ARGONAUTE10 and ARGONAUTE1 Regulate the Termination of Floral Stem Cells through Two MicroRNAs in Arabidopsis Lijuan Ji1, Xigang Liu1, Jun Yan 2 , Wenming Wang 3 , Rae Eden Yumul1,4, Yun Ju Kim1, Thanh Theresa Dinh1,4, Jun Liu1, Xia Cui5, Binglian Zheng 1 , Manu Agarwal1, Chunyan Liu5, Xiaofeng Cao 5 , Guiliang Tang 2 , Xuemei Chen1* 1 Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California Riverside, Riverside, California, United States of America, 2 Gene Suppression Laboratory, Department of Plant and Soil Sciences and KTRDC, University of Kentucky, Lexington, Kentucky, United States of America, 3 Rice Institute, Sichuan Agricultural University, Chengdu, China, 4 ChemGen IGERT program, Center for Plant Cell Biology, Institute of Integrative Genome Biology, University of California Riverside, Riverside, California, United States of America, 5 State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Abstract 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. Citation: Ji L, Liu X, Yan J, Wang W, Yumul RE, et al. (2011) ARGONAUTE10 and ARGONAUTE1 Regulate the Termination of Floral Stem Cells through Two MicroRNAs in Arabidopsis. PLoS Genet 7(3): e1001358. doi:10.1371/journal.pgen.1001358 Editor: Li-Jia Qu, Peking University, China Received August 15, 2010; Accepted February 28, 2011; Published March 31, 2011 Copyright: 2011 Ji et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by a grant from National Institutes of Health (GM61146) to X Chen, by grants from National Basic Research Program of China (2007CB948202) to C Liu and (2009CB941500) to X Cao, by National Natural Science Foundation of China (30828002) to X Chen and X Cao, and by Transgenic Projects Funds 2008ZX08009-001 and 2011ZX08009-001 to C Liu and 2011ZX08009-001 and 2009ZX08009-39B to X Cui. TT Dihn and RE Yumul were supported by an NSF ChemGen IGERT training grant (DGE0504249). G Tang is supported by KTRDC, KSEF (# KSTC-144-401-08-029), USDA (2006-35301-17115 and 2006-35100- 17433), and NSF (MCB-0718029, Subaward no. S-00000260 and IOS-1048216). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: xuemei.chen@ucr.edu Introduction Stem cells are key to morphogenesis in multicellular organisms and understanding the mechanisms governing their maintenance or termination is a major goal in developmental biology. In plants, small populations of pluripotent stem cells are found in meristems located at the tips of roots, shoots, or developing flowers. The stem cells in the shoot apical meristem (SAM) continuously provide new cells for organogenesis to give rise to the entire above-ground portion of the plant. The SAM generates leaf primordia during vegetative growth and, after floral transition, produces floral meristems on its flanks. Arabidopsis floral meristems produce four concentric whorls of floral organs of fixed numbers, namely four sepals, four petals, six stamens and two fused carpels. In contrast to the SAM, floral meristems are genetically programmed to terminate after the primordia of the female reproductive organs (carpels) are formed (reviewed in [1]). The termination of floral stem cells is temporally precisely regulated to coincide with the formation of the female reproductive organs to ensure successful reproduction of plants. This property of floral stem cells, i.e. determinacy, offers an opportunity to understand the temporal regulation of stem cell maintenance. A key factor in stem cell maintenance in plants is the WUSCHEL (WUS) gene encoding a homeodomain transcription factor. In both the SAM and the floral meristems, WUS is expressed in a small group of cells named the organizing center PLoS Genetics | www.plosgenetics.org 1 March 2011 | Volume 7 | Issue 3 | e1001358