AGO10 and Floral Stem Cell Termination Figure 4. Genetic interactions between AGO10 and meristem regulators. (A) A hua1 hua2 ago10-12 wus-1 flower. wus-1 was completely epistatic to hua1 hua2 ago10-12 in floral determinacy such that the quadruple mutant flower had an incomplete set of floral organs as in a wus-1 flower. (B) An ag-1 flower. (C) An ag-1 pnh-1 flower that had apparently more floral organs than an ag-1 flower and showed signs of fasciation. (D,E) ap2-2 partially suppressed the floral determinacy defects of hua1 hua2 ago10-12 and ag-10 ago10-13. (D) A representative of the non-bulged gynoecia of some hua1 hua2 ago10-12 ap2-2 flowers. The petaloid stamens in hua1 hua2 ago10-12 were rescued back to stamens (arrow) by the ap2- 2 mutation. (E) A representative of ag-10 ago10-13 ap2-2 gynoecia, which are longer than ag-10 ago10-13 gynoecia (Figure 2E). (F–H) phb-1d and phv- 5d each enhanced the determinacy defects of ag-10. (F) ag-10 phb-1d/+ siliques. Ectopic floral organs, indicated by the arrow, were found in the siliques of most ag-10 phb-1d/+ flowers. (G) ag-10 phv-5d siliques were bulged or contained ectopic floral organs, as indicated by the arrow. (H) A longitudinal section of an ag-10 phb-1d/+ gynoecium. An ectopic floral meristem (indicated by the arrow) could be observed inside the carpels. (I–L) ag-10 plants harboring transgenic amiR165/166 exhibited loss of floral determinacy. (I–J) Weaker determinacy defects of some ag-10 amiR165/166 plants. The gynoecia were bulged as in ag-10 ago10-13. (K–L) Stronger determinacy defects of some ag-10 amiR165/166 plants. The gynoecia were replaced by an internal flower. Scale bars, 50 mm in (H), and 1 mm in all other panels. ca, capel; se, sepal. doi:10.1371/journal.pgen.1001358.g004 triple mutant were restored to stamens in the quadruple mutant (Figure 4D). While the third whorl organs in hua1 hua2 ago10-12 had patches of cone-shaped cells reminiscent of petal epidermal cells (Figure S3I, S3J), the third whorl organs in hua1 hua2 ago10-12 ap2-2 had epidermal cells with the shapes of interlocking puzzle pieces resembling anther epidermal cells (Figure S3K,S3L). This indicates that the homeotic transformation in hua1 hua2 ago10-12 flowers is due to de-repressed AP2 and that AGO10 mediates the function of miR172 in repressing AP2. Second, ap2-2 partially suppressed the floral determinacy defects of hua1 hua2 ago10-12 and ag-10 ago10-13 flowers. ap2-2 did not affect the shapes of hua1 hua2 or ag-10 gynoecia ([31]; Figure S3M), but caused hua1 hua2 ago10-12 and ag-10 ago10-13 gynoecia to be less bulged (compare Figure 4D to Figure 1B and Figure 4E to Figure 2E), suggesting that the determinacy defects were not as severe as in hua1 hua2 ago10-12 or ag-10 ago10-13. To quantify this effect, we dissected gynoecia to observe the presence/absence of ectopic organs inside. Compared with hua1 hua2 ago10-12 and ag-10 ago10-13 plants that had ectopic floral organs in 91% and 100% of the siliques, respectively, ap2-2 reduced the percentage of siliques with internal organs to 41% and 86% in hua1 hua2 ago10-12 and ag-10 ago10-13, respectively (Table S1). Although the suppression effect for the latter genotype was not large, we found that ap2-2 completely suppressed the presence of an elongated gynophore in ag-10 ago10- 13 (Figure S3N-S3P). An elongated gynophore probably reflects a partial conversion of the floral meristem to an inflorescence meristem, hence, prolonged stem cell activity. Therefore, the floral stem cell defects of ago10 mutants were partially rescued by ap2-2, indicating that AP2 is likely de-repressed in ago10 mutants and that the de-repression is partially responsible for the floral stem cell defects. This conclusion is consistent with the similar genetic behavior of AP2m3 and ago10 in that both enhanced ag-1 ([12]; Figure 4B,4C). Despite the genetic evidence supporting de-repressed AP2 expression in ago10 mutants, we were unable to consistently detect an increase in AP2 protein levels in ag-10 ago10-13 vs. ag-10 or PLoS Genetics | www.plosgenetics.org 7 March 2011 | Volume 7 | Issue 3 | e1001358