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Histone Deacetylases and ASYMMETRIC LEAVES2Are Involved in the Establishment of Polarity inLeaves of Arabidopsis W
Yoshihisa Ueno,a Takaaki Ishikawa,a,b Keiro Watanabe,c Shinji Terakura,a Hidekazu Iwakawa,a,b
Kiyotaka Okada,c,d Chiyoko Machida,b,d and Yasunori Machidaa,1
a Division of Biological Sciences, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8602, Japanb College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japanc Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japand CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 322-0012, Japan
We show that two Arabidopsis thaliana genes for histone deacetylases (HDACs), HDT1/HD2A and HDT2/HD2B, are required
to establish leaf polarity in the presence of mutant ASYMMETRIC LEAVES2 (AS2) or AS1. Treatment of as1 or as2 plants with
inhibitors of HDACs resulted in abaxialized filamentous leaves and aberrant distribution of microRNA165 and/or micro-
RNA166 (miR165/166) in leaves. Knockdown mutations of these two HDACs by RNA interference resulted in phenotypes like
those observed in the as2 background. Nuclear localization of overproduced AS2 resulted in decreased levels of mature
miR165/166 in leaves. This abnormality was abolished by HDAC inhibitors, suggesting that HDACs are required for AS2
action. A loss-of-function mutation in HASTY, encoding a positive regulator of miRNA levels, and a gain-of-function muta-
tion in PHABULOSA, encoding a determinant of adaxialization, suppressed the generation of abaxialized filamentous leaves
by inhibition of HDACs in the as1 or as2 background. AS2 and AS1 were colocalized in subnuclear bodies adjacent to the
nucleolus where HDT1/HD2A and HDT2/HD2B were also found. Our results suggest that these HDACs and both AS2 and
AS1 act independently to control levels and/or patterns of miR165/166 distribution and the development of adaxial-abaxial
leaf polarity and that there may be interactions between HDACs and AS2 (AS1) in the generation of those miRNAs.
INTRODUCTION
The determination of cell fate along the adaxial-abaxial axis is
essential for the development of leaves (Sussex, 1955; Waites and
Hudson, 1995). Previous genetic analyses in Arabidopsis thaliana
revealed that FILAMENTOUS FLOWER (FIL), YABBY3, KANADI1
(KAN1), KAN2, KAN3, ETTIN/AUXIN RESPOSE FACTOR3 (ARF3),
and ARF4 play important roles in the determination of abaxial cell
fate and the subsequent expansion of the lamina (Eshed et al., 1999,
2001, 2004; Sawa et al., 1999; Siegfried et al., 1999; Kerstetter
et al., 2001; Pekker et al., 2005). By contrast, the extensive and
enhanced accumulation in Arabidopsis leaf cells of the tran-
scripts of the PHABULOSA (PHB), PHAVOLUTA (PHV), and
REVOLUTA (REV) genes, in which target sites for microRNA165
and/or microRNA166 (miR165/166) had been disrupted, resulted
in changes in cell fate from abaxial to adaxial (McConnell et al.,
2001; Emery et al., 2003; Mallory et al., 2004). Therefore, these
latter genes appear to be redundant positive regulators that are
sufficient for the determination of adaxial cell fate, and the activ-
ity of these genes appears to be regulated by microRNA (miRNA).
Kidner and Martienssen (2004) proposed that the adaxially re-
stricted distribution of transcripts of PHB, PHV, and REV can be
ascribed to that of miR165/166 on the abaxial side of leaf primor-
dia. Surgical isolation of leaf primordia results in the outgrowth of
abaxialized radial organs, suggesting that a meristem-derived
signal directs adjacent regions of leaf primordia to adopt an
adaxial fate (Sussex, 1955). Therefore, one of the most important
cues for the establishment of adaxial-abaxial polarity in the devel-
opment of leaves appears to be the regulation of the distribution
of miR165/166. Thus, the existence has been proposed of a reg-
ulatory mechanism that involves miR165/166 on the adaxial side
of leaves.
A loss-of-function mutation of the PHANTASTICA (PHAN)
gene in Antirrhinum majus, in combination with low-temperature
treatment or with the handlebars mutation, results in a defect
in the adaxial development of leaves (Waites and Hudson, 1995,
2001). Arabidopsis plants with the recessive mutations asym-
metric leaves1 (as1) and as2 exhibit various defects in leaf devel-
opment (Redei and Hirono, 1964; Serrano-Cartagena et al.,
1999; Byrne et al., 2000; Semiarti et al., 2001; Xu et al., 2003).
Moreover, the phenotype of as1 as2 double mutants is similar to
that of each single mutant, suggesting that AS1 and AS2 function
within the same pathway (Serrano-Cartagena et al., 1999). AS1
encodes a MYB protein, which is a homolog of the products of
1 To whom correspondence should be addressed. E-mail [email protected]; fax 81-52-789-2966.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Yasunori Machida([email protected]).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.106.042325
The Plant Cell, Vol. 19: 445–457, February 2007, www.plantcell.org ª 2007 American Society of Plant Biologists
the PHAN and ROUGH SHEATH2 (RS2) genes in maize (Zea
mays; Byrne et al., 2000). AS2 encodes a protein that contains an
AS2/LATERAL ORGAN BOUNDARY (LOB) domain (Iwakawa
et al., 2002). In Arabidopsis plants with an erecta (er) background
and an as1 or as2 mutation, leaf development is partially deficient
on the adaxial side, suggesting that, similarly to PHAN, AS1 and
AS2 are involved in the regulation of adaxial cell-fate determi-
nation (Xu et al., 2003). The involvement of AS2 in the adaxial-
abaxial polarity has been also reported by Lin et al. (2003). The
accumulation of miR165/166 is enhanced in as1 er and as2 er
double mutants grown at high temperature (Li et al., 2005).
However, we still do not know how AS1 and AS2 regulate the
accumulation of miR165/166 and how they determine, in this
way, fate of leaf cells.
The pickle (pkl) mutation enhances the severity of the abnormal
phenotype of as1 and as2 mutants (Ori et al., 2000). PKL encodes
an ortholog of Mi-2, which belongs to the family of SWI2/SNF2
ATPase chromatin-remodeling factors (Eshed et al., 1999; Ogas
et al., 1999). Moreover, Mi-2 is also a component of the NuRD
complex that is involved in transcriptional repression and in-
cludes two histone deacetylases (HDACs; Ahringer, 2000).
In this report, we show that the HDACs HDT1/HD2A and
HDT2/HD2B are involved in the control of miRNA expression and
the determination of cell fate in leaves of as2 mutant plants. We
propose a model, based on our results, for the mechanism that
underlies the establishment of polarity in Arabidopsis leaves.
RESULTS
Inhibition of HDAC Activity Results in Abaxialized
Filamentous Leaves in as1 and as2 Mutant Plants
We treated as1 and as2 mutant plants with specific inhibitors of
HDACs, namely, trichostatin A (TSA) and 4-(dimethylamino)-
N-[6-(hydroxyamino)-6-oxohexyl]-benzamide (CAY10398; CAY)
at concentrations from 0 to 10 mM and 0 to 100 mM, respectively.
On medium that contained 1 to 3 mM TSA or 10 to 30 mM CAY,
the adaxial-abaxial polarity of leaves of wild-type plants was
unaffected or only slightly affected. By contrast, as1 and as2
mutant plants frequently produced filamentous leaves with re-
duced numbers of trichomes (Figures 1A to 1F, Table 1) and
trumpet-shaped leaves. This effect was dependent on the dose
of inhibitor and the mutation (as1 or as2; Figures 1G and 1H) and
was nonspecific to the allelic series examined (Table 1). Protein
gel blot analysis revealed a stepwise increase in overall levels of
acetylation of histone H4 in as2 plants after treatment with 1 to
3 mM TSA for 3 h (Figure 1I). These results were correlated with the
severity of the mutant phenotype. Similar results were obtained
with wild-type plants and as1 mutant plants (see Supplemental
Figure 1 online). Medium containing 10 mM TSA or 100 mM CAY
was lethal to wild-type, as1, and as2 plants. These results
indicated that the efficacy of each drug was unaffected by the
as1 and as2 mutations and that inhibition of HDAC activity
enhanced the severity of the mutant phenotype of as1 and as2
mutant leaves specifically.
In the filamentous leaves, there was no obvious xylem system
of the type that is normally localized adaxially in leaf vascular
bundles (cf. Figure 1D to 1F). Strong signals from green fluo-
rescent protein (GFP), encoded by a gene for GFP under the
control of the FIL promoter (Watanabe and Okada, 2003) and
designated FILp:GFP, were detected in cells at peripheral po-
sitions in such filamentous leaves (Figures 1K and 1L), whereas
no signals were detected in cells on the adaxial side of leaves of
wild-type plants or of as2 mutant plants in the absence of these
inhibitors (Watanabe and Okada, 2003; Figure 1J). We obtained
similar results with the as1 mutant (Figure 1M). Furthermore,
scanning electron microscopy revealed that the epidermal cells
of filamentous leaves on as2-1 mutant plants grown in the pres-
ence of TSA were long and narrow (Figure 1N), resembling those
of transgenic plants in which KAN2 is ectopically expressed
(Eshed et al., 2001) and of as2 er double mutant plants grown
at high temperature (Xu et al., 2003). These results suggested
that the filamentous leaves that formed on as1 and as2 mutant
plants in the presence of HDAC inhibitors had been abaxialized
(Figure 1O).
The Distribution of miR165/166 Is Aberrant in Leaves of
as1 and as2 Mutant Plants Grown on Medium with TSA
To monitor the accumulation and activity of miR165/166 in situ,
we constructed a reporter gene (Figure 2). This reporter gene
included erG3GFP (Tamai and Meshi, 2001) for targeting to the
endoplasmic reticulum and a sequence complementary to
miR165 (c165) in the 39 untranslated region. The gene was fused
to the 35S promoter of Cauliflower mosaic virus (35S; Figure 2A).
We also constructed two control reporter genes. One of them
had a point mutation that mimics that of phb-3D (McConnell
et al., 2001) in the region of c165 and the other did not include
c165. The constructs were designated 35S:erG3GFP:c165,
35S:erG3GFP:mc165, and 35S:erG3GFP, and each was intro-
duced into wild-type plants (Columbia-0 [Col-0]).
In sections of a 35S:erG3GFP plant (line #3.2), the signal due to
GFP was detected constitutively (Figure 2D). At a less-sensitive
level of detection (Figures 2B and 2C), the signal was relatively
strong in vascular bundles and the epidermis, and no difference
between the adaxial and abaxial sides of the leaf was evident.
Similar results were obtained for 35S:erG3GFP:mc165. By con-
trast, the signal on the adaxial side was stronger than on the
abaxial side of the leaf in a 35S:erG3GFP:c165 plant (line #2.1)
at three different levels of sensitivity (Figures 2E to 2G), suggest-
ing the prominent accumulation of miR165/166 on the abaxial
side of wild-type leaves. Similar patterns of fluorescence were ob-
served in another Col-0 transgenic line (line #2.2) of 35S:erG3GFP:
c165 plants and independent lines of as2-1 35S:erG3GFP:c165
plants (described below), suggesting that these patterns of GFP
fluorescence were not specific to the first-mentioned transgenic
line.
Signals due to GFP in cells of young leaves of as2-1 35S:
erG3GFP:c165 plants (line #1.1), grown on medium without
inhibitors, were also much stronger on the adaxial than on the
abaxial side (Figure 2H), resembling those of 35S:erG3GFP:
c165 plants (Figures 2E to 2G). By contrast, signals from the
filamentous leaves of as2-1 35S:erG3GFP:c165 plants, grown on
medium with TSA, were weaker on the adaxial side and limited to
central regions (Figure 2I). Similar results were obtained with
446 The Plant Cell
another line of as2-1 35S:erG3GFP:c165 plants (line #1.2) and
with independent transgenic as2-1 35S:GUS:c165 lines (lines
#G1.1 and #G3.1; see Supplemental Figures 2A to 2D online),
suggesting that the ectopic localization of miR165/166 occurred
on the adaxial side of young leaves as a result of the combination
of the as2 mutation and inhibition of HDACs.
AS2 Protein Functions in Nuclei
To investigate the function of AS2 in the nuclei (Iwakawa et al.,
2002), we introduced, into Col-0 plants, a chimeric gene that
encoded the ligand binding domain of the glucocorticoid recep-
tor (GR) fused to the C terminus of AS2, in frame, under the con-
trol of the 35S promoter. We designated this gene 35S:AS2-GR
(Figure 3A). The nuclear localization of AS2-GR was induced
by dexamethazone (Dex) and by mifepristone (RU486; RU). A
chimeric protein, in which GFP and GR were fused to the C
terminus of AS2, designated AS2-GFP-GR, was concentrated in
the nucleus after treatment with Dex of tobacco (Nicotiana
tabacum) BY-2 cultured cells that had been transformed with
35S:AS2-GFP-GR (see Supplemental Figure 3 online). We gen-
erated 35S:AS2-GR transgenic Arabidopsis plants (line #5.4)
and introduced the 35S:AS2-GR construct into as2-1 plants by
genetic cross. The resulting plant was designated as2-1 35S:
AS2-GR (line #5.4). After treatment with Dex, as2-1 35S:AS2-GR
plants had normal leaves (see Supplemental Figure 4 online).
When the conserved Gly residue (Iwakawa et al., 2002) in the
AS2-GR encoded by the transgene was replaced by Glu, res-
toration of normal leaf shape did not occur (see Supplemental
Figure 4 online). Therefore, we concluded that the AS2-GR
fusion protein was functional in the nuclei of as2-1 35S:AS2-GR
plants.
Figure 1. Inhibitors of HDACs Induce Formation of Abaxialized Filamentous Leaves in as2 Plants.
(A) to (F) as2-1 plants were grown on medium without (mock-treated; [E] and [F]) or with 30 mM CAY ([A] and [B]) or 3 mM TSA ([C] and [D]). (A) and (B)
Cleared specimens; (C) to (F) transverse sections of leaves. The image shown in (C) was obtained from the midzone of a filamentous leaf that formed on
an as2-1 mutant plant in the presence of TSA. The section was cut from approximately the same region as that indicated by the red line in (B). Arrows in
(F) indicate xylem. (B), (D), and (F) Magnified views of boxed areas in (A), (C), and (E), respectively. (A) and (B) An obvious xylem system, visible as white
lines, was not observed in filamentous leaves. Bars ¼ 1 mm in (A), 100 mm in (B) and (E), 50 mm in (C), and 10 mm in (D) and (F).
(G) and (H) Dose-dependent formation of filamentous leaves on as2-1 and as1-1 mutants. Col-0 (squares), as1-1 (circles), and as2-1 (diamonds) plants
were grown for 17 d on medium that contained TSA or CAY as indicated on horizontal axes. The filamentous leaves on each plant were counted.
(I) Dose-dependent accumulation of hyperacetylated histone H4 in as2-1 mutants. Protein gel blot analysis of plants exposed to indicated doses of TSA
was performed with antibodies specific for acetylated histone H4 (AcH4) and histone H4 (H4).
(J) to (M) as2-1 FILp:GFP ([J] to [L]) and as1-1 FILp:GFP (M) plants were grown on medium without (mock treated; [J]) or with 3 mM TSA (K) or 30 mM
CAY ([L] and [M]). Transverse sections of leaves are shown. Green, signals from GFP; red, autofluorescence. Bars ¼ 50 mm.
(N) A filamentous leaf formed on an as2-1 mutant plant in the presence of 3 mM TSA. Bar ¼ 100 mm.
(O) Schematic representation of the results derived from (A) to (N). Leaves of the wild type, regardless of treatment with inhibitors, and of as1 and as2
mutant plants without inhibitors were expanded, and asymmetric polarity along an adaxial-abaxial axis was established. Radialized and unexpanded
leaves of as1 and as2 mutant plants grown with TSA or CAY lacked the adaxial domain. The ellipse represents a transverse section from an expanded
leaf; the circle represents a transverse section from a filamentous leaf. ad, adaxial domain; ab, abaxial domain.
HDACs and AS2 Regulate Leaf Polarity 447
Nuclear Localization of AS2-GR Protein Represses the
Accumulation of miR165/166
Ectopic expression of AS2 results in upwardly curling leaves
(Iwakawa et al., 2002; Lin et al., 2003). The phenotype resembles
that of plants with mutations in genes for either the biogenesis
ofmiRNAs or the targetsites for miR165/166.Toexamine the effect
of nuclear localization of AS2-GR protein on the distribution of
miR165/166, we generated 35S:AS2-GR (from line #5.4) 35S:
erG3GFP:c165 (from line#2.1)double-transgenicplantsbygenetic
cross. In leaves of the 35S:AS2-GR 35S:erG3GFP:c165 plants that
had been treated with RU, strong signals due to expression of GFP
extended to the abaxial side of leaves (Figures 3B and 3C),
suggesting that levels of miR165/166 had been reduced.
Transcripts of PHB were confined to the adaxial side of young
leaf primordia in wild-type plants (Figure 3D). They were also
found on the abaxial side of leaf primordia of 35S:AS2-GR plants
treated with RU (line #5.4; Figure 3E). These results suggested
that the nuclear localization of AS2-GR has suppressed expres-
sion of miR165/166 even on the abaxial side of leaves, with
resultant ectopic accumulation of PHB transcripts.
Upward Curling of Leaves and the Reduction in Levels of
miR165/166 Observed in 35S:AS2-GR Transgenic Plants
Depend on Endogenous AS1 and HDAC Activity
In the presence of 10 nM Dex or 1 mM RU, 35S:AS2-GR
transgenic plants (line #5.4) had severely upwardly curled leaves
(Figure 4A), while 100 nM Dex was lethal. Similar results were
obtained from four other independent transgenic lines (lines #1,
2, 3.4, and 4). To examine whether the phenotype depended on
AS1, we introduced 35S:AS2-GR (from line #5.4) into as1-1 mu-
tants by genetic cross. In the presence of 10 nM Dex, leaves of
as1-1 35S:AS2-GR plants did not curl upwards (Figure 4B),
unlike those of 35S:AS2-GR plants (Figure 4A), but 100 nM Dex
was still lethal. TSA also reduced the number of upwardly curling
leaves in 35S:AS2-GR plants (line #5.4; Figure 4C). These results
suggested that upwardly curling leaves required both endoge-
nous AS1 and HDAC activity.
Table 1. Frequencies of Radialized Leaves on Plants after Growth on
Medium That Contained an Inhibitor of HDACs
Numbers of Radialized Leaves per Plant
Genotype 0 1 2 3 >3 Total (n)
Mock Treatment
Col-0 105 (100%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 105
as1-1 118 (100%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 118
as2-1 128 (100%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 128
as2-4 132 (100%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 132
Ler 155 (100%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 155
as2-5 124 (100%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 124
Plus 3 mM TSA
Col-0 85 (93%) 4 (4%) 2 (2%) 0 (0%) 0 (0%) 91
as1-1 27 (22%) 34 (28%) 47 (39%) 7 (6%) 6 (5%) 121
as2-1 21 (19%) 34 (31%) 38 (35%) 11 (10%) 4 (4%) 108
as2-4 13 (13%) 22 (21%) 58 (56%) 10 (10%) 1 (1%) 104
Ler 117 (96%) 4 (3%) 1 (1%) 0 (0%) 0 (0%) 122
as2-5 33 (37%) 23 (26%) 18 (20%) 6 (7%) 10 (11%) 90
Plus 30 mM CAY10398
Col-0 87 (91%) 7 (7%) 2 (2%) 0 (0%) 0 (0%) 96
as1-1 22 (21%) 27 (25%) 28 (26%) 26 (25%) 3 (3%) 106
as2-1 16 (16%) 20 (20%) 27 (27%) 13 (13%) 23 (23%) 99
as2-4 5 (3%) 22 (14%) 96 (63%) 22 (14%) 7 (5%) 152
Ler 34 (94%) 1 (3%) 1 (3%) 0 (0%) 0 (0%) 36
as2-5 7 (8%) 7 (8%) 11 (13%) 5 (6%) 56 (65%) 86
The filamentous or trumpet-shaped leaves on each plant were counted
under a stereomicroscope 17 to 21 d after imbibition of seeds. Ler,
Landsberg erecta.
Figure 2. The 35S:erG3GFP:c165 Reporter Gene, Which Revealed the
Distribution of miR165/166, Demonstrated the Aberrant Accumulation of
miR165/166 in as2 Mutant Plants That Had Been Treated with TSA.
(A) Schematic representation of the 35S:erG3GFP:c165 and 35S:
erG3GFP:mc165 constructs. Sequences of c165, mc165, miR165, and
miR166 are shown. In the mc165 sequence, the adenine (A) that replaced
guanine (G) in the c165 sequence is underlined.
(B) to (G) Fluorescent signals due to 35S:erG3GFP (line #3.2; [B] to [D])
and 35S:erG3GFP:c165 (line #2.1; [E] to [G]) transgenic plants with a
Col-0 background. Three pictures were taken of individual sections from
each panel at different sensitivities. The sensitivity for detection of
fluorescence due to GFP was increased gradually from the top to the
bottom photograph in each column to allow visualization of overall
patterns of fluorescence. Bars ¼ 50 mm.
(H) and (I) as2-1 35S:erG3GFP:c165 (line #1.1) grown on medium without
(H) or with (I) 3 mM TSA. Signals due to GFP that were restricted to the
central region of a young leaf of an as2-1 35S:erG3GFP:c165 plant
treated with TSA are indicated by an arrow. Green, fluorescence due to
GFP; red, autofluorescence. Bars ¼ 50 mm.
448 The Plant Cell
As suggested by the results obtained with the 35S:erG3GFP:
c165 reporter gene in the 35S:AS2-GR plants (Figure 3C), levels
of endogenous miR165/166 were indeed depressed in the
35S:AS2-GR plants after treatment with RU or Dex (Figure 4D).
The as1 and TSA each restored normal levels of miR165/166 in
the 35S:AS2-GR plants (line #5.4; Figure 4D). We obtained
similar results with another transgenic line (line #3.4). Thus,
AS2-GR in the nucleus requires AS1 for a reduction in levels of
mature miR165/166, as does endogenous AS2. Accumulation of
the precursors to miR165/166 (pri-miR165/166) in 35S:AS2-GR
plants was unaffected by treatment with RU (Figure 4E). We also
examined levels of miR171, miR172, and miR173 in 35S:AS2-GR
plants grown in the presence of RU. The accumulation of miR171
and miR173 was unaffected in 35S:AS2-GR plants, but that of
miR172 was reduced (see Supplemental Figure 5 online).
We determined how quickly changes in miR165/166 occur
following Dex treatment of 35S:AS2-GR plants. Incubation of
these plants in a liquid culture containing 10 mM Dex for 24 h did
not affect levels of miR165/166. Subsequently, we examined
whether longer incubations of 35S:AS2-GR plants on a solid
medium may affect levels of miR165/166. Results of this exper-
iment showed that the levels decreased 4 d after the incubation
(see Supplemental Figure 6 online), suggesting that the effect on
decrease of miR165/166 by the nuclear localization of AS2-GR is
likely to be indirect.
Identification of HDT1/HD2A and HDT2/HD2B as
the Relevant HDACs
The Arabidopsis genome includes 12, four, and two genes for
members of the RPD3/HDA1, HD2, and SIR2 families, respec-
tively, of HDACs (Pandey et al., 2002). Proteins belonging to the
SIR2 family are insensitive to TSA, while members of the RPD3/
HDA1 and HD2 families are very susceptible to TSA and CAY
(Jung et al., 1997). To identify the HDAC(s) responsible for the
determination of the leaf polarity, we constructed RNA interfer-
ence (RNAi) libraries specific for the genes in the RPD3/HDA1
and HD2 families, designating our libraries the HDA-RNAi library
and the HDT-RNAi library, respectively. Then, we transformed
as2-1 35S:AS2-GR (from line #5.4) plants, generated by genetic
cross, with each library as described in Methods.
We looked for knockdown transformants with radialized
(filamentous or trumpet-shaped) leaves (Figure 5A). Among
20 independent transformants obtained with the HDT-RNAi
library, two knockdown lines (lines #1 and #301) had produced
clearly radialized leaves in the T1 generation (as shown by
the example in Figure 5B). A total of 104 of 129 plants (81%) and
42 of 83 plants (51%) of knockdown lines #1 and #301, respec-
tively, produced radialized leaves in the T2 generation (Table 2).
Analysis by PCR revealed that RNAi constructs specific for
HDT1/HD2A and HDT2/HD2B and for HDT2/HD2B were pres-
ent in the genomes of lines #1 and #301, respectively (Table 2).
DNA analysis of other lines that formed radialized leaves in
the T2 generation revealed that these lines contained T-DNA
for RNAi of HDT1/HD2A (HDT1i) or T-DNA for RNAi of HDT2/
HD2B (HDT2i) (Table 2). Moreover, endogenous levels of HDT1/
HD2A and HDT2/HD2B transcripts in knockdown line #1 were
lower than those in control host plants and in siblings in the T2
generation without radialized leaves, whereas those of HDT3/
HD2C and HDT4/HD2D were not depressed (Figure 5C). We
backcrossed knockdown line #1 to a Col-0 plant to examine
the phenotype on a wild-type AS2 background. All F2 siblings,
which were indistinguishable from wild-type plants, lacked
radialized leaves. Therefore, we concluded that the phenotype
was dependent on as2. However, both HDT1i and HDT2i were
tightly linked to the radialized-leaf phenotype (see Supple-
mental Table 1 online). These results indicated that the forma-
tion of radialized leaves depended on HDT1i, HDT2i, or both in
line #1.
To confirm our results, we reintroduced HDT1i and HDT2i into
as2-1 35S:AS2-GR plants. Among seven lines of newly isolated
transformants, one transformant (T1 generation), which was
sterile, had filamentous leaves similar to those of line #1. From
9 to 23% of T2 siblings with radialized leaves were segregated
from another three lines among the seven lines. These results
suggested that HDT1/HD2A and/or HDT2/HD2B might be the
relevant factor(s). Moreover, they were consistent with our earlier
observation that the generation of filamentous leaves depended
on both the as2 or as1 mutation and the dose of HDAC inhibitor
(Figure 1, Table 1).
Figure 3. Ectopic Overproduction of AS2-GR in Nuclei Reduced the
Accumulation of miR165/166.
(A) Schematic representation of the 35S:AS2-GR construct. t3A, the
poly(A) signal sequence from the gene for ribulose-1,5-bis-phosphate
carboxylase/oxygenase 3A from pea (Pisum sativum).
(B) and (C) 35S:AS2-GR (from line #5.4) 35S:erG3GFP:c165 (from line
#2.1) double-transgenic plants were grown on medium without (mock
treated; [B]) or with (C) 1 mM RU. Green, fluorescence due to GFP; red,
autofluorescence.
(D) and (E) Localization of PHB transcripts (purple signals indicated by
arrowheads) in Col-0 (D) and in a 35S:AS2-GR (line #5.4) plant grown in
the presence of 1 mM RU (E).
Bars ¼ 50 mm.
HDACs and AS2 Regulate Leaf Polarity 449
Among 67 independent transgenic lines obtained from the
HDA-RNAi library, we found no knockdown transformants with
radialized leaves in the T1 and T2 generations.
HDT1/HD2A and HDT2/HD2B Are Involved in the
Determination of Adaxial-Abaxial Polarity
We examined the expression of genes involved in the determi-
nation of adaxial-abaxial polarity by RT-PCR in knockdown line
#1 (as2-1 35S:AS2-GR [from line #5.4] hdt1-RNAi hdt2-RNAi). In
T2 siblings that formed filamentous leaves, the levels of tran-
scripts of PHB and FIL were lower and higher, respectively, than
those in the normal (nonfilamentous) siblings (Figure 5C). Taken
together, these observations suggested that the levels of tran-
scripts of the PHB and FIL genes were affected positively and
negatively, respectively, by HDT1/HD2A and HDT2/HD2B in
leaves with the as2 mutant background.
On the surfaces of filamentous leaves on plants of knockdown
lines #1 and #301, the epidermal cells were long and narrow, with
reduced numbers of trichomes (Figures 5D and 5E). These surface
characteristics of filamentous leaves were strikingly similar to
those of as2-1 plants that had been exposed to TSA (Figure 1N).
Transcripts of the FIL gene accumulate throughout younger leaf
primordial, and their levels on theadaxial sidediminish asprimordia
grow (Sawa et al., 1999; Siegfried et al., 1999). The decrease in the
accumulation of FIL transcripts that we observed in Col-0 (Figure
5F) was not evident in knockdown line #301 at the same stage
(Figure 5G). A higher number of strong signals were observed
preferentially on the adaxial side of the leaf primordium. Similar
results were obtained from knockdown line #1. These results
indicated that the filamentous leaves that were formed on plants of
knockdown lines #1 and #301 had been abaxialized. Therefore, we
concluded that HDT2/HD2B (and also HDT1/HD2A) were involved
in the establishment of adaxial fate on the as2 mutant background.
Genetic Interaction between AS2 and Genes Related to the
Function of miR165/166
We examined the effects of TSA on as2-1 hst-1, as2-1 phb-1D,
and as1-1 hst-1 double mutants. In hst mutants, the levels of
Figure 4. Upward Curling of Leaves and the Depressed Accumulation of Mature miR165/166 in 35S:AS2-GR Transgenic Plants Depended on
Endogenous AS1 and HDAC Activity.
(A) and (B) Upward curling of leaves on 35S:AS2-GR transgenic plants depended on endogenous AS1. 35S:AS2-GR (line #5.4; [A]) and as1-1
35S:AS2-GR (line #5.4; [B]) plants were grown on medium that contained 10 nM Dex. Bar ¼ 1 mm.
(C) Upward curling of leaves on 35S:AS2-GR transgenic plants was prevented by inhibition of HDAC activity. The frequency of upward curling of leaves of
35S:AS2-GR (line #5.4) plants in response to Dex isshown. Plants weregrown on medium that contained 3 or 10 nM Dex without or with 1 mM TSA, as indicated.
(D) The depressed accumulation of miR165/166, observed in 35S:AS2-GR transgenic plants, depended on endogenous AS1 and HDAC activity. RNA
gel blot analysis of miRNAs in Col-0, 35S:AS2-GR (line #5.4), and as1-1 35S:AS2-GR (line #5.4) plants, grown for 10 d with drugs as indicated. The
relative intensity of each signal is indicated below the corresponding band. EtBr, ethidium bromide.
(E) Levels of precursors to miR165 (pri-miR165) and miR166 (pri-miR166) were not significantly affected in 35S:AS2-GR plants exposed to RU. RT-PCR
was performed using poly(A)þ RNA from 35S:AS2-GR plants that had been grown for 10 d on medium with (þ) or without (�) RU after reverse
transcription (þRT for miR165 and miR166) or left untranscribed as controls (�RT for miR165), with primer pairs that allowed amplification of cDNAs for
both miR165a and miR165b and for several cDNA for miR166. Levels of pri-miR165a and pri-miR166 were the same in the presence and absence of RU.
No pri-miR165b was detected under either condition. Genomic DNA from a Col-0 plant was used as the control template (DNA).
450 The Plant Cell
many miRNAs, including those of miR165/166, were lower than
those in wild-type plants (Park et al., 2005). On medium without
TSA, as2-1 hst-1 double mutants had undulating but nearly flat
leaves, while as2-1 single mutants had downwardly curling
leaves, and hst-1 mutants had upwardly curling leaves (Figures
6A to 6C). Even on medium with TSA, on which most as2-1
single-mutant plants formed abaxialized filamentous leaves,
as2-1 hst-1 double mutants had flat leaves, as did hst-1 single
mutants (Figures 6D to 6F). These observations showed that hst
suppressed, at least to some extent, the effects of the as2
mutation and the inhibition of HDAC activity. The as1-1 hst-1
double mutant plants had a similar phenotype. Our results sug-
gested that the formation of filamentous leaves by mutation of
AS1 or AS2 and by TSA requires the activity of HST.
Regulation by miR165/166 of the expression of phb-1D tran-
scripts is eliminated by disruption of the complementary se-
quences in the transcripts (McConnell et al., 2001; Mallory et al.,
2004). Our as2-1 phb-1D double mutants formed filamentous
leaves with many trichomes, and these leaves were indistin-
guishable from leaves of phb-1D single mutants, even in the
presence of TSA (Figures 6G and 6H). On the surfaces of
filamentous leaves on as2-1 phb-1D plants, the shapes and
organization of epidermal cells were somewhat irregular (Figure
6I), resembling those on the adaxialized leaves of phb-1D mu-
tants (McConnell and Barton, 1998), as compared with those on
abaxialized leaves on as2-1 plants in the presence of TSA and on
HDT2/HD2B knockdown lines with the as2-1 background (Fig-
ures 1N, 5D, and 5E). Thus, the phb-1D allele was epistatic to the
as2-1 allele regardless of the presence of TSA.
Subnuclear Localization of HDT1/HD2A, AS2, and AS1
To examine the subnuclear localization of AS2 and AS1, we
prepared plasmids that encoded the following constructs: yellow
fluorescent protein (YFP) fused to the C terminus of AS2, in
frame, (AS2-YFP) under the control of an estrogen-inducible
promoter (ER:AS2-YFP); GFP fused to the N terminus of AS1, in
frame, (GFP-AS1) under the control of the 35S promoter
(35S:GFP-AS1); YFP fused to the N terminus of HDT1/HD2A, in
Figure 5. Knockdown of HDT1/HD2A and HDT2/HD2B in Combination
with the as2 Mutation Resulted in Abaxialized Filamentous Leaves.
(A) Schematic representation of constructs used for generation of RNAi
libraries and protocol for identification of relevant HDACs.
(B) Knockdown line #1 (T1 generation) isolated using the HDT-RNAi
library.
(C) Alteration of gene expression by HDT1i and HDT2i in as2 mutant plants.
RNA was prepared from five 19-d-old T2 siblings of knockdown line #1 with
(lane 1) and without (lane 2) filamentous leaves and subjected to analysis by
RT-PCR. Levels of transcripts of HDT, PHB, and FIL genes and of a gene for
hygromycin phosphotransferase (HPT), as a marker for the presence of
T-DNA, were examined. F, filamentous; N, not filamentous.
(D) to (G) Filamentous leaves in RNAi lines specific for HDT1/HD2A and
for HDT2/HD2B in combination with the as2 mutation were abaxialized.
(D) and (E) Scanning electron micrographs.
(D) A filamentous leaf of a line #1 plant with the as2-1 mutation, HDT1i
and HDT2i, which had been backcrossed once to Col-0 and from which
the 35S:AS2-GR transgene had been eliminated.
(E) A filamentous leaf of a T2 sibling of line #301 carrying the as2-1
mutation and HDT2i.
(F) and (G) Accumulation of FIL transcripts in a leaf primordium of Col-0
(F) and a T2 sibling of line #301 (G). Diminished and normal accumulation
of transcripts in the adaxial domain of the leaf primordium from the Col-0
plant and the #301 plant, respectively, is shown by arrowheads. The leaf
primordium is outlined in red. An asterisk indicates the shoot apical
meristem.
Bars ¼ 0.5 mm in (B), 100 mm in (D) and (E), and 50 mm in (F) and (G).
Table 2. Generation of Radialized Leaves and T-DNAs Carried
by Knockdown Lines Isolated after Transfection with the
HDT-RNAi Library
Line No.
Radialized Leaves
on T1 Plants T-DNA for RNAi
Frequency of
Plants with
Radialized
Leaves among
the T2 Progeny
1 Filamentous and
trumpet-shaped
HDT1 and HDT2 81% (n ¼ 129)
3 Unclear HDT3 and HDT4 0% (n > 30)
4 Unclear HDT2, HDT3,
and HDT4
5% (n ¼ 59)
5 Unclear HDT1 0% (n ¼ 55)
6 Unclear HDT4 0% (n > 30)
7 Unclear HDT4 0% (n > 30)
8 Unclear HDT3 0% (n ¼ 37)
9 Unclear HDT3 0% (n > 30)
11 Unclear HDT3 0% (n > 30)
12 Unclear HDT1 and HDT4 9% (n ¼ 34)
301 Trumpet-shaped HDT2 51% (n ¼ 83)
Both the analysis of the DNA of T1 plants and the segregation analysis
of T2 siblings were performed with 11 of 20 transgenic lines. No obvious
abnormalities, apart from filamentous or trumpet-shaped leaves, were
observed in the T1 and T2 generations of the various knockdown lines.
HDACs and AS2 Regulate Leaf Polarity 451
frame, (YFP-HDT1) under the control of the 35S promoter
(35S:YFP-HDT1); and GFP fused to the C terminus of nucleolin,
in frame, (Nuc.-GFP) under the control of the 35S promoter
(35S:Nucleolin-GFP), as a marker of nucleoli.
Fluorescent signals due to AS2-YFP protein were localized as
subnuclear bodies around the periphery of nucleoli in the leaf
epidermal cells of ER:AS2-YFP (from line #TI1.1) 35S:Nucleolin-
GFP (from line #TI9) double-transgenic plants (98% [n¼ 47]; see
examples in Figures 7B and 7C). Similar results were obtained
from another transgenic line ER:AS2-YFP (#TI10.5) plants. Fluo-
rescent signals due to GFP-AS1 protein were localized as one
to several subnuclear bodies in the leaf epidermal cells of
35S:GFP-AS1 (lines #TI1 and #TI2) transgenic plants (86% [n ¼29]). The largest bodies were localized around the periphery of
nucleoli (see Supplemental Figure 7 online). As reported previ-
ously by Lawrence et al. (2004), our results also showed that
fluorescent signals due to YFP-HDT1 were localized in the
nucleoli in 35S:YFP-HDT1 (lines #TI3 and #TI5) transgenic plants
(see Supplemental Figure 7 online). When cells that were positive
for both GFP and YFP signals in the ER:AS2-YFP (from line
#TI1.1) 35S:GFP-AS1 (from lines #TI17) double-transgenic
plants were examined, fluorescent signals from GFP-AS1 and
AS2-YFP were colocalized around the periphery of nucleoli
(100%; Figures 7D to 7F). The proportion of such double-positive
cells was 20% of AS2-YFP positive cells and variable in GFP-AS1
positive cells depending on conditions for the induction of AS2-
YFP gene expression. Using another double-transgenic line,
ER:AS2-YFP (from line #TI1.1) 35S:GFP-AS1 (from line #TI22),
similar results were obtained.
The subnuclear accumulation of AS1/AS2 appeared to be
separate from the nucleolus, in which HDT1/HD2A and HDT2/
HD2B were concentrated (Figure 7F; Lawrence et al., 2004; Zhou
et al., 2004).
DISCUSSION
HDACs and AS2 (AS1) Are Involved Independently in
the Establishment of the Adaxial-Abaxial Polarity
of Leaves in Arabidopsis
In this study, we found that loss of function of two genes for
HDAC, HDT1/HD2A and HDT2/HD2B, on the as1 or as2 mutant
background resulted in abaxialized filamentous leaves and ab-
errant distribution in leaves of miR165/166 (Figures 1, 2, and 5).
These observations suggest that these HDACs are involved in the
determination of adaxial-abaxial polarity in leaves, as are AS1
and AS2 (Xu et al., 2003). Inducible nuclear localization of over-
produced AS2-GR in Arabidopsis resulted in upwardly curled
leaves, a reduction in levels of miR165/166, and the aberrant
accumulation of transcripts of PHB in the abaxial domain (Figures
3 and 4). These abnormalities were suppressed by inhibition of
HDACs and the as1 mutation. Thus, the phenotype generated by
AS2 seems to require the functions of the HDACs and AS1. It was
demonstrated recently that AS2 transcripts accumulate in the
Figure 6. Interaction between AS2 and Genes Related to the Role of miR165/166.
(A) to (H) as2-1 ([A] and [D]), hst-1 ([B] and [E]), as2-1 hst-1 ([C] and [F]), phb-1D (G), and as2-1 phb-1D (H) plants were grown on medium without
(mock-treated; [A] to [C]) or with ([D] to [H]) 3 mM TSA. Bars ¼ 1 mm.
(I) An adaxialized filamentous leaf of the as2-1 phb-1D mutant. Bars ¼ 100 mm.
452 The Plant Cell
adaxial domain of leaves, including the epidermis, and that AS2
acts to repress cell division on the adaxial side, contributing to
the upward curling of leaves that is found upon overproduction
of AS2 and explaining the downward curling in as2 mutants
(H. Iwakawa, Y. Ueno, C. Machida, and Y. Machida, unpublished
data). Our data suggest that the HDACs and AS2 (also AS1) are
involved independently in the determination of the appropriate
distribution and levels of miR165/166 and in depression of the
rate of cell division in the adaxial domain, as well as in the
determination of adaxial polarity.
Upward Curling of Leaves Seems to Be Closely Related to
the Biogenesis and/or Functions of Some miRNAs
Upward curling is one of the most obvious phenotypic abnor-
malities associated with the nuclear import of overproduced AS2
(Figure 4; Iwakawa et al., 2002; unpublished data). Since loss-of-
function mutations in the AS2 genes result in downward curling
of leaves (Semiarti et al., 2001; Iwakawa et al., 2002; unpublished
data) and since overexpression of some other members of the
AS2/LOB family does not induce such conspicuous upward
curling while most members of this family have no effect at all on
the curling status of leaves (Shuai et al., 2002; Nakazawa et al.,
2003; Chalfun-Junior et al., 2005), the upward curling observed
upon overproduction and nuclear localization of AS2 seems to be
attributable to an intrinsic function of AS2.
Several genes are known that are related to the formation of
upwardly curled leaves. Loss-of-function mutations in the HST, HUA
ENHANCER1 (HEN1), DCL1/CARPEL FACTORY, HYPONASTIC
LEAVES1 (HYL1), SERRATE, and ARGONAUTE1 (AGO1) genes, all
of which are related to the formation or functions of miRNA, result in
the upward curling of leaves (Telfer and Poethig, 1998; Jacobsen
et al., 1999; Lu and Fedoroff, 2000; Chen et al., 2002; Han et al.,
2004; Vaucheret et al., 2004; Grigg et al., 2005; Mallory and
Vaucheret, 2006). The mutations reduce levels of miRNAs and
increase levels of transcripts of the PHB, PHV, and REV genes,
which are targets of miR165/166 (Reinhart et al., 2002; Vaucheret
et al., 2004; Vazquez et al., 2004; Grigg et al., 2005; Park et al., 2005;
Yang et al., 2006). A point mutation, located at the site of cleavage
mediated by miR165/166 within the INCURVATA4/CORONA/
ATHB-15 gene, also resulted in upcurled leaves (Ochando et al.,
2006). These observations suggest that products of genes that are
involved in the production and functioning of miR165/166 regulate
leaf flatness, apparently opposing the functions of AS2 and AS1.
The Mechanism whereby HDACs and AS2 (AS1) Control
Adaxial-Abaxial Polarity
HDACs Are Involved in Establishing the Adaxial Domain
by Affecting the Level and Patterns of Distribution of
miR165/166 during Leaf Development in as2 Plants
Three types of model might explain how HDACs establish the
adaxialization of leaves and control the distribution of miR165/
166 in the as2 mutant. (1) The HDACs could affect the distribution
of miR165/166, which could in turn establish adaxialization. (2)
The HDACs could directly control leaf polarity, which could in
turn alter the distribution of miR165/166. (3) The HDACs could
control both processes separately. The second and third models
seem less plausible than the first since the formation of abax-
ialized filamentous leaves, which was induced by inhibitors of
HDACs and the as2 mutation, was suppressed by the hst-1
mutation (Figure 6). In addition, when double mutant (as2-1
phb-1D) plants, with a dominant allele associated with resis-
tance to degradation of mRNA by miR165/166, were treated with
inhibitors of HDACs, adaxialized filamentous leaves were formed
instead of abaxialized leaves (Figure 6). Thus, the hst-1 and
phb-1D mutations were epistatic to the inhibition of HDACs in
the as2 mutant. It seems likely that the HDACs are involved in
establishing the adaxial domain via effects on the levels and
distribution of miR165/166 during leaf development in the as2
mutant. However, it remains unknown whether such effects in
as2 mutants are directly or indirectly attributable to HDACs.
Since overexpression of AS2, as2 mutations, and application
of inhibitors of HDACs had no obvious effects on levels of
transcripts of AGO1, DCL1, HEN1, HST, and HYL1, which are
required for biogenesis of miRNAs (our unpublished data), it
seems to be unlikely that HDACs and AS2 are involved in the
transcription of these genes or in the processes that are medi-
ated by their products.
Possible Control by HDACs and AS2 (AS1) of
Levels of miRNA165/166
Our results indicate that two genes for HDACs, HDT1/HD2A and
HDT2/HD2B, function in establishment of the adaxial domain of
leaves in Arabidopsis with the as2 mutation. The AS2 gene also
plays a similar role in leaf development when the activity of the
HDACs is reduced by an inhibitor or by knockdown of genes for
Figure 7. AS2-YFP and GFP-AS1 Were Colocalized in Subnuclear
Bodies at the Periphery of the Nucleolus.
Fluorescence due to GFP and YFP of 35S:Nucleolin-GFP ER:AS2-YFP
([A] to [C]) and 35S:GFP-AS1 ER:AS2-YFP ([D] to [F]) double-transgenic
plants was monitored by confocal laser scanning microscopy. (A) and (D)
Signals due to GFP; (B) and (E) signals due to YFP; (C) and (F) merged
views of (A) and (B) and of (D) and (E), respectively. The profile of the
nucleus is indicated by a dashed line in (C) and (F). Numbers under
panels represent ratios of positive cells that exhibit localization in nucleoli
(A) or subnuclear bodies ([B] and [D]). The ratio in panel (D) represents
an average value because it depended on concentrations of the inducer
(estradiol). Bars ¼ 3 mm.
HDACs and AS2 Regulate Leaf Polarity 453
HDACs by RNAi. As discussed above, it is likely that HDACs and
AS2 are involved independently in the accumulation and/or
distribution of miR165/166 in leaves of wild-type Arabidopsis. A
similar function for AS2 was proposed by Li et al. (2005). Since
nuclear localization of overproduced AS2 proteins did not affect
levels of pri-miR165 or pri-miR166 but reduced levels of mature
miR165/166 (Figure 4), it seems possible that AS2 might be
responsible for posttranscriptional processing that generates
mature miR165/166.
It is generally accepted that HDACs are involved in the control
of transcription via the deacetylation of histones in chromatin.
HDT1/HD2A is involved in the deacetylation of nucleosomal
histone H3 and in modulating transcription of genes for rRNA in
genetic hybrids (Lawrence et al., 2004). In addition, HDT1/HD2A
and HDT2/HD2B have been shown to repress the transcription of
reporter genes (Wu et al., 2000, 2003). We found a relationship
between the formation of abaxialized filamentous leaves and an
increase in the level of acetylation of histone H4, bothof whichwere
induced by the application of TSA, an inhibitor of HDACs (Figure1I).
In addition, application of TSA to the as2 mutant resulted in a slight
increase in levels and a change in the distribution of mature
miR165/166 (Figure 2). It seems plausible that HDACs might
influence levels and patterns of distribution of these miRNAs by
controlling the histone-deacetylation status of the chromatin.
The subnuclear localization of HDT1/HD2A, HDT2/HD2B, AS2,
and AS1 allows us to speculate about possible functional rela-
tionships among these proteins. GFP-tagged AS1 and YFP-
tagged AS2 were colocalized in subnuclear bodies adjacent to
the nucleolus of epidermal cells of Arabidopsis leaves (Figure 7).
The GFP-fused RS2 protein, a maize homolog of AS1, exhibits a
similar pattern of subnuclear localization in Arabidopsis root cells
(Theodoris et al., 2003). HDT1/HD2A and HDT2/HD2B are con-
centrated in the nucleoli of epidermal cells in Arabidopsis leaves
(Zhou et al., 2004). Recently, factors such as RNA polymerase
IVb and ARGONAUTE4 that are required for gene silencing–
related chromatin modification guided by small interfering RNAs
(siRNAs) have been reported to colocalize with siRNAs within
specific bodies in the nucleolus, which are proposed to function
as a center for the assembly of complexes, facilitating siRNA-
directed gene silencing at target loci (Li et al., 2006; Pontes et al.,
2006). These HDACs and AS2 (AS1) were concentrated in the
nucleolus and juxtaposedsubnuclear bodies, respectively. The re-
pression of miR165/166 accumulation by AS2-GR required the
incubation of the 35S:AS2-GR plants for 4 d (see Supplemental
Figure 6 online), suggesting that the repression was probably
indirect. The repression might occur at the posttranscriptional
level (Figure 4E). Other unknown mechanisms (e.g., siRNA-
directed gene silencing) might also mediate the repression of
miRNA accumulation by AS2 (AS1) and HDACs. We need now to
investigate the roles of the nucleolus and the subnuclear bodies
in the mechanism that controls the adaxial-abaxial polarity.
METHODS
Plant Materials and Growth Conditions
Arabidopsis thaliana hst-1 and phb-1D mutants were obtained from the
ABRC. Other plant materials used in this study are listed in Supplemental
Table 2 online. Plants were grown on Murashige and Skoog (MS) medium
with and without TSA (Wako), CAY10398 (Cayman Chemical), Dex
(Sigma-Aldrich), or RU486 (Sigma-Aldrich) under previously described
conditions (Semiarti et al., 2001) except during screening after introduc-
tion of the HDA-RNAi or HDT-RNAi library.
Histological Analyses
Thin sections and cleared specimens were prepared as described
previously (Semiarti et al., 2001). For analysis of fluorescence due to
GFP, hand-cut sections were examined by confocal microscopy as
described previously (Watanabe and Okada, 2003). For staining of
b-glucuronidase (GUS) activity, tissues were incubated at 308C for
;16 h with staining solution (2 mg/mL 5-bromo-4-chloro-3-indolyl-b-D-
glucuronide, 10% [v/v] dimethylformamide, 50 mM sodium phosphate
buffer, pH 7.0, 2 mM potassium ferrocyanide, 2 mM potassium ferricy-
anide, 0.2% [w/v] Triton X-100, and 1 mM EDTA). Samples were
dehydrated through an ethanol series and embedded in Technovit 7100
resin (Heraeus Kulzer) and sectioned at a thickness of 4 mm with a
microtome. Scanning electron microscopy was performed as described
previously (Semiarti et al., 2001).
Detection of Acetylation of Histone H4 by Protein Gel Blot Analysis
Seeds of as2-1, as1-1, and Col-0 plants were allowed to germinated on
solid MS medium for 24 h in the light and then for 72 h in darkness.
Etiolated seedlings were dipped in liquid MS medium that contained 2%
(w/v) sucrose and 0 to 3 mM TSA for 3 h. The histone-enriched fraction
was prepared as described by Moehs et al. (1988). Antibodies against
acetylated histone H4 (#06-866) and against histone H4 (#07-108) were
purchased from Upstate.
RNA Gel Blot Analysis of miRNA
Total RNA was prepared as described elsewhere (Kim et al., 2005), and
other procedures were performed according to the instruction manual
provided by Ambion with the UltraHybOligo hybridization buffer and a
locked nucleic acid (LNA)–containing oligonucleotide probe (Valoczi
et al., 2004; Thermo Fisher Scientific), which had been labeled with T4
polynucleotide kinase and [g-32P]ATP. The sequences of LNA-containing
probes were as follows: pU695 for miR165/166, 59-GGgGGAtGAAg-
CCTgGTCcGA-39; pU706 for miR171, 59-GAtATTgGCGcGGCtCAAtCA-39;
pU743 for miR172, 59-ATgCAGcATCaTCAaGATtCT-39; and pU744 for
miR173, 59-GTgATTtCTCtCTGcAAGcGAA-39. Upper- and lowercase let-
ters indicate DNA and LNA, respectively.
RNA Hybridization in Situ
RNA hybridization in situ was performed as described previously (Kaya
et al., 2001) with some modifications. The plasmids for the preparation of
the PHB and FIL probes were generous gifts from Hirokazu Tanaka
(Universitat Tubingen) and Shinichiro Sawa (University of Tokyo), respec-
tively. Antisense probes for PHB and FIL were synthesized using T3 and T7
RNA polymerase, respectively, and digoxigenin-11-UTP (DIG-11-UTP).
After hybridization at 508C, sections were washed twice for 1 h at 558C in
0.23 SSC. After the second wash, sections were treated with 20 mg/mL
RNaseA in NTE (0.5 M NaCl, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) for
30 min at 378C and washed twice again for 5 min each in NTE at 378C.
Alkaline phosphatase–conjugated antibodies against DIG (Roche) and
WesternBlue (Promega) were used for immunological detection of DIG.
Construction of RNAi Libraries
HDA10 and HDA17, which belong to the RPD3/HDA1 family, lack cata-
lytic domains (Pandey et al., 2002). Thus, 14 HDACs in the RPD3/HDA1
454 The Plant Cell
plus HD2 families are targets for TSA and CAY, as described in Results.
We constructed HDA-RNAi and HDT-RNAi libraries that corresponded to
10 genes in the RPD3/HDA1 family and four genes in the HD2 family,
respectively. The DNA sequences of HDA9, HDA10, and HDA17, as
annotated by The Arabidopsis Information Resource (TAIR; http://
www.arabidopsis.org), are strongly homologous. We postulated that an
RNAi construct specific for one of them would repress the expression of
all three. Since the cDNA that encoded HDA17 was cloned so efficiently,
we employed it instead of cDNA for HDA9 in the HDA-RNAi library. We
used a partial genomic fragment for HDA7 since we were unable to clone
the cognate cDNA. Each cDNA encoding the translation unit for an HDAC
in the RPD3/HDA1 family, with the exception of HDA7, or for an HDAC in
the HD2 family, as annotated by TAIR, was amplified by RT-PCR with
appropriate primers (see Supplemental Table 3 online) and cloned into
pENTR-D-TOPO (Invitrogen). Each construct was subcloned into the
binary vector pYU501 (see Supplemental Methods online) for RNAi and
introduced into Agrobacterium tumefaciens strain GV3101. After intro-
duction of each plasmid into Agrobacterium, we transformed as2-1
35S:AS2-GR plants by the floral dip method.
Identification of the Relevant HDACs Using an RNAi Library
Transgenic plants carrying the RNAi library were screened on MS
medium, without sucrose, that contained 3 mg/L thiamine, 5 mg/L
nicotinic acid, 0.5 mg/L pyridoxine, 0.5 g/L MES-KOH, pH 5.7, 0.5%
(w/v) Gellan gum (Wako), and 15 mg/mL hygromycin B (Wako). The
screenings were performed by reference to phenotype as described in
Results. The introduced T-DNAs were identified by PCR using the
appropriate vector primers (Rv59 [59-CAGGAAACAGCTATGACCAT-
GATTAC-39] for HDT1, HDT2, and HDT4; and pU104 [59-CTATCCTTCG-
CAAGACCCTTCC-39] for HDT3) and the primer specific to each HDT
gene (pU508 for HDT1; pU510 for HDT2; pU512 for HDT3; and pU514 for
HDT4). To confirm the nature of the T-DNA specific for RNAi of HDT3,
we performed nested PCR using pU803 (59-GGGAGAGAGTAAGAA-
CAATGT-39) and pU804 (59-CAGGCTTCTTTGGGGTTTCCT-39). The se-
quences of primers that are not given here can be found in Supplemental
Table 2 online.
RT-PCR
RT-PCR was performed as described previously (Semiarti et al., 2001).
The primers used for RT-PCR were as follows: for a-tubulin (TUBA; as a
control), pU51 and pU52 (Semiarti et al., 2001); for HDT1/HD2A, pU723
(59-ACATTTTCAGCTGCTCATAAACCCT-39) and pU508 (see Supple-
mental Table 3 online); for HDT2/HD2B, pU771 (59-CCTCTCTTCTCTC-
TTCCTCGTTCAACAACA-39) and pU772 (59-CCCAAAACCTCTCCTTG-
ATCCACGTCC-39); for HPT, HPT1 (59-AGCCTGAACTCACCGCGACGT-39)
and HPT2 (59-TGCCCTCGGACGAGTGCTGGG-39); for HDT3/HD2C
and HDT4/HD2D, the primers used to clone the cDNA for each HDT (see
Supplemental Table 2 online); for PHB, pU579 (59-ATGATGATGGTC-
CATTCGATGAGCAGA-39) and pU580 (59-GGGCCTCCTCTGCTATA-
GAAAGGAGTCCT-39); for FIL, pU221 (59-ATGTCTATGTCGTCTATGT-
CCTCCCC-39) and pU222 (59-AATAAGGAGTCACACCAACGTTAGC-
AGC-39); for pri-miR165, pU663 (59-CATCATTCCCTCATCATAACACCAT-
CATCAC-39) and pU664 (59-GGGGGATGAAGCCTGGTCCGA-39); and for
pri-miR166, 6F and 7R (Juarez et al., 2004).
Localization of Proteins
Synthesis of AS2-YFP, encoded by ER:AS2-YFP, was induced by treat-
ment of appropriate transgenic plants with 5 mM estradiol for 20 min 1 d
prior to observations. Fluorescence due to GFP and YFP in leaf epidermal
cells was observed by confocal laser scanning microscopy (LSM 510
META; Carl Zeiss).
Note
Plasmids were constructed by standard techniques. Details are provided
in Supplemental Methods online.
While RU acts antagonistically against the endogenous glucocorticoid
in mammals, RU is used only as a weak agonist in this study because no
GR is encoded in the Arabidopsis genome.
For screening of HDACs using the RNAi-library, we employed as2-1
35S:AS2-GR plants as a host instead of as2-1 mutants since these plants
would also allow us to screen plants by reference to the suppression of
overproduction of AS2-GR. However, no such screening was performed
since we obtained transformants with a strong phenotype similar to that
of as2-1 35S:AS2-GR plants, which resembled that of as2 mutant plants
treated with inhibitors of HDACs.
Accession Numbers
GenBank/EMBL accession numbers and Arabidopsis Genome Initiative
locus identifiers for the genes mentioned in this article are as follows:
HDT1/HD2A (AT3G44750), NM_114344; HDT2/HD2B (AT5G22650),
NM_122171; AS1 (AT2G37630), NM_129319; and AS2 (AT1G65620),
AB080802, NM_105235.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Methods. Construction of Plasmids
Supplemental Figure 1. Acetylation Levels of Histone H4 Were Not
Affected in as1 and as2 Mutants.
Supplemental Figure 2. Patterns of Expression of Reporter Genes
from a Second 35S:erG3GFP:c165 Transgenic Line and a 35S:
GUS:c165 Plant.
Supplemental Figure 3. AS2-GFP-GR Fusion Proteins Were Local-
ized in Nuclei of BY-2 Cells Treated with Dex.
Supplemental Figure 4. The 35S:AS2-GR Transgene Can Restore
a Flat and Symmetric Leaf Shape in as2-1 Mutants in the Presence
of Dex.
Supplemental Figure 5. Ectopic Overproduction of AS2-GR Re-
duces the Accumulation of miR165/166 and miR172.
Supplemental Figure 6. Repressive Accumulation of miR165/166 in
35S:AS2-GR Plants after Treatment with RU.
Supplemental Figure 7. Subnuclear Localization of GFP-AS1 and
YFP-HDT1 Proteins.
Supplemental Table 1. Association between the Presence of Fila-
mentous Leaves and the as2 Allele, HDT1i, and HDT2i.
Supplemental Table 2. Plant Materials and Chemicals Used in
This Study.
Supplemental Table 3. Primers Used for Cloning the cDNAs for
HDAC-Specific RNAi Libraries.
ACKNOWLEDGMENTS
We thank Yasushi Yoshioka for critical reading of the original manuscript,
helpful suggestions, and encouragement. We also thank Atsushi Tamai
and Tetsuo Meshi for providing the erG3GFP; Kenzo Nakamura for
providing the nucleolin-GFP construct; Nam-Hai Chua for providing
pER8; the John Innes Centre for providing pGreen and marker cassettes;
the ABRC for providing hst-1 and phb-1D seeds; Bekir Ulker and Imre E.
Somssich for providing pJawohl8; Hirokazu Tanaka for providing the PHB
HDACs and AS2 Regulate Leaf Polarity 455
plasmid; Shinichiro Sawa for providing the FIL plasmid; and Satoshi Araki,
Michiko Sasabe, Miki Muranaka, and other members of their laboratory for
helpful discussions and technical assistance. This work was supported in
part by a Grant-in-Aid for Scientific Research on Priority Areas (14036216)
from the Japanese Ministry of Education, Culture, Sports, Science, and
Technology. Y.U. is the recipient of research fellowships from the NISSAN
Science Foundation and TOKAI Gakujutsu Syoureikai. T.I. is supported by
a grant from the Program for Promotion of Basic Research Activities for
Innovative Biosciences. S.T. is supported by a grant from the 21st Century
Circle of Excellence Program (Systems Biology).
Received March 7, 2006; revised December 29, 2006; accepted January
26, 2007; published February 9, 2007.
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HDACs and AS2 Regulate Leaf Polarity 457
DOI 10.1105/tpc.106.042325; originally published online February 9, 2007; 2007;19;445-457Plant Cell
Okada, Chiyoko Machida and Yasunori MachidaYoshihisa Ueno, Takaaki Ishikawa, Keiro Watanabe, Shinji Terakura, Hidekazu Iwakawa, Kiyotaka
ArabidopsisPolarity in Leaves of Histone Deacetylases and ASYMMETRIC LEAVES2 Are Involved in the Establishment of
This information is current as of June 17, 2020
Supplemental Data /content/suppl/2007/02/09/tpc.106.042325.DC1.html
References /content/19/2/445.full.html#ref-list-1
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