Download ARGONAUTE1 Acts in Arabidopsis Root Radial

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.
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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.
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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
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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.
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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
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MGP
Relative expression levels
(normalized by ACT7 expression)
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0
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-10
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-10
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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
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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,
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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.
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(Received November 27, 2008; Accepted January 28, 2009)