hua2 hen6 were rescued by AGO10 genomic DNA and the fully rescued plants appeared morphologically identical to hua1 hua2 (Figure 1I). Therefore, hen6 was renamed ago10-12. While many previously isolated ago10 alleles show SAM defects with incomplete penetrance, the ago10-12 single mutant had no obvious SAM defects, suggesting that the L674F mutation does not completely compromise AGO10 function. hen7 strongly enhanced hua1 hua2 such that hua1 hua2 hen7 flowers resembled those of ag mutants. Stamens were transformed to petals and the floral meristems failed to terminate (Figure 1A,1C). Rough mapping showed that hen7 was linked to AG, and sequencing of AG revealed a G-to-A mutation resulting in an glutamate (E)-to- lysine (K) substitution at the 144th amino acid (Figure S1A). To show that AG could rescue the hen7 mutant phenotypes, we utilized the fertile hua1 hen7 double mutant. hua1 hen7 flowers had no organ identity defects, but the gynoecia were bulged with secondary floral organs inside the primary carpels (Figure 1F; Table S1). The floral determinacy defects of hua1 hen7 flowers could be rescued by a pAG::AG-GFP construct (Figure 1F). Therefore, hen7 is an ag allele, which we named ag-10. The ag-10 single mutant flowers had a normal complement of floral organs as in wild type (Figure 2A); as such, it is the weakest ag allele known to date. Most siliques on an ag- 10 plant, however, can be distinguished from wild type by their curvature (Figure 2D). A small proportion of siliques were slightly bulged with additional organs inside (Figure 2D; Table S1). This contrasted with hua1 ag-10 plants, in which all siliques were bulged with internal floral organs inside (Table S1). Therefore, ag-10 was mildly defective in floral determinacy and this defect was drastically enhanced by hua1. We performed another EMS screen in the hua1 ag-10 background to identify mutants with enhanced floral determinacy defects. One mutant had a flowers-within-flower phenotype reminiscent of ag mutants. After removal of the hua1 mutation by crossing, the double mutant of ag-10 and the enhancer mutation still exhibited strong floral determinacy defects; all siliques on the double mutant were severely bulged and ectopic floral organs were present inside the primary carpels (Figure 2B,2E; Table S1). Flowers of the double mutant did not exhibit any homeotic transformation and were fertile. We noticed that seedlings of the double mutant exhibited SAM defects at a low penetrance similar to ago10 alleles. This, together with the mapping of the mutation to the top of chromosome 5, led us to suspect that the enhancer mutation was in AGO10. Sequencing AGO10 from the double mutant identified a G-to-A mutation that introduced a premature stop codon close to the end of the second exon of AGO10 (Figure S1A). Introduction of a pAGO10::AGO10- FLAG construct into the double mutant fully rescued the floral determinacy defect (Figure 2C). Therefore, we named this mutation ago10-13. From longitudinal sections, it was clear that the floral meristems persisted well beyond stage 6 in all ag-10 ago10-13 flowers but not in most ag-10 flowers (Figure 2G,2H), suggesting that AGO10 is required for floral stem cell termination. Like other ago10 alleles, the gynoecia and siliques of the ago10-13 single mutant were shorter and wider than wild type (Figure S3A,S3B). In an EMS screen in the ag-10 background, we identified yet another ago10 allele, ago10-14, which harbored a G-to-A mutation that changes the 731th amino acid from aspartic acid (D) to asparagines (N) (Figure S1A). Flowers of ag-10 ago10-14 resembled those of ag-10 ago10-13 in that the gynoecia were bulged (data not shown). The following genetic and molecular studies were performed mainly with ago10-12 and ago10-13 alleles. Given that ago10-12 enhanced hua1 hua2 and both ago10-13 and ago10-14 enhanced ag-10 in terms of floral determinacy, we AGO10 and Floral Stem Cell Termination conclude that AGO10 promotes floral stem cell termination. Consistent with such a role, the YFP-ZLL fusion protein from the functional pZLL::YFP-ZLL transgene [36] was found in the center of stages 5-6 floral meristems by immunolocalization (Figure 3A). By stage 7, YFP-ZLL was found on the adaxial side of carpel primordia (Figure 3B). AGO10 terminates floral stem cells through repression of WUS expression To investigate how AGO10 regulates floral stem cells, we examined the genetic interactions between ago10 alleles and mutations in key stem cell regulators. In wus-1, the floral meristems are terminated prematurely, resulting in incomplete flowers with four sepals, four petals, and typically a single stamen [2] (Figure S3E). hua1 hua2 ago10-12 wus-1 flowers had identical phenotypes to those of wus-1 in terms of floral determinacy (Figure 4A), indicating that wus-1 was epistatic to hua1 hua2 ago10-12. Unlike wus-1 (Figure S3E), the single stamen in hua1 hua2 ago10-12 wus-1 was petaloid as in hua1 hua2 ago10-12 (Figure 4A), indicating that the homeotic transformation of stamens into petaloid stamens in hua1 hua2 ago10-12 was independent of WUS. Similarly, wus-1 was epistatic to ag-10 ago10-13 in terms of floral determinacy (data not shown). Therefore, AGO10 acts through WUS to regulate floral meristem activity. This conclusion was also supported by the prolonged expression of WUS in floral meristems caused by ago10 mutations. Using in situ hybridization, we found that WUS expression in hua1 hua2 ago10-12 flowers persisted well beyond floral stage 6, when WUS expression was terminated in hua1 hua2 flowers (Figure 3E,3F). Similarly, WUS expression was detected in very old ag-10 ago10-13 but not ag-10 flowers (Figure 3G,3H). CLV3 controls the spatial domain of WUS expression and the size of the stem cell domain [37]. clv3-1 flowers contain extra floral organs of all types, particularly stamens and carpels, due to enlarged floral meristems [38]. The combination of hua1 hua2 ago10-12 and clv3-1 had a synergistic effect on floral determinacy. The gynoecia of the quadruple mutant generated a massive amount of stigmatic tissue bursting out of the primary carpels (Figure S3F,S3G). Similar phenotypes were observed for ag-10 ago10-13 clv3-1 flowers (data not shown), implying that AGO10 and CLV3 act largely independently. Since AG is crucial in the termination of floral stem cells, we evaluated the relationship between AG and AGO10 through extensive genetic interaction studies. We first introduced the ag-1 allele into hua1 hua2 ago10-12. The quadruple mutant flowers were initially similar to ag-1 flowers, but the meristems continued to develop and appeared fasciated in old flowers (data not shown). Given that ago10-12 was a weak allele, we next combined the stronger ago10 alleles pnh-1 [25] or ago10-13 with ag-1. Most flowers of the ag-1 pnh-1 and ag-1 ago1-13 double mutants appeared more fasciated than ag-1 flowers (Figure 4B,4C; data not shown). The mild enhancement implies that AGO10 acts through the AG pathway to repress WUS expression but that AGO10 also has an AG-independent function in floral stem cell regulation. We conducted in situ hybridization to examine the patterns of AG expression in ag-10 and ag-10 ago10-13 flowers. AG transcripts were detected in the inner two whorls of ag-10 and ag- 10 ago10-13 floral meristems (Figure 3C,3D). Therefore, ago10-13 did not affect the spatial domain of AG expression or lead to obvious changes in AG mRNA levels in floral meristems. AGO1 is also required for floral stem cell termination Since AGO1 is the most closely related to AGO10 among the ten Arabidopsis AGO genes, we sought to determine whether AGO1 is also required for floral stem cell termination. We introduced the PLoS Genetics | www.plosgenetics.org 4 March 2011 | Volume 7 | Issue 3 | e1001358