APETALA2 antagonizes the transcriptional activity of AGAMOUS in regulating floral stem cells in Arabidopsis thaliana.

APETALA2 (AP2) is best known for its function in the outer two floral whorls, where it specifies the identities of sepals and petals by restricting the expression of AGAMOUS (AG) to the inner two whorls in Arabidopsis thaliana. Here, we describe a role of AP2 in promoting the maintenance of floral stem cell fate, not by repressing AG transcription, but by antagonizing AG activity in the center of the flower. We performed a genetic screen with ag-10 plants, which exhibit a weak floral determinacy defect, and isolated a mutant with a strong floral determinacy defect. This mutant was found to harbor another mutation in AG and was named ag-11. We performed a genetic screen in the ag-11 background to isolate mutations that suppress the floral determinacy defect. Two suppressor mutants were found to harbor mutations in AP2. While AG is known to shut down the expression of the stem cell maintenance gene WUSCHEL (WUS) to terminate floral stem cell fate, AP2 promotes the expression of WUS. AP2 does not repress the transcription of AG in the inner two whorls, but instead counteracts AG activity

AGAMOUS Terminates Floral Stem Cell Maintenance in Arabidopsis by Directly Repressing WUSCHEL through Recruitment of Polycomb Group Proteins

Floral stem cells produce a defined number of floral organs before ceasing to be maintained as stem cells. Therefore, floral stem cells offer an ideal model to study the temporal control of stem cell maintenance within a developmental context. AGAMOUS (AG), a MADS domain transcription factor essential for the termination of floral stem cell fate, has long been thought to repress the stem cell maintenance gene WUSCHEL (WUS) indirectly. Here, we uncover a role of Polycomb Group (PcG) genes in the temporally precise repression of WUS expression and termination of floral stem cell fate. We show that AG directly represses WUS expression by binding to the WUS locus and recruiting, directly or indirectly, PcG that methylates histone H3 Lys-27 at WUS. We also show that PcG acts downstream of AG and probably in parallel with the known AG target KNUCKLES to terminate floral stem cell fate. Our studies identify core components of the network governing the temporal program of floral stem cells

ARGONAUTE10 and ARGONAUTE1 Regulate the Termination of Floral Stem Cells through Two MicroRNAs in Arabidopsis

Stem cells are crucial in morphogenesis in plants and animals. Much is known about the mechanisms that maintain stem cell fates or trigger their terminal differentiation. However, little is known about how developmental time impacts stem cell fates. Using Arabidopsis floral stem cells as a model, we show that stem cells can undergo precise temporal regulation governed by mechanisms that are distinct from, but integrated with, those that specify cell fates. We show that two microRNAs, miR172 and miR165/166, through targeting APETALA2 and type III homeodomain-leucine zipper (HD-Zip) genes, respectively, regulate the temporal program of floral stem cells. In particular, we reveal a role of the type III HD-Zip genes, previously known to specify lateral organ polarity, in stem cell termination. Both reduction in HD-Zip expression by over-expression of miR165/166 and mis-expression of HD-Zip genes by rendering them resistant to miR165/166 lead to prolonged floral stem cell activity, indicating that the expression of HD-Zip genes needs to be precisely controlled to achieve floral stem cell termination. We also show that both the ubiquitously expressed ARGONAUTE1 (AGO1) gene and its homolog AGO10, which exhibits highly restricted spatial expression patterns, are required to maintain the correct temporal program of floral stem cells. We provide evidence that AGO10, like AGO1, associates with miR172 and miR165/166 in vivo and exhibits “slicer” activity in vitro. Despite the common biological functions and similar biochemical activities, AGO1 and AGO10 exert different effects on miR165/166 in vivo. This work establishes a network of microRNAs and transcription factors governing the temporal program of floral stem cells and sheds light on the relationships among different AGO genes, which tend to exist in gene families in multicellular organisms

<em>POWERDRESS</em> and Diversified Expression of the <em>MIR172</em> Gene Family Bolster the Floral Stem Cell Network

<div><p>Termination of the stem cells in the floral meristem (also known as floral determinacy) is critical for the reproductive success of plants, and the molecular activities regulating floral determinacy are precisely orchestrated during the course of floral development. In <em>Arabidopsis thaliana</em>, regulators of floral determinacy include several transcription factor genes, such as <em>APETALA2</em> (<em>AP2</em>), <em>AGAMOUS</em> (<em>AG</em>), <em>SUPERMAN</em> (<em>SUP</em>), and <em>CRABSCLAW</em> (<em>CRC</em>), as well as a microRNA (miRNA), miR172, which targets <em>AP2</em>. How the transcription factor and miRNA genes are coordinately regulated to achieve floral determinacy is unknown. A mutation in <em>POWERDRESS</em> (<em>PWR</em>), a previously uncharacterized gene encoding a SANT-domain-containing protein, was isolated in this study as an enhancer of the weakly indeterminate <em>ag-10</em> allele. <em>PWR</em> was found to promote the transcription of <em>CRC</em>, <em>MIR172a</em>, <em>b</em>, and <em>c</em> and/or enhance Pol II occupancy at their promoters, without affecting <em>MIR172d</em> or <em>e</em>. A mutation in mature miR172d was additionally found to enhance the determinacy defects of <em>ag-10</em> in an <em>AP2</em>-dependent manner, providing direct evidence that miR172d is functional in repressing <em>AP2</em> and thereby contributes to floral determinacy. Thus, while <em>PWR</em> promotes floral determinacy by enhancing the expression of three of the five <em>MIR172</em> members as well as <em>CRC</em>, <em>MIR172d</em>, whose expression is <em>PWR</em>-independent, also functions in floral stem cell termination. Taken together, these findings demonstrate how transcriptional diversification and functional redundancy of a miRNA family along with <em>PWR</em>-mediated co-regulation of miRNA and transcription factor genes contribute to the robustness of the floral determinacy network.</p> </div

A summary of the floral determinacy gene network highlighting the function of <i>PWR</i>.

<p><i>AP2</i> and <i>AG</i> act antagonistically in terms of the regulation of <i>WUS</i> expression: <i>AP2</i> promotes <i>WUS</i> expression and stem cell maintenance, while <i>AG</i> represses <i>WUS</i> expression to elicit stem cell termination. <i>CRC</i> acts downstream of <i>AG</i>, and miR172 represses <i>AP2</i> expression. <i>PWR</i> promotes floral stem cell termination by enhancing the expression of <i>CRC</i> and three of the five <i>MIR172</i> genes. <i>MIR172d</i>, whose expression is independent of <i>PWR</i>, is functional in the repression of <i>AP2</i> expression and the control of floral determinacy.</p

miRNA abundance in <i>pwr-1</i> and <i>pwr-2</i>.

<p>(A) Abundance of miR159, miR172, miR173, miR166, and miR390 in L<i>er</i>, <i>pwr-1</i>, Col, and <i>pwr-2</i> detected by small RNA northern blotting. The abundance of miR159, miR172, and miR173 (underlined) was reduced in both <i>pwr</i> alleles relative to their respective controls. Decreased accumulation was not observed for miR166 or miR390 in either allele. (B) Additional miRNAs tested in <i>pwr-1</i> did not have reduced abundance compared to L<i>er</i>. In (A) and (B), values indicate the relative abundance of the indicated miRNA species in <i>pwr-1</i> compared to L<i>er</i> or in <i>pwr-2</i> compared to Col. For all blots, ImageJ signal intensity analysis was used for quantification. The numbers above the miRNA blots indicate the relative miRNA abundance between the mutants and wild type.</p

pri–miRNA abundance and Pol II occupancy at <i>MIR</i> genes in <i>pwr-1</i> and <i>pwr-2</i>.

<p>(A) Transcript levels of pri-miR172a-e in <i>pwr-1</i>. pri-miR172a and pri-miR172b transcript levels were reduced in <i>pwr-1</i>, while no significant changes were observed for pri-miR172c, pri-miR172d, and pri-miR172e. (B) Transcript levels of pri-miR172a in <i>PWR⁺</i> (<i>PWR</i>/<i>PWR</i> and <i>PWR</i>/<i>pwr-1</i>) and <i>pwr-1</i> plants harboring a <i>p35S:MIR172a</i> transgene. (C) Real-time RT-PCR analysis of other pri-miRNAs. A general decrease was observed in pri-miRNA transcript levels in both <i>pwr</i> alleles. For all pri-miRNAs tested, transcript levels were normalized to <i>UBIQUITIN 5</i> and compared to the respective wild-type control. In (A) to (C), error bars indicate the standard deviation for triplicate technical replicates. Three biological replicates gave similar results. (D) Pol II occupancy at <i>MIR</i> loci determined by ChIP using anti-RPB2 antibodies in L<i>er</i> and <i>pwr-1</i>. Error bars indicate the standard deviation for triplicate technical replicates. Pol II C1, located in the intergenic region between At2g17470 and At2g17460, has no appreciable Pol II occupancy as determined in a previous study <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003218#pgen.1003218-Chodavarapu1" target="_blank">[51]</a> and is used as a negative control.</p

Phenotypes of <i>pwr-1</i> and <i>pwr-2</i> and transcript levels of genes involved in floral development.

<p>(A) L<i>er</i> (left) and <i>pwr-1</i> (right) siliques. (B) Col (left) and <i>pwr-2</i> (right) siliques. In (A) and (B), arrows indicate the bulged carpel tips of <i>pwr-1</i> and <i>pwr-2</i> siliques, and scale bars = 5 mm. (C) RT-PCR for <i>PWR</i> using cDNA obtained from L<i>er</i> roots, rosette leaves, cauline leaves, and inflorescences. <i>UBIQUITIN 5</i> was used as the control. The highest abundance of the <i>PWR</i> transcript was observed in inflorescences. (D) Real-time RT-PCR analysis of the transcript levels of <i>AP3</i>, <i>PI</i>, <i>CRC</i>, and <i>FT</i>. For <i>AP3</i> and <i>PI</i>, two biological replicates are shown for <i>pwr-2</i> compared to Col. For <i>CRC</i> and <i>FT</i>, data is shown for both <i>pwr</i> alleles. For all genes tested, transcript levels were normalized to <i>UBIQUITIN 5</i> and compared to the respective wild-type control. Error bars indicate the standard deviation for triplicate technical replicates. (E) Pol II occupancy at the <i>CRC</i> promoter determined by ChIP using anti-RPB2 antibodies in L<i>er</i> and <i>pwr-1</i>. Pol II C1, located in the intergenic region between At2g17470 and At2g17460, has no appreciable Pol II occupancy as determined in a previous study <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003218#pgen.1003218-Chodavarapu1" target="_blank">[51]</a> and is used as a negative control. (F) Col (left) and <i>pwr-2</i> (right) plants grown side by side under 24-hour light conditions.</p