2, which promotes WUS expression. Both AGO1 and AGO10 are associated with miR172 in vivo and genetic evidence supports a role of AGO10 in mediating the activities of miR172. miR165/166-mediated repression of PHB and PHV is also necessary for floral stem cell termination. AGO1 and AGO10 exert opposite effects on miR165/166. While AGO1 mediates the activities of miR165/166, AGO10 represses the expression of miR165/166. The relationship between PHB/PHV and WUS is unknown. Interactions that are potentially indirect are represented by dotted lines. doi:10.1371/journal.pgen.1001358.g006 AGO10 and Floral Stem Cell Termination miR172 in floral meristems such that AGO10, which is specifically expressed in certain cells in the floral meristem, is necessary to act upon the same miRNA. Intriguingly, AGO1 and AGO10 exert opposite effects on miR165/166 accumulation and the expression of the HD-Zip genes. The increased miR165/166 levels and reduced expression of HD-Zip genes in ago10 mutants suggest that AGO10 promotes HD-Zip gene expression by reducing miR165/ 166 levels. How AGO10 does so is currently unknown. Despite the opposite effects on HD-Zip expression, AGO1 and AGO10 both promote floral determinacy. This can be reconciled by the fact that both reduced expression and de-repression of HD-Zip genes compromise floral determinacy. SAM and floral meristems—differences in stem cell maintenance It has not escaped our attention that loss of function of AGO10 results in opposite effects in stem cell regulation between the SAM and the floral meristems. While ago10 mutations lead to premature termination of stem cells in the SAM, they result in prolonged floral stem cell maintenance. A previous study found that over expression of miR165/166 recapitulates the SAM defects of ago10 mutants [49]. In this study, we show that over expression of miR165/166 recapitulates not only the SAM defects but also the floral stem cell defects of ago10 mutants. Therefore, over accumulation of miR165/166 and reduced expression of the HD-Zip genes probably underlie the failure to maintain the SAM and to terminate the floral stem cells. How reduced expression of the HD-Zip genes leads to opposite effects in the two types of meristems is currently unknown. Materials and Methods Plant strains and EMS mutagenesis The plant strains used in this study are all in the Landsberg erecta (Ler) ecotype except for ago1-36 pAGO1::FLAG-AGO1 [20], which is in the Columbia (Col) background. All plants were grown at 23uC under continuous light except for those containing the phb-1d mutation, which were grown at 17uC. zll-1 pZLL::YFP-ZLL [36], clv3-1 [38], wus-1 [2], ap2-2 [50], phb-1d [42], hua1-1 [31], hua2-1 [31], and pnh-1 [25] were all previously characterized. ag-10, ago10-12, ago10-13, ago10-14 and phv-5d were isolated in this study. An EMS mutagenesis screen was carried out in the hua1 hua2 background as described [32]. Single M2 families were screened and two independent mutants, hua1 hua2 ago10-12 and hua1 hua2 ag-10, with bulged gynoecia and ag mutant-like phenotypes, respectively, were identified. The ag-10 mutation was recovered from fertile siblings. The two lines were backcrossed to hua1 hua2 three times to clean up the genetic backgrounds. They were also crossed to either hua1 or hua2 to generate hua1 ago10-12, hua2 ago10-12, hua1 ag-10 and hua2 ag-10 double mutants. The ago10-12 or ag-10 single mutant was obtained by crossing hua1 ago10-12 or hua1 ag-10 to wild type. The ago10-13 allele was isolated in a separate EMS mutagenesis screen in the hua1 ag-10 background as a mutation that enhanced the floral determinacy phenotypes of hua1 ago-10. The mutant was backcrossed to ag-10 three times to obtain the ag-10 ago10-13 double mutant, with which most subsequent analyses were conducted. The ago10-13 single mutant was obtained by crossing ag-10 ago10-13 to Ler. The ago10-14 and phv-5d alleles were isolated in an EMS screen in the ag-10 background as enhancers of its weak determinacy defects. Construction of amiR165/166 The amiR165/166 plasmid contains modified pre-miR165 and pre-miR166, two stem-loop structures placed in tandem and PLoS Genetics | www.plosgenetics.org 11 March 2011 | Volume 7 | Issue 3 | e1001358