known to do so on other miRNA target genes [26]. If this were the case, the levels of HD-Zip proteins would be increased in ago10-13 despite the reduction in the mRNA levels. The lack of antibodies against the HD-Zip proteins prevented us from directly determin- ing the levels of HD-Zip proteins in ago10 mutants. We used the mRNA levels of LITTLE ZIPPER3 (ZPR3), which is a direct target of PHB [44], as a proxy for PHB protein levels. It was shown that ZPR3 mRNA levels are reduced in HD-Zip loss-of-function mutants and increased in miRNA165/166-resistant HD-Zip mutants [44]. Realtime RT-PCR showed that ZPR3 levels were severely reduced in ago10-13 (Figure S5), suggesting that HD-Zip protein levels were low in ago10-13. Next, we investigated whether reduced HD-Zip gene expression could result in loss of floral determinacy. ag-10 plants were transformed with an artificial miR165/166 construct in which pre-miR165 and pre-miR166 were placed in tandem and driven by the 35S promoter (Figure S6). Among 64 primary transformants, 16 exhibited premature termination of the SAM, a phenotype reminiscent of ago10 mutants (Figure S3T). A few of them couldn’t survive to generate true leaves. 12 plants exhibited bulged gynoecia similar to ag-10 ago10 and 4 plants had more severe floral determinacy defects in that the gynoecia were replaced by an internal flower (Figure 4I– 4L). This demonstrated that down-regulation of HD-Zip gene expression by over-expression of miR165/166 compromises floral determinacy. Discussion AGO10 and AGO1 have similar small RNA–binding specificities but can act differently on miRNAs in vivo Among the ten argonaute proteins in Arabidopsis, AGO10 is the most similar in sequence to AGO1, the major miRNA effector. Genetic evidence indicates that AGO10 and AGO1 have redun- dant, overlapping, or even antagonistic functions in development and in RNA silencing [23,27]. For several tested miRNA target genes, protein but not mRNA levels are elevated in ago10 mutants, suggesting that AGO10 is necessary for miRNA-mediated translational repression of targets [26]. However, direct molecular evidence supporting AGO10 as a miRNA effector has been lacking, perhaps owing to the spatially highly restricted expression of AGO10, which makes it challenging to assay small RNA binding and cleavage activities of AGO10. In this study, we provide direct evidence that AGO10 associates with miRNAs in vivo and possesses miRNA-directed cleavage activity in vitro. So far, three other Arabidopsis AGO proteins, AGO1, AGO4 and AGO7, have been shown to possess ‘‘slicer’’ activity [18,20,45]. AGO1 preferentially associates with most miRNAs, ta-siRNAs and transgene siRNAs and mediates target mRNA cleavage and translational inhibition [17,20,26]. AGO7 specifically binds and mediates the function of miR390 in ta-siRNA production through both cleavage and non- cleavage functions [18]. AGO4 binds 24 nt endogenous siRNAs and causes transcriptional silencing of target loci in either a slicer- dependent or slicer-independent manner [45]. From our limited examination of small RNAs bound by AGO10, it appears that AGO10 has similar small RNA binding specificities as AGO1 in that it binds all examined miRNAs except for miR390 and it does not bind 24 nt endogenous siRNAs. Despite the similar small RNA binding specificities of the two proteins, mutations in the two genes can have different effects on miRNA accumulation and the expression of miRNA target genes. The abundance of many miRNAs is reduced in severe ago1 mutants [46] but not affected in ago10 alleles ([26] and this study). While many miRNA target mRNAs are increased in abundance in ago1 mutants [21], most assayed miRNA targets are AGO10 and Floral Stem Cell Termination not significantly affected in ago10 mutants ([26] and this study). miR165/166 is an extreme example of the different effects of ago1 and ago10 mutations. While miR165/166 levels are reduced in ago1-11, they are increased in ago10 mutants. The targets of miR165/166 are also affected in opposite directions in the two mutants – they are de-repressed in ago1-11 and further repressed in ago10. This shows that AGO1 is the major slicer acting upon the HD-Zip mRNAs in vivo, although AGO10 has slicer activity in vitro. While the two argonaute proteins act differently on miR165/166, they both mediate the activities of miR172. Both ago1-11 and ago10-12 result in petaloid stamens in the hua1 hua2 background, suggesting that both ago1 and ago10 mutations lead to de- repression of AP2. Therefore, AGO1 and AGO10 mediate the activities of some miRNAs but also have different effects on others. Floral stem cell termination versus differentiation Floral stem cell termination is tightly coupled to the formation of carpel primordia in flower development. AG, which specifies carpel identities, acts to terminate the floral stem cells. It may thus be presumed that the termination of floral stem cells is simply via differentiation of these cells into carpel cells. However, the fact that ag-10 ago10-13 and hua1 hua2 ago10-12 flowers have 4th whorl carpels but are indeterminate indicates that carpel identity specification and floral stem cell termination, although both directed by AG, are two separable processes. It is expected that factors acting downstream of AG in the two processes must be distinct. KNUCKLES, for example, is a target of AG that is only required for floral determinacy specification [47]. HD-Zip genes, targets of miR165/166, are crucial for the temporal program of floral stem cells Our previous studies show that both miR172 and its target AP2 regulate the temporal program of floral stem cells. Plants expressing miR172-resistant AP2 cDNA have prolonged WUS expression and indeterminate floral meristems that produce numerous stamens [12]. In this study, we show that the HD-Zip genes, which are targets of miR165/166 and which are previously known to specify adaxial identity in lateral organs, are also crucial factors in floral stem cell regulation. Over expression of miR165/ 166 in the ag-10 background results in indeterminate flowers. Consistent with this, the phb phv cna triple mutant was observed to have occasional flowers with enlarged gynoecia containing ectopic carpels inside [48]. On the other hand, ag-10 phb-1d/+ and ag-10 phv-5d gynoecia are also indeterminate. Given the similar phenotypes of de-repressed PHB or PHV and loss of function in PHB, PHV and CNA in terms of floral determinacy, it is likely that either too much or too little HD-Zip activity is detrimental to the precise regulation of floral stem cells. Given that the HD-Zip genes promote adaxial identities of lateral organs, our findings raise the intriguing possibility that floral stem cell termination depends on correct polarity specification of the fourth whorl organs. This hypothesis will be tested in the future. AGO10 and AGO1 promote floral stem cell termination via similar and different mechanisms The relationship between AGO10 and AGO1 varies depending on the developmental processes. During embryogenesis, AGO10 and AGO1 share overlapping functions in maintaining STM expression [23]. In the weak zll-15 mutant background, increasing the dosage of AGO1 suppressed the defects in embryonic stem cells, while reducing AGO1 dosage enhanced the defects [27]. Our studies provide the molecular evidence supporting the overlapping biological roles of AGO1 and AGO10 by showing that they have PLoS Genetics | www.plosgenetics.org 10 March 2011 | Volume 7 | Issue 3 | e1001358