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ARGONAUTE1 Acts in Arabidopsis Root Radial Pattern Formation Independently of the SHR/SCR Pathway Shunsuke Miyashima, Takashi Hashimoto and Keiji Nakajima* Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192 Japan Regular Paper The formation of radially symmetric tissue patterns is one of the most basic processes in the development of vascular plants. In Arabidopsis thaliana, plant-specific GRAS-type transcription factors, SHORT-ROOT (SHR) and SCARECROW (SCR), are required for asymmetric cell divisions that separate two ground tissue cell layers, the endodermis and cortex, as well as for endodermal cell fate specification. While loss of SHR or SCR results in a singlelayered ground tissue, radially symmetric cellular patterns are still maintained, suggesting that unknown regulatory mechanisms act independently of the SHR/SCR-dependent pathway. In this study, we identified a novel root radial pattern mutant and found that it is a new argonaute1 (ago1) allele. Multiple ago1 mutant alleles contained supernumerary ground tissue cell layers lacking a concentric organization, while expression patterns of SHR and SCR were not affected, revealing a previously unreported role for AGO1 in root ground tissue patterning. Analyses of ago1 scr double mutants demonstrated that the simultaneous loss of the two pathways caused a dramatic reduction in cellular organization and ground tissue identity as compared with the single mutants. Based on these results, we propose that highly symmetric root ground tissue patterns are maintained by the actions of two independent pathways, one using post-transcriptional regulation mediated by AGO1 and the other using the SHR/SCR transcriptional regulator. Keywords: Arabidopsis thaliana • ARGONAUTE Differentiation • MicroRNA • Pattern formation • Root. • Abbreviations: ACT, ACTIN; AGO1, ARGONAUTE1; CEI, cortex/endodermis initial; CLSM, confocal laser scanning microscopy; dpg, days post-germination; EMS, ethylmethane sulfonate; GFP, green fluorescent protein; HYL1, HYPONASTIC LEAVES1; LRC, lateral root cap; MiRNA, microRNA; MGP, MAGPIE; NUC, NUTCRACKER; qRT–PCR, quantitative reverse transcription–PCR; SCR, SCARECROW; SHR, SHORT-ROOT Introduction Pattern formation along the central–peripheral axis is a fundamental process in the development of land plants where axial organs with a cylindrical tissue organization are prevalent. Identification of genetic mechanisms underlying radial pattern formation is therefore essential to understanding the basic elements of plant development. Arabidopsis roots have been used as a model system to study radial pattern formation because of their simplicity and amenability to microscopic and genetic analyses (Dolan et al. 1993). In cross-sections of Arabidopsis roots, each single layer of epidermis, cortex, endodermis and pericycle forms concentric rings that surround the central vascular tissue (Fig. 1A). The cortex and endodermis are collectively called ground tissue. In the distal part, a few layers of lateral root cap (LRC) are located outside the epidermis (Fig. 1A). LRC is exceptional in that it forms a spiral rather than a concentric organization (Baum and Rost 1996). This radial pattern is first laid down during embryogenesis and maintained by stereotyped cell divisions of the stem cells located in the root meristem (Scheres et al. 1994). It has been shown that plant-specific GRAS-type transcription factors, SHORTROOT (SHR) and SCARECROW (SCR), control the asymmetric divisions of the cortex/endodermis initial (CEI) daughter cells, as well as endodermal cell fate specification (Di Laurenzio et al. 1996, Helariutta et al. 2000, Heidstra et al. 2004, Sena et al. 2004). SHR proteins move from the stele (pericycle and vascular tissue) into neighboring cells, where they interact with SCR to form a nuclear-localized protein *Corresponding author: E-mail, [email protected]; Fax, +81-743-72-5529. Plant Cell Physiol. 50(3): 626–634 (2009) doi:10.1093/pcp/pcp020, available online at www.pcp.oxfordjournals.org © The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] 626 Plant Cell Physiol. 50(3): 626–634 (2009) doi:10.1093/pcp/pcp020 © The Author 2009. ARGONAUTE1 in root radial pattern formation A G ago1-102 ago1-101 PAZ domain H 5gf32 TGG TGA 607W STOP PIWI domain 01 02 1-1 go1-1 gf32 a 5 WT ago Quiescent Center Epidermis Pericycle Columella Cortex Vascular Tissue Lateral Root Cap Endodermis AGO1 ACT8 Cortex/Endodermis Initial (CEI) B D C E F Fig. 1 miRNA-mediated gene regulation is required for Arabidopsis root radial patterning. (A) Diagrams showing the tissue organization of Arabidopsis roots. (B, C) Longitudinal confocal sections of 4 dpg wild-type (B) and 5gf32 (C) roots. GT, ground tissue; Epi, epidermis; Co, cortex; En, endodermis. Right panels show higher magnification images of the boxed regions. (D–F) Cross-sections of wild-type (D), 5gf32 (E) and hyl1-2 (F) roots. Asterisks indicate cells in the cortex. (G) Schematic representation of AGO1 gene structure and positions of mutant lesions. Thick lines indicate the positions of conserved PAZ and PIWI domains. Triangles indicate the positions of the primers used in the RT–PCR shown in (H). (H) RT–PCR analysis of AGO1 in ago1 mutants and wild-type plants. ACTIN8 (ACT8) was used as a control. Scale bars, 20 µm (B–F). complex (Nakajima et al. 2001, Cui et al. 2007). The SHR– SCR protein complex thus formed specifies a single layer of endodermis by regulating a number of genes including SCR itself (Cui et al. 2007). While loss of either SHR or SCR results in a single-layered ground tissue, radial symmetry along the central–peripheral axis is still maintained in such a genetic background (Scheres et al. 1995), suggesting that the radial tissue organization is controlled by as yet unknown mechanisms independent of SHR and SCR. Recent advances in other aspects of plant developmental studies have highlighted the importance of microRNAs (miRNAs) in the post-transcriptional control of several patterning genes (Willmann and Poethig 2007). miRNAs are small regulatory RNAs encoded in both plant and animal Plant Cell Physiol. 50(3): 626–634 (2009) doi:10.1093/pcp/pcp020 © The Author 2009. 627 S. Miyashima et al. genomes. In Arabidopsis, initial cloning experiments identified 19 miRNAs which fell into 15 families (Llave et al. 2002a, Mette et al. 2002, Park et al. 2002, Reinhart et al. 2002). Largescale sequencing approaches have revealed numerous miRNAs and their target genes (Rajagopalan et al. 2006, Fahlgren et al. 2007). Processed from imperfectly complementary stem–loop precursor RNAs (Bartel 2004), miRNAs regulate gene expression by targeting their complementary mRNA for cleavage or translational inhibition (Tang et al. 2003, Chen 2004). In the miRNA-dependent regulatory mechanisms, ARGONAUTE (AGO) proteins play integral roles in all known pathways regulated by small RNAs (Hutvagner and Simard 2008). The Arabidopsis genome encodes 10 AGO proteins (Vaucheret 2008). Biochemical analyses have revealed that AGO1 cleaves miRNA-targeted mRNA in vitro (Baumberger and Baulcombe 2005, Qi et al. 2005). Immunoprecipitation experiments demonstrated that AGO1 preferentially associates with small RNAs that have a uridine nucleotide at their 5′ termini (Mi et al. 2008). The occurrence of 5′ uridine in most miRNA species suggests that AGO1 preferentially contributes to the miRNA-dependent gene regulation in Arabidopsis and, indeed, the accumulation of miRNA-targeted transcripts was increased in ago1 mutants (Vaucheret et al. 2004). Several loss-of-function ago1 alleles have been characterized so far. Although their pleiotropic effects on the establishment of the shoot apical meristem in embryogenesis, formation of adventitious roots and adaxial–abaxial polarity of lateral organs have been reported (Bohmert et al. 1998, Lynn et al. 1999, Kidner and Martienssen 2004, Sorin et al. 2005), the roles of AGO1 in root radial patterning have not been described. In this study, we report a novel function of AGO1 in root radial patterning. Loss of function of AGO1 perturbed the radial symmetry of Arabidopsis root ground tissue. Analyses of ago1 scr and ago1 shr double mutant combinations revealed novel patterning defects that were not apparent in each single mutant. Based on histological and marker analyses, we propose that AGO1-dependent post-transcriptional regulation acts in parallel with SHR/SCR-dependent transcriptional regulation and that the concerted activities of the two pathways are required to establish the highly symmetric Arabidopsis root radial pattern. Results 628 radial patterning, it is necessary to screen mutants by direct observation of their cellular patterns. To this end, we screened ethylmethane sulfonate (EMS)-mutagenized Arabidopsis plants for aberrant root cellular patterns by observing each root by confocal laser scanning microscopy (CLSM). This strategy allowed us to identify a mutant 5gf32 that exhibited a disorganized root ground tissue pattern. In contrast to wild-type roots in which ground tissue consists of two concentric layers, the inner endodermis and outer cortex (Fig. 1A, B, D), 5gf32 contained a three-layered ground tissue that was not organized concentrically (Fig. 1C, E). Because 5gf32 shoots formed unexpanded pointed cotyledons reminiscent of previously characterized ago1 mutants (data not shown) (Bohmert et al. 1998), we sequenced the AGO1-coding region of 5gf32 and identified a nonsense mutation within the conserved PIWI domain (Fig. 1G). Reverse transcription–PCR (RT–PCR) analysis revealed an accumulation of the AGO1 transcript in 5gf32 (Fig. 1H). However, based on the notion that the PIWI domain is essential for AGO1 to function in miRNA-targeted mRNA cleavage (Baumberger and Baulcombe 2005), AGO1 in 5gf32 probably represents a loss-of-function allele. Two T-DNA insertion mutants of AGO1 (SALK_035319 and SALK_096625, hereafter called ago1-101 and ago1-102, respectively) showed almost a complete loss of AGO1 transcripts (Fig. 1H) and the same patterning defects as in 5gf32 (Figs. 1C, E, and 3C, H). These results indicate that all the three ago1 alleles are hypomorphic and that AGO1 is required for the highly organized root ground tissue patterning. The identification of AGO1 as the causal gene for the 5gf32 phenotype suggested the possible involvement of miRNAmediated regulation in root ground tissue patterning. We therefore tested whether disruption of miRNA biogenesis could also disturb root cell patterning. HYPONASTIC LEAVES 1 (HYL1) encodes a double-stranded RNA (dsRNA)-binding protein required for proper accumulation of miRNAs in Arabidopsis (Han et al. 2004, Vazquez et al. 2004). In hyl1 roots, cortex layers were not arranged concentrically (Fig. 1F), similarly to the situation in ago1. This observation confirmed that miRNA-mediated gene regulation is required for proper radial patterning of Arabidopsis roots. Loss of AGO1 affects root ground tissue patterning Loss of AGO1 does not affect expression of SHR and SCR Well-known root radial pattern mutants such as scarecrow (scr) and short-root (shr) are also defective in root stem cell maintenance (Di Laurenzio et al. 1996, Helariutta et al. 2000, Sabatini et al. 2003). It has been realized, however, that mutations in some genes disturb root radial patterning without significantly affecting root growth (Welch et al. 2007). Therefore, in order to identify novel genes responsible for root Because ago1 mutants were defective in the cellular arrangement of root ground tissue, we analyzed expression patterns of two GRAS family transcription factors, SHR and SCR, in ago1 mutants. SHR and SCR are known to regulate asymmetric cell divisions of the CEI daughter cells and differentiation of the endodermis (Di Laurenzio et al. 1996, Helariutta et al. 2000, Heidstra et al. 2004, Sena et al. 2004). In wild-type Plant Cell Physiol. 50(3): 626–634 (2009) doi:10.1093/pcp/pcp020 © The Author 2009. ARGONAUTE1 in root radial pattern formation root stele, the SHR–green fluorescent protein (GFP) fusion protein expressed by the SHR promoter localized to both cytoplasm and nuclei, while it localized exclusively to the nuclei in endodermis, as described previously (Fig. 2A) (Nakajima et al. 2001). This expression pattern was essentially the same in ago1-101 roots, with SHR–GFP detected in stele cells and the innermost layer of ground tissue (Fig. 2B). Similarly, the expression of SCR in ago1-101 was also the same as in the wild type, with GFP–SCR expressed by the SCR promoter accumulated in the endodermis (Fig. 2C, D). These observations suggest that neither SHR nor SCR expression is regulated by AGO1. AGO1 acts in ground tissue patterning independently of SHR and SCR Fig. 2 Loss of AGO1 function does not affect expression of SHR and SCR. Expression of SHR–GFP (A, B) and GFP–SCR (C, D) fusion proteins in 4 dpg wild-type (A, C) and ago1-101 (B, D) roots. Scale bars, 50 µm. To analyze the relationship between AGO1 and SCR in root radial patterning, we generated ago1-101 scr-3 double mutants and examined their cell arrangement at 4 days post-germination (dpg). In wild-type longitudinal sections, two ground tissue cell layers, the cortex and endodermis, appeared as well-defined cell files (Fig. 3A). Similarly, in the case of scr-3 or ago1-101 single mutants, although the number of ground tissue cell layers was either decreased (scr-3) or increased (ago1-101), the ground tissue could still be recognized as defined cell layers (Fig. 3B, C) (Di Laurenzio et al. 1996). In contrast, root ground tissue in ago1-101 scr-3 double mutants was highly disorganized to the level that cell layers were hardly discernible (Fig. 3D). In wild-type root cross-sections, cells in ground tissue exhibited shapes typical of their differentiation status; an ellipsoidal shape for cortex and a rectangular shape for endodermis (Fig. 3E). In scr-3 and shr-2 cross-sections, cells in the single-layered ground tissue exhibited an ellipsoidal shape similar to cells in the wild-type cortex (Fig. 3F, G). In ago1-101, although ground tissue layers were not arranged concentrically, they were still made up of cells with typical shapes for ground tissue; Fig. 3 Loss of AGO1 in the shr or scr mutant background resulted in novel radial patterning defects. (A–D) Longitudinal confocal sections of 4 dpg roots of the wild type (A), scr-3 (B), ago1-101 (C) and ago1-101 scr-3 (D). Note that cellular patterns in ago1-101 scr-3 double mutants were highly disorganized to the level that ground tissue was hardly discernible. (E–J) Root cross-sections of 4 dpg wild type (E), scr-3 (F), shr-2 (G), ago1-101 (H), ago1-101 scr-3 (I) and ago1-101 shr-2 (J). Cell type labels are the same as in Fig. 1. Scale bars, 20 µm. Plant Cell Physiol. 50(3): 626–634 (2009) doi:10.1093/pcp/pcp020 © The Author 2009. 629 S. Miyashima et al. ellipsoidal cells in the outer layer and rectangular cells in the inner layers (Fig. 3H). In contrast, the corresponding region of ago1-101 scr-3 roots was filled with spherical cells with less radial stratification (Fig. 3I). Similar defects were observed in ago1-101 shr-2 double mutant roots (Fig. 3J). Other than the root radial patterning defects, phenotypes were similar to those described for each single mutant (Di Laurenzio et al. 1996, Bohmert et al. 1998, Helariutta et al. 2000). In summary, the simultaneous loss of AGO1 and SHR/SCR resulted in novel defects in radial patterning that were not observed in each single mutant. To investigate the differentiation status of the ground tissue in ago101 scr-3, we examined the expression of two genes, MAGPIE (MGP) and NUTCRACKER (NUC), which encode C2H2-type zinc finger proteins expressed in ground tissue (Levesque et al. 2006). We compared the accumulation of their transcripts in each genotype using real-time quantitative RT–PCR (qRT–PCR). Transcript levels of both MGP and NUC were reduced in ago-101 and scr-3 single mutants compared with the wild type, and were even more reduced in ago1-101 scr-3 (Fig. 4A, B). We also examined pMGP::mGFP5ER expression in these genotypes. In wild-type roots, pMGP::mGFP5ER was expressed in ground tissue and the pericycle (Fig. 4C). This expression almost matched previously reported in situ hybridization data (Levesque et al. 2006). In either scr-3 or ago1-101, the GFP signal was observed in the mutant ground tissue layer and pericycle (Fig. 4D, E). In contrast, in ago1-101 scr-3, a weak GFP signal was detected in the basal region and no signal was detected in the meristematic region (Fig. 4F). Additionally, we analyzed the expression of an endodermis marker En7 in each mutant background (Fig. 4G–J) (Heidstra et al. 2004). In scr-3 and ago1-101, En7 was expressed in the mutant ground tissue and the innermost ground tissue layer, respectively (Fig. 4H, I). In contrast, most ago1-101 scr-3 roots failed to express the En7 marker (75.8%, n = 34) (Fig. 4J) or expressed it only weakly. Taken together, these results indicate that loss of AGO1 function in the scr mutant background causes further loss of the ground tissue identity in roots. AGO1 regulates ground tissue patterning postembryonically Ground tissue identity is initially established during embryogenesis (Scheres et al. 1994, Wysocka-Diller et al. 2000). To assess whether loss of AGO1 affects the establishment of ground tissue in embryogenesis, we examined cell division and marker expression patterns in embryos. In torpedo stage embryos where single-layered CEIs and two-layered ground tissue formed normally, the CEIs in most ago1 embryos divided periclinally (81.3%, n = 16) (arrows in Fig. 5B, compare with Fig. 5A). Sporadic periclinal divisions were also observed in the endodermal cell layer of ago1 embryos (arrowheads in Fig. 5B, F). In contrast, single-layered ground tissue was formed in ago1-101 scr-3 embryos similarly to scr-3 embryos (Fig. 5C, D). These observations indicate that scr-3 is epistatic to ago1-101 for the extra cell divisions in A 1.2 1.0 MGP Relative expression levels (normalized by ACT7 expression) 0.8 0.6 0.4 0.2 0 B 1 -10 l-0 01 Co o1-1 scr-3 ago1 3 g a scr 1.2 1.0 NUC 0.8 0.6 0.4 0.2 0 1 -10 01 l-0 Co o1-1 scr-3 ago1 3 g a scr Fig. 4 Simultaneous loss of AGO1 and SCR eliminates root ground tissue identity. (A, B) MGP (A) and NUC (B) transcript levels measured by real-time qRT–PCR. Quantification was normalized to the ACTIN7 (ACT7) transcript levels. Values relative to the wild-type measurements are shown. (C–J) Expression of pMGP::mGFP5ER (C–F) and the En7 marker (G–J) in the wild type (C, G), ago1-101 (D, H), scr-3 (E, I) and ago1-101 scr-3 (F, J). Scale bars, 50 µm 630 Plant Cell Physiol. 50(3): 626–634 (2009) doi:10.1093/pcp/pcp020 © The Author 2009. ARGONAUTE1 in root radial pattern formation Fig. 5 The contribution of AGO1 to ground tissue patterning is negligible in embryos. (A–H) Longitudinal confocal sections of torpedo stage embryos of the wild type (A, E), ago1-101 (B, F), scr-3 (C, G) and ago1-101 scr-3 (D, H). pMGP::mGFP5ER expression patterns are shown in E and F. Lower panels in A–D show higher magnification images of the boxed regions. Pe, pericycle. Other cell type labels are the same as in Fig. 1. Arrows and arrowheads indicate the periclinal division of CEI and endodermis, respectively. Scale bars, 50 µm. embryos, in contrast to their synergistic effects observed in mature roots. Expression patterns of pMGP::mGFP5ER in torpedo stage embryos were not significantly different among the genotypes. In wild-type embryos, pMGP::mGFP5ER was expressed in the pericycle, endodermis and cortex, the same pattern as in roots (Fig. 5E). This expression pattern was essentially unaffected in scr-3, ago1-101 or scr-3 ago1-101 embryos (Fig. 5F–H). The maintenance of pMGP::mGFP5ER expression in scr-3 ago1-101 embryos is noteworthy, because it is mostly lost in scr-3 ago1-101 roots (Fig. 4F). Taken together, these observations suggest that, in ground tissue patterning, AGO1-dependent regulatory mechanisms operate mostly after embryogenesis, while SHR/SCR-dependent regulation acts both in the initial patterning process in embryos and in post-embryonic maintenance. Discussion Previous studies have shown that SHR and SCR are required for the asymmetric cell division that separates the two ground tissue cell layers, endodermis and cortex, as well as for endodermal cell fate specification (Di Laurenzio et al. 1996, Helariutta et al. 2000, Heidstra et al. 2004, Sena et al. 2004). However, both shr and scr mutants maintained singlelayered ground tissue, indicating that the formation and maintenance of ground tissue per se are controlled by as yet known pathways independent of SHR and SCR. In this study, we demonstrated that direct transcriptional targets of the SHR–SCR complex, MGP and NUC, as well as a marker of endodermis, En7, were still expressed in the ground tissue of scr, supporting the notion that the formation of root ground tissue is not solely dependent on SHR/SCR-mediated transcriptional control. Mutant screening based on direct observations of cellular patterns by CLSM led us to identify AGO1 as a novel regulator of root ground tissue patterning. In ago1 root cross-sections, the number of ground tissue layers was increased to three in contrast to the two-layered ground tissue typical of wildtype Arabidopsis roots. This phenotype is reminiscent of transgenic plants ectopically expressing SHR that contained multiple layers of endodermis (Helariutta et al. 2000, Nakajima et al. 2001). However, En7 expression indicates that only the innermost ground tissue of ago1 is differentiated as endodermis. The independence of AGO1- and SHR/SCR-mediated ground tissue patterning was further demonstrated by the observation that expression of SHR and SCR was not affected in ago1 mutants. This is also consistent with the fact that no miRNA species in the Arabidopsis genome have so far been reported to match the SHR- or SCR-coding sequences (Gustafson et al. 2005). Therefore, we conclude that AGO1 regulates root ground tissue patterning independently of the SHR/SCR pathway. Analyses of ago1 scr and ago1 shr double mutants revealed novel patterning defects that were not apparent in any of the single mutants. In the double mutant backgrounds, Plant Cell Physiol. 50(3): 626–634 (2009) doi:10.1093/pcp/pcp020 © The Author 2009. 631 S. Miyashima et al. cells taking typical shapes of endodermis or cortex were missing. Instead, most cells in root cross-sections appeared uniform both in size and in shape. Expression levels of MGP, NUC and En7 were also dramatically reduced in the double mutants as compared with each single mutant. These results indicate that ground tissue cell patterning is controlled by at least two independent pathways; one using AGO1-mediated post-transcriptional regulation and the other using SHR/ SCR-mediated gene transcription. It would be interesting to determine whether the spherical cells in ago1 scr double mutants have taken the identity of cells other than those in ground tissue, such as provascular or epidermal cells. The observed synergistic effects of the two pathways could be explained in several ways. In a simple scenario, the two pathways independently promote ground tissue identity, but later the output of one pathway acts to stabilize the effect of the other. However, based on the postulated functions of AGO1 in the negative regulation of mRNA accumulation, a more plausible explanation would be that SHR/SCR and an unknown third pathway promote ground tissue identity and that these activities are antagonized by AGO1 targets. In the absence of SHR/SCR, ground tissue identity can still be maintained by the third pathway while a functional AGO1 effectively suppresses the antagonizing effects. In the absence of AGO1, misexpressed target genes attenuate ground tissue identity to some extent. If the functions of both SHR/SCR and AGO1 are lost, the third pathway alone cannot withstand the effects of misexpressed AGO1 targets. Although roles for AGO1 in embryogenesis have been described for the adaxial–abaxial polarity of cotyledons (Lynn et al. 1999), its contribution to embryonic ground tissue patterning seems to be relatively small. While ago1 mutant embryos showed a weak defect in cell division, this was repressed by the scr-3 mutation. Moreover, ago1 scr double mutant embryos retained expression of MGP in contrast to its severe down-regulation in roots. So far, the mechanism behind the different epistatic relationship is unclear. Based on previous reports (Lynn et al. 1999, Birnbaum et al. 2003) and our reporter analysis (S.M., T.H. and K.N. unpublished data), AGO1 is expressed throughout the embryos and roots. It is possible that the weak cell division defect seen in the ago1 embryos resulted from the misregulation of miRNA targets different from those in post-embryonic roots, and that only the former could be effectively suppressed by the scr mutation. What genes then are subjected to AGO1-dependent degradation to create a precise ground tissue pattern? Among GRAS-type transcription factors, SCARECROW-LIKE 6 is known to be regulated by miRNA170/171 (Llave et al. 2002b). A previous report showed that miR171-mediated regulation was active in restricted tissues of aerial organs but not in roots (Parizotto et al. 2004). A search of our microarray data set comparing transcript levels in ago1 and wild-type roots, 632 combined with information from published lists of miRNAregulated genes (Fahlgren et al. 2007), revealed about 50 candidate genes for AGO1-mediated regulation in root ground tissue patterning (data not shown). Analysis of their expression patterns in the wild-type and ago1 backgrounds, together with the transgenic expression of miRNA-resistant versions, will allow us to identify the target genes for AGO1mediated root radial patterning. Materials and Methods Plant materials and growth conditions Arabidopsis thaliana Columbia ecotype (accession Col-0) was used as the wild type. The 5gf32 mutant was isolated from M2 plants derived from EMS-mutagenized Arabidopsis seeds harboring a WUSCHEL RELATED HOMEOBOX5 (WOX5)-GFP marker gene (Sarkar et al. 2007). 5gf32 was backcrossed twice with Col-0. ago1-101 (SALK_035319), ago1-102 (SALK_096625) and hyl1-2 (CS859864) (Vazquez et al. 2004) were obtained from the Arabidopsis Biological Resource Center. T-DNA insertion positions of ago1-101 and ago1-102 were determined as 1,261 and 597 bp 3′ downstream from the predicted translation start site, respectively. shr-2, scr-3 and hyl1-2 mutants as well as SHR-GFP and GFPSCR marker lines have been described (Fukaki et al. 1998, Helariutta et al, 2000, Nakajima et al. 2001, Gallagher et al. 2004). Arabidopsis seeds were allowed to germinate on plates containing 0.5× Arabidopsis nutrient solution (Haughn and Somerville 1986) supplemented with 1% (w/v) sucrose and 1% (w/v) agar. Plants were grown at 23°C with 16 h light/8 h dark cycles. Expression analysis For the AGO1 expression analysis in ago1 mutants, total RNA was extracted from 10 dpg seedlings with an RNAeasy Plant Minikit (Qiagen, Valencia, CA, USA). First-strand cDNA was synthesized by Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with oligo(dT) primers, and used as a template for PCR amplification. For MGP and NUC expression analyses, total RNA was extracted from 4 dpg roots, and first-strand cDNA was synthesized as described above. qRT– PCR for MGP, NUC and ACT7 was performed using the LightCycler system (Roche Diagnostics, Basel, Switzerland) with SYBR Premix Ex Taq (TAKARA BIO INC., Shiga, Japan). Sequences of the primers used are as follows: MGPf, 5′tccagaagctgaggtcatag-3′; MGPr, 5′-tggactgcatatctcttggc-3′; NUCf, 5′-aaatctccctggaaatcctga-3′; NUCr, 5′-cctttgccaca tacctcaca-3′; ACT7f, 5′-cgctgcttctcgaatcttct-3′; ACT7r, 5′-ccatt ccagttccattgtca-3′; AGO1f, 5′-attgttgaaggccagcggta-3′; AGO1r, 5′-TCCATTGACCAACTTGTGGC-3′; ACT8f, 5′-ATGAAGA TTAAGGTCGTGGCA-3′; and ACT8r, 5′-TCCGAGTTTGAA GAGGCTAC-3′. Plant Cell Physiol. 50(3): 626–634 (2009) doi:10.1093/pcp/pcp020 © The Author 2009. ARGONAUTE1 in root radial pattern formation Plasmid construction and generation of transgenic plants To construct the pMGP::mGFP5ER reporter, a 3,790 bp promoter fragment of MGP (At1g03480) was amplified by PCR with the primers MGP(-)3790, 5′-gtggaaaatggttggaaagc-3′ and MGPproEND, 5′-gtcttcttcttggacaaaagttttg-3′. This fragment was ligated with the mGFP5ER-coding sequence (a gift from Jim Haseloff, Cambridge, UK) and inserted in a binary vector pBIN19AN (Hirota et al. 2007) carrying a hygromycin resistance marker. Microscopy CLSM was performed with a C1-ECLIPSE E600 confocal laser scanning microscope (Nikon, Tokyo, Japan). Samples were stained with 10 µM propidium iodide. For dissected embryos, 7% (w/v) glucose was included in the staining solution. Root cross-sections were prepared as basically described (Furutani et al. 2000). The contrast of some pictures was enhanced with the PHOTOSHOP program (Adobe Systems, San Jose, CA, USA). Funding The Japan Society for the Promotion of Science (JSPS) Grant-in-aid for Scientific Research (to K.N. and S.M.); JSPS Research Fellowships for Young Scientists (to S.M.).. Acknowledgments We thank Ben Scheres, Renze Heidstra, Philip Benfey, Hidehiro Fukaki, Masao Tasaka, Jim Haseloff and the Arabidopsis Biological Resource Center for materials. References Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism and function. Cell 116: 281–297. Baum, S.F. and Rost, T.L. (1996) Root apical organization in Arabidopsis thaliana. 1. Root cap and protoderm. Protoplasma 192: 178–188. Baumberger, N. and Baulcombe, D.C. (2005) Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc. Natl Acad. Sci. USA 102: 11928–11933. Birnbaum, K., Shasha, D.E., Wang, J.Y., Jung, J.W., Lambert, G.M., Galbraith, D.W., et al. (2003) A gene expression map of the Arabidopsis root. Science 302: 1956–1960. Bohmert, K., Camus, I., Bellini, C., Bouchez, D., Caboche, M. and Benning, C. (1998) AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J. 17: 170–180. Chen, X. (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303: 2022–2025. Cui, H., Levesque, M.P., Vernoux, T., Jung, J.W., Paquette, A.J., Gallagher, K.L., et al. (2007) An evolutionarily conserved mechanism delimiting SHR movement defines a single layer of endodermis in plants. Science 316: 421–425. Di Laurenzio, L., Wysocka-Diller, J., Malamy, J.E., Pysh, L., Helariutta, Y., Freshour, G., et al. (1996) The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 86: 423–433. Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, S., Roberts, K., et al. (1993) Cellular organisation of the Arabidopsis thaliana root. Development 119: 71–84. Fahlgren, N., Howell, M.D., Kasschau, K.D., Chapman, E.J., Sullivan, C.M., Cumbie, J.S., et al. (2007) High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS ONE 2: e219. Fukaki, H., Wysocka-Diller, J., Kato, T., Fujisawa, H., Benfey, P.N. and Tasaka, M. (1998) Genetic evidence that the endodermis is essential for shoot gravitropism in Arabidopsis thaliana. Plant J. 14: 425–430. Furutani, I., Watanabe, Y., Prieto, R., Masukawa, M., Suzuki, K., Naoi, K., et al. (2000) The SPIRAL genes are required for directional control of cell elongation in Arabidopsis thaliana. Development 127: 4443–4453. Gallagher, K.L., Paquette, A.J., Nakajima, K. and Benfey, P.N. (2004) Mechanisms regulating SHORT-ROOT intercellular movement. Curr. Biol. 14: 1847–1851. Gustafson, A.M., Allen, E., Givan, S., Smith, D., Carrington, J.C. and Kasschau, K.D. (2005) ASRP: the Arabidopsis Small RNA Project Database. Nucleic Acids Res. 33: D637–640. Han, M.-H., Goud, S., Song, L. and Fedoroff, N. (2004) The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc. Natl Acad. Sci. USA 101: 1093–1098. Haughn, G.W. and Somerville, C. (1986) Sulfonylurea-resistant mutants of Arabidopsis thaliana. Mol. Gen. Genet. 204: 430–434. Heidstra, R., Welch, D. and Scheres, B. (2004) Mosaic analyses using marked activation and deletion clones dissect Arabidopsis SCARECROW action in asymmetric cell division. Genes Dev. 18: 1964–1969. Helariutta, Y., Fukaki, H., Wysocka-Diller, J., Nakajima, K., Jung, J., Sena, G., et al. (2000) The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell 101: 555–567. Hirota, A., Kato, T., Fukaki, H., Aida, M. and Tasaka, M. (2007) The auxin-regulated AP2/EREBP gene PUCHI is required for morphogenesis in the early lateral root primordium of Arabidopsis. Plant Cell 19: 2156–2168. Hutvagner, G. and Simard, M.J. (2008) Argonaute proteins: key players in RNA silencing. Nat. Rev. Mol. Cell Biol. 9: 22–32. Kidner, C.A. and Martienssen, R.A. (2004) Spatially restricted microRNA directs leaf polarity through ARGONAUTE1. Nature 428: 81–84. Levesque, M.P., Vernoux, T., Busch, W., Cui, H., Wang, J.Y., Blilou, I., et al. (2006) Whole-genome analysis of the SHORT-ROOT developmental pathway in Arabidopsis. PLoS Biol. 4: e143 EP. Llave, C., Kasschau, K.D., Rector, M.A. and Carrington, J.C. (2002a) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14: 1605–1619. Llave, C., Xie, Z., Kasschau, K.D. and Carrington, J.C. (2002b) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297: 2053–2056. Lynn, K., Fernandez, A., Aida, M., Sedbrook, J., Tasaka, M., Masson, P., et al. (1999) The PINHEAD/ZWILLE gene acts pleiotropically in Plant Cell Physiol. 50(3): 626–634 (2009) doi:10.1093/pcp/pcp020 © The Author 2009. 633 S. Miyashima et al. Arabidopsis development and has overlapping functions with the ARGONAUTE1 gene. Development 126: 469–481. Mette, M.F., van der Winden, J., Matzke, M. and Matzke, A.J. (2002) Short RNAs can identify new candidate transposable element families in Arabidopsis. Plant Physiol. 130: 6–9. Mi, S., Cai, T., Hu, Y., Chen, Y., Hodges, E., Ni, F., et al. (2008) Sorting of small RNAs into Arabidopsis Argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133: 116–127. Nakajima, K., Sena, G., Nawy, T. and Benfey, P.N. (2001) Intercellular movement of the putative transcription factor SHR in root patterning. Nature 413: 307–311. Parizotto, E.A., Dunoyer, P., Rahm, N., Himber, C. and Voinnet, O. (2004) In vivo investigation of the transcription, processing, endonucleolytic activity and functional relevance of the spatial distribution of a plant miRNA. Genes Dev. 18: 2237–2242. Park, W., Li, J., Song, R., Messing, J. and Chen, X. (2002) CARPEL FACTORY, a Dicer homolog and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr. Biol. 12: 1484–1495. Qi, Y., Denli, A.M. and Hannon, G.J. (2005) Biochemical specialization within Arabidopsis RNA silencing pathways. Mol. Cell 19: 421–428. Rajagopalan, R., Vaucheret, H., Trejo, J. and Bartel, D.P. (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev. 20: 3407–3425. Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B. and Bartel, D.P. (2002) MicroRNAs in plants. Genes Dev. 16: 1616–1626. Sabatini, S., Heidstra, R., Wildwater, M. and Scheres, B. (2003) SCARECROW is involved in positioning the stem cell niche in the Arabidopsis root meristem. Genes Dev. 17: 354–358. Sarkar, A.K., Luijten, M., Miyashima, S., Lenhard, M., Hashimoto, T., Nakajima, K., et al. (2007). Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers. Nature 446: 811–814. Scheres, B., Wolkenfelt, W., Willemsen, V., Terlouw, M., Lawson, E., Dean, C., et al. (1994) Embryonic origin of the Arabidopsis primary root and root meristem initials. Development 120: 2475–2487. 634 Scheres, B., Di Laurenzio, L., Willemsen, V., Hauser, M.T., Janmaat, K., Weisbeek, P., et al. (1995) Mutations affecting the radial organisation of the Arabidopsis root display specific defects throughout the embryonic axis. Development 121: 53–62. Sena, G., Jung, J.W. and Benfey, P.N. (2004) A broad competence to respond to SHORT ROOT revealed by tissue-specific ectopic expression. Development 131: 2817–2826. Sorin, C., Bussell, J.D., Camus, I., Ljung, K., Kowalczyk, M., Geiss, G., et al. (2005) Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1. Plant Cell 17: 1343–1359. Tang, G., Reinhart, B.J., Bartel, D.P. and Zamore, P.D. (2003) A biochemical framework for RNA silencing in plants. Genes Dev. 17: 49–63. Vaucheret, H. (2008) Plant ARGONAUTES. Trends Plant Sci. 13: 350–358. Vaucheret, H., Vazquez, F., Crete, P. and Bartel, D.P. (2004) The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev. 18: 1187–1197. Vazquez, F., Gasciolli, V., Crete, P. and Vaucheret, H. (2004) The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Curr. Biol. 14: 346–351. Welch, D., Hassan, H., Blilou, I., Immink, R., Heidstra, R. and Scheres, B. (2007) Arabidopsis JACKDAW and MAGPIE zinc finger proteins delimit asymmetric cell division and stabilize tissue boundaries by restricting SHORT-ROOT action. Genes Dev. 21: 2196–2204. Willmann, M.R. and Poethig, R.S. (2007) Conservation and evolution of miRNA regulatory programs in plant development. Curr. Opin. Plant Biol 10: 503–511. Wysocka-Diller, J., Helariutta, Y., Fukaki, H., Malamy, J. and Benfey, P. (2000) Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot. Development 127: 595–603. Plant Cell Physiol. 50(3): 626–634 (2009) doi:10.1093/pcp/pcp020 © The Author 2009. (Received November 27, 2008; Accepted January 28, 2009)