AGO10 and Floral Stem Cell Termination of stage 12 ag-10 and ag-10 ago10-13 flowers. The floral meristem was not visible in most ag-10 flowers (G), whereas it was still active in ag-10 ago10- 13 flowers to result in extra whorls of organs inside the fourth whorl carpels (H). ca, carpel; st, stamen. (I) A scanning electron micrograph of an ago1- 11 ago10-13/+ flower. The severe floral determinacy defects were reflected by the presence of numerous floral organs inside unfused primary carpels (indicated by arrows). (J–L) ago1-11 enhanced the floral determinacy defects of hua1 hua2 and ag-10. (J) An ago1-11 flower. (K) A hua1 hua2 ago1-11 flower with bulged gynoecia and petaloid stamens (arrows). (L) A representative ag-10 ago1-11 flower with a severely bulged gynoecium. In (A), (B), (C), and (J), some outer whorl floral organs were removed to expose the gynoecia. Scale bars, 50 mm in (G) and (H), 500 mm in (I), and 1 mm in all other panels. doi:10.1371/journal.pgen.1001358.g002 partial loss-of-function ago1-11 mutation [39] into hua1 hua2 and ag-10. The infertile ago1-11 flowers develop all four types of floral organs, although the organs are abnormal in morphology (Figure 2J; Figure S3C). In hua1 hua2 ago1-11 flowers, either the gynoecia were severely bulged or the carpels were completely unfused with ectopic flowers developing internal to the primary carpels (Figure 2K). Similarly, indeterminate phenotypes were observed in the ag-10 ago1-11 double mutant (Figure 2L). Therefore, AGO1, like AGO10, is required for floral stem cell termination. Genetic studies conducted by others as well as by ourselves show that AGO1 and AGO10 have overlapping functions in terminating floral meristems. First, it was previously shown that combining the strong ago1-7 and pnh-2 (an ago10 allele) alleles resulted in embryo lethality [23]. But ago1-7/+ pnh-2/pnh-2 and ago1-7/ago1-7 pnh-2/+ flowers showed an increase in the number of floral organs as well as ectopic growth of tissues inside the primary carpels (i.e., loss of floral determinacy). Second, we crossed the weak ago1-11 allele with the strong ago10-13 allele but also failed to obtain viable double mutants. The ago1-11 ago10-13/+ plants were smaller than ago1-11 in stature. Some inflorescences harbored flowers with filamentous floral organs and obviously enlarged floral meristems (Figure S3D). When carpels were formed, they were unfused and additional floral organs were observed inside the primary carpels (Figure 2I). In ago10-13 ago1-11/+ plants, the gynoecia were bulged and additional floral organs were found in some of the gynoecia upon dissection (Figure 2F). In summary, both ago1 ago10/+ or ago1/+ ago10 plants were more severe in floral determinacy defects than the corresponding single mutants. The dosage effects of ago1 and ago10 alleles indicate that the two genes have overlapping functions in floral stem cell termination. AGO10 acts partly through miR172 to promote floral determinacy miR172 negatively regulates AP2 mainly through translational repression [10,11] to result in proper floral patterning including the termination of floral stem cells [12] and stamen identity specification [32]. In the hua1 hua2 background in which AG expression is reduced, loss of function in HEN1, a player in miRNA biogenesis [40], results in stamen-to-petal transformation. The homeotic transformation is mainly due to de-repressed AP2 expression since the ap2-2 mutation restores stamen identity to hua1 hua2 hen1-1 flowers [32]. To determine whether AGO10 acts through miR172 in floral patterning, we examined the genetic interactions between ago10 and ap2 mutations by generating hua1 hua2 ago10-12 ap2-2 and ag- 10 ago10-13 ap2-2. First, ap2-2 suppressed the homeotic defects of hua1 hua2 ago10-12 in that the third whorl petaloid stamens in the Figure 3. Expression patterns of AGO10, AG, and WUS in floral meristems. (A, B) Immunolocalization of AGO10 protein using anti-GFP antibodies in zll-1 pZLL::YFP-ZLL flowers. The grey signals represent AGO10. Before floral stage 6, AGO10 was predominantly localized at the floral meristem and in provascular tissue (A); AGO10 was adaxialized in carpels beyond stage 6 (B). (C, D) The ago10-13 mutation did not alter the spatial patterns of AG expression in developing flowers. AG mRNA was similarly present in the inner two whorls of an ag-10 (C) and an ag-10 ago10-13 (D) flower at stages 6–7. (E–H) ago10 mutations led to prolonged WUS expression. In hua1 hua2, the latest stage when WUS mRNA could be detected was stage 6 (E). WUS expression persisted to stages 8-9 in hua1 hua2 ago10-12 flowers (arrow in F). WUS expression was diminished by stage 7 in ag-10 (G). In ag-10 ago10-13, WUS expression could be observed in stages 8–9 flowers (arrow in H). Scale bars, 50 mm. ca, carpel; se, sepal; st, stamen. doi:10.1371/journal.pgen.1001358.g003 PLoS Genetics | www.plosgenetics.org 6 March 2011 | Volume 7 | Issue 3 | e1001358