1 Plant Gene Expression Center, USDA/UC Berkeley, 800 Buchanan Street, Albany,
CA 94710, USA
2 Dept of Molecular, Cellular and Developmental Biology, UCLA, Los Angeles, CA
90095, USA
Author for correspondence (e-mail:
fletcher{at}nature.berkeley.edu)
Accepted 13 June 2005
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SUMMARY |
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Key words: microRNA, Arabidopsis thaliana, shoot apical meristem, polarity, HD-ZIP
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Introduction |
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miRNAs appear to regulate gene expression by binding to complementary
sequences in the mRNA transcripts produced by their target genes
(Bartel, 2004). While animal
miRNAs tend to have several mismatches with their target mRNA sequences, plant
miRNAs are characterized by their near-perfect complementarity with their
targets (Rhoades et al.,
2002
). Interaction of the miRNA with its target mRNA sequence is
recognized by an RNA-induced silencing complex (RISC), leading either to
specific cleavage of the mRNA via RISC endonuclease activity, or to
translational repression (reviewed by
Bartel, 2004
). The RISC has
been purified from fly and human cells
(Hammond et al., 2000
;
Martinez et al., 2002
), and in
both cases contains a member of the ARGONAUTE (AGO) family of PPD proteins
(Cerutti et al., 2000
).
Arabidopsis plants with reduced AGO1 gene activity
accumulate mRNAs that are normally targeted for miRNA-mediated cleavage,
confirming a role for AGO1 in the miRNA regulatory pathway
(Vaucheret et al., 2004
).
Mutants that lack DCL1 activity are embryo lethal
(Schauer et al., 2002),
revealing that miRNA metabolism is essential for normal plant development.
Similarly, plants carrying weaker dcl1 alleles survive embryogenesis
but display a wide spectrum of developmental defects
(Schauer et al., 2002
), while
hen1 and hyl1 null mutants show reduced miRNA levels and
morphological phenotypes that overlap with those of weak dcl1 alleles
(Han et al., 2004
;
Park et al., 2002
;
Vazquez et al., 2004
).
ago1 null mutants are viable but have pleiotropic developmental
phenotypes (Bohmert et al.,
1998
), while ago1 hypomorphic mutants exhibit
morphological defects similar to those of dcl1, hen1 and
hyl1 mutants (Vaucheret et al.,
2004
).
Consistent with the demonstration that activity of the miRNA pathway is
important for plant development, it has been observed that a large fraction of
the predicted target transcripts of plant miRNAs encode members of
transcription factor families (Rhoades et
al., 2002). Many of these transcription factors have defined or
predicted roles in developmental patterning, phase transition and/or cell fate
control (Bartel, 2004
). A
direct role for miRNAs in regulating different aspects of Arabidopsis
development has been experimentally demonstrated in a number of cases
(Achard et al., 2004
;
Aukerman and Sakai, 2003
;
Baker et al., 2005
;
Chen, 2004
;
Emery et al., 2003
;
Mallory et al., 2004
;
Palatnik et al., 2003
;
Zhong and Ye, 2004
).
Among the Arabidopsis developmental regulators targeted by miRNAs
are five members of the class III homeodomain-leucine zipper (HD-ZIP) family
of transcription factors (Sessa et al.,
1998), REVOLUTA (REV), PHABULOSA
(PHB), PHAVOLUTA (PHV), CORONA
(CNA) and ATHB8. Loss-of-function phb, phv, cna and
athb8 mutants are aphenotypic
(Baima et al., 2001
;
Prigge et al., 2005
), but
rev mutants form defective lateral and floral meristems and develop
aberrant stem vasculature (Otsuga et al.,
2001
; Talbert et al.,
1995
). rev phb phv triple mutants fail to establish a
shoot apical meristem and produce abaxialized cotyledons, indicating that
these three genes play overlapping roles in regulating SAM formation, leaf
polarity and radial patterning (Emery et
al., 2003
; Prigge et al.,
2005
). PHB, PHV and CNA have overlapping
functions in regulating meristem size, lateral organ polarity and vascular
development that are distinct from REV
(McConnell and Barton, 1998
;
McConnell et al., 2001
;
Prigge et al., 2005
).
ATHB8 has been proposed to play a role in vascular development
(Baima et al., 1995
;
Baima et al., 2001
), and acts
redundantly with CNA to promote post-embryonic meristem activity
(Prigge et al., 2005
).
The five class III HD-ZIP gene transcripts are targeted by miRNAs
from the miR165/166 group. Two MIR165 genes and seven
MIR166 genes are encoded in the Arabidopsis genome, and the
mature miR165 and miR166 sequences differ from one another
by a single nucleotide (Reinhart et al.,
2002). In wheat germ extracts, miR165/166 guides the RISC
to efficiently cleave wild-type PHV mRNA
(Tang et al., 2003
) and
CNA/ATHB15 mRNA (Kim et al.,
2005
). Increased expression of miR166a in an
activation-tagged line causes a reduction in PHB, PHV and
CNA transcript levels, leading to an expansion of xylem tissue and
the interfascicular region, indicative of accelerated vascular cell
differentiation (Kim et al.,
2005
). Stem fasciation and SAM enlargement are also reported in
the miR166a over-expression line. Conversely, phv, phb and
rev gain-of-function alleles that alter the miR165/166
complementary site are resistant to mRNA cleavage mediated by
miRNA165/166 (Emery et al.,
2003
; Tang et al.,
2003
; Mallory et al.,
2004
; Zhong and Ye,
2004
), and they confer specific patterning phenotypes as a result
of ectopic expression of their target gene products
(Emery et al., 2003
;
McConnell and Barton, 1998
;
McConnell et al., 2001
;
Zhong and Ye, 2004
).
Using an activation tagging approach, we demonstrate that miR166g causes de novo SAM formation and disrupts the morphogenesis of leaves, vascular bundles and gynoecia when over-expressed in Arabidopsis. We show that jba-1D meristem cells express much higher than normal levels of WUS mRNA, and that WUS activity is required to obtain the jba-1D meristem phenotype. We find that miR166 is expressed in a dynamic pattern in developing wild-type and jba-1D embryos, being largely complementary to its HD-ZIP target transcripts in early stages but coincident with them in later stages. Over-expression of miR166g causes reduced accumulation of PHB, PHV and CNA transcripts in jba-1D seedlings and inflorescence apices. Increased accumulation of REV transcripts is also detected, but is not sufficient to account for the meristem defects. We propose that down-regulation of PHB, PHV and CNA mRNAs in jba-1D plants leads to up-regulation of WUS transcription in the SAM organizing center, which results in splitting and fasciation of the primary shoot apex.
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Materials and methods |
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Microscopy
Scanning electron microscopy was performed as described previously
(Bowman et al., 1989) using a
Hitachi 4700 scanning electron microscope with digital imaging capability.
Confocal scanning electron microscopy analysis was performed as described
(Running et al., 1995
) using a
LSM Zeiss 510 confocal microscope.
Histology
Tissues were fixed, embedded in Technovit 7100 resin, sectioned at 5 µm
thick and stained in Toluidine Blue solution as described previously
(Smith and Hake, 2003).
GUS staining
The GUS staining reaction and subsequent tissue embedding and sectioning
were performed as described previously
(Sieburth and Meyerowitz,
1997) using 2 mM 5-bromo-4-chloro-3-indolyl-ß-D glucoronide
(X-GLUC; Bioworld, Dublin, OH, USA).
In situ hybridization
Tissue fixation and in situ hybridization were performed as described
previously (Jackson, 1992).
Probes for in situ hybridization were transcribed using the digoxigenin
labeling mix (Roche). For the REV probe, the full-length cDNA was
used as a template. For the CNA and PHB probes, the
nucleotide regions 1230-2511 and 1285-2554, respectively, relative to the ATG,
were used as templates. For the miR166 probe, four concatamers of
sense or antisense sequences were synthesized as oligonucleotides and cloned
into pBS-SK. Hybridization with the miR166 probe was carried out at
42°C overnight. After hybridization, the slides were washed four times at
40°C for 10 minutes each.
Real-time and RT-PCR
Aerial parts of 28-day Col and jba-1D homozygous plants were used
for real-time PCR analysis. cDNA synthesis was carried out in a 20 µl
reaction using 1 µg DNase I-treated total RNA by the reverse transcription
system (Promega). The cDNA reaction mixture was diluted 1:10 using DNase-,
RNase-free Milli-Q water and 1 µl was taken for real-time amplifications.
Amplifications were carried out in duplicate on 96-well plates in a 25 µl
reaction volume containing 12.5 µl 2x SYBR Green Supermix (Bio-Rad),
0.25 µM each of forward and reverse primers and 1 µl of the 1:10-diluted
cDNA. All reactions were performed independently twice on iCycler (Bio-Rad)
and once on DNA Engine Opticon (MJ Research, Hercules, CA, USA) to ensure
reproducibility. For all samples the cDNA levels were normalized using a
ubiquitin control. For RT-PCR analysis, total RNA was isolated from 9-day-old
seedlings and inflorescences using the RNeasy plant kit (Qiagen). The samples
were treated with RQ1 RNase-free DNase (Promega) for 30 minutes at 37°C
and then purified with phenol/chloroform. First strand cDNA synthesis was
performed on 5 mg of total RNA using Superscript III RNase H
reverse transcriptase (RT) (Gibco-BRL, Life Technologies) and an oligo(dT)
primer according to the manufacturer's instructions. Of the 20 µl of the RT
reaction 1 µl was used for each PCR reaction. For CNA, PHB, PHV
and ATHB8, 30 cycles of PCR were performed; for REV, 25
cycles of PCR were performed. The annealing temperature was 62°C for all
genes. The set of primers used for all five genes flank the miR166
recognition sequence: (REV S-ggattgctctcaatcgcagagg,
AS-ctcacaaactgagaagctgaagc; PHB S-ggactcctttctatagcagaggagg,
AS-aaagtttgaagaaggtggcccag; PHV S-ttgcggaggagaccttggcg,
AS-gatagtaccaccatttccagtg; ATHB8 S-cttgacccctcaacatcagcctc,
AS-gcaagcacgagcagcgattccc; CNA S-caattggcatctcaaaatcctcag
AS-gggccaatgtagttggtgcatag). The primers used to amplify the control
EF1 gene are S-caggctgattgtgctgttcttatcat and
AS-cttgtagacatcctgaagtgtggaaga.
Small RNA isolation and blot analysis
RNA isolation and RNA filter hybridization were performed as described
previously (Chen, 2004). Blots
were hybridized using miR166 antisense end-labeled oligonucleotides.
As a ladder, a mixture of 0.1 µM miR166 sense oligonucleotides and
0.1 µM miR166 sense plus 13 nucleotides was loaded on a gel.
Primer sequences are available on request.
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Results |
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Analysis of hemizygous and homozygous jba-1D plants revealed three distinct morphological phenotypes (Fig. 1). The most dramatic jba-1D/+ phenotype was extreme fasciation of the flowering shoot apical meristem (inflorescence meristem), which caused the stem to grow as a wide strap-like structure rather than as a point (Fig. 1B). The second phenotype was a reduced or filamentous gynoecium, which rendered the hemizygous plants less fertile than their wild-type siblings (Fig. 1G). The third phenotype was epinastic leaf morphology (Fig. 1K). Homozygous jba-1D plants had more dramatic leaf phenotypes, producing severely downward curling and radialized leaves (Fig. 1I,L,M). jba-1D plants also displayed more extreme inflorescence meristem and flower phenotypes than jba-1D/+ plants, and were completely sterile (Fig. 1C,H). Thus the developmental phenotypes appeared to be dose-dependent a single copy of this dominant mutation was sufficient to confer the phenotypes, but two copies made the phenotypes more severe. Pollen dehiscence was also reduced in jba hemizygous and homozygous plants, but all other aspects of development appeared to be unaffected by the mutation.
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We confirmed the jba-1D SAM splitting phenomenon by using pSTM::GUS as a marker for the meristem boundary. This construct drives GUS expression at the SAM periphery, between the central region of the SAM and the developing lateral organ primordia (Fig. 2I). We found that after 11 days of vegetative growth, some jba-1D shoot apices consisted of two well-defined meristems rather than one single primary meristem. Each meristem was marked by pSTM::GUS expression at the periphery (Fig. 2J). Multiple meristems were never observed in jba-1D embryos, indicating that this phenomenon occurs de novo during post-embryonic development.
Cell fate in the SAM is controlled via a regulatory pathway involving the
WUSCHEL (WUS) and CLAVATA (CLV) gene products (reviewed by
Carles and Fletcher, 2003).
The WUS gene is expressed in the central, interior region of the SAM,
and encodes a homeodomain transcription factor that confers stem cell fate on
the overlying cell population (Mayer et
al., 1998
; Schoof et al.,
2000
). CLV3 is expressed in the stem cells, and encodes a
small, secreted polypeptide that limits the size of the WUS
expression domain (Brand et al.,
2000
; Fletcher et al.,
1999
; Rojo et al.,
2002
). Using promoter-GUS constructs we determined that the
WUS expression domain (Fig.
3A-D) and the CLV3 expression domain
(Fig. 3E-F) enlarged
coordinately in jba-1D SAMs, indicating that the fasciated meristem
phenotype of jba-1D plants is due to the presence of many excess stem
cells. In addition, we found that the distribution of pWUS::GUS
expression was not uniform, and in some cases we observed two adjacent foci of
WUS expression within the primary meristem
(Fig. 3D). The appearance of
such foci of WUS expression in jba-1D plants can explain the
observed meristem phenotype, where independent domains of WUS
expression lead to the formation of discrete, ectopic meristems. In serial
longitudinal sections through entire 11-day old jba-1D seedlings, no
WUS expression was detected outside of the shoot apical meristem.
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We further examined the jba-1D fasciation phenotype by analyzing
the stem vascular patterning. In wild-type Arabidopsis plants, the
vascular tissue in the stem is present as a ring of approximately five to
eight separate bundles (Fig.
4A). The vascular bundles are collateral, with xylem positioned
close to the center of the stem and phloem in more peripheral positions
(Turner and Sieburth, 2001).
Cross sections of the inflorescence stem revealed three types of alterations
in the vascular patterning of jba-1D plants. First, jba-1D
stems had increased numbers of discrete vascular bundles in peripheral
positions along the stem (Fig.
4C). In the most extremely fasciated jba-1D stems, as
many as 29 individual bundles could be observed. Second, jba-1D
mutant stems displayed defects in the organization of the vascular cell types
within these bundles. The wild-type collateral pattern of xylem toward the
inside and phloem toward the outside (Fig.
4B) was disrupted in jba-1D stems by the appearance of
ectopic xylem elements close to the periphery
(Fig. 4D,F). This suggests a
defect in the jba-1D plants in the production, reception or
interpretation of positional signals that pattern the vasculature. Third,
jba-1D mutants formed extra bundles abnormally located in the center
of the stem (Fig. 4C). These
bundles exhibited an amphivasal arrangement, with xylem surrounding phloem
(Fig. 4E), and might represent
veins from the multiply splitting meristems and/or from the radial leaves that
protrude from the shoot apex. These results reveal that the jba-1D
mutation affects the number, positioning and polarity of stem vascular
bundles.
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Since jba-1D plants show very similar shoot meristem and vascular
phenotypes to those of the REV gain-of-function mutants avb1
and rev-10D (Emery et al.,
2003; Zhong and Ye,
2004
), we tested the contribution of REV to the
jba-1D phenotype by crossing jba-1D plants to previously
described rev-6 mutant plants
(Otsuga et al., 2001
). The
REV locus and the jba-1D T-DNA insertion site are tightly
linked on chromosome 5 with an estimated recombination frequency of 3.2%
(
5 cM). From 250 F2 plants that were selected on plates for
the jba-1D T-DNA insertion, we were able to identify three
jba-1D/+ rev/rev plants. Analysis of these plants showed that the
rev-6 mutation does not suppress the jba-1D/+ stem
fasciation phenotype (see Fig. S2 in supplementary material). This result
shows that wild-type REV function is not essential for the stem
fasciation of jba plants. In contrast, radial leaves are not observed
in the jba-1D/+ rev/rev plants, indicating that REV does
play a role in conditioning the jba leaf phenotypes.
Over-expression of miR166g causes the jba-1D phenotypes
The jba-1D T-DNA insertion falls in an intergenic region on
chromosome 5 (Weigel et al.,
2000). We determined that the T-DNA element was inserted 1890 base
pairs (bp) downstream of At5g63710 and 1861 bp upstream of At5g63720
(Fig. 5A). Using RT-PCR we
found that the At5g63720 gene is over-expressed in jba-1D mutant
plants. However, transgenic Arabidopsis plants over-expressing
At5g63720 under the control of the CaMV 35S promoter displayed a wild-type
phenotype, indicating that this gene does not cause the jba
phenotypes. Searching for other potential genes in the region we found a known
microRNA locus, MIR166g, located 394 bp downstream of the T-DNA
insertion site (Fig. 5A).
MIR166g potentially targets members of the class III
homeodomain-leucine zipper (HD-ZIP) family of transcription factors
(Rhoades et al., 2002
).
Alignment of the mature miR166g RNA sequence with the members of the
Arabidopsis class III HD-ZIP gene family shows 18 bp of
complementarity with all five sequences
(Fig. 5B). The miRNA
complementarity site is found within the highly conserved putative
sterol/lipid-binding START domain. Plants carrying dominant mutations in this
region that reduce miRNA complementarity but do not change the amino acid
sequence exhibit severe developmental phenotypes, suggesting that the
gain-of-function phenotypes may be due to altered miRNA binding rather than
altered protein function (Emery et al.,
2003
; Tang et al.,
2003
).
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The over-expression of miR166 in jba-1D plants was confirmed by hybridizing a low-molecular mass RNA blot with a 21-nucleotide probe complementary to the mature miR166 sequence (Fig. 5C). In jba-1D seedlings and inflorescence meristems, the level of the 21-nucleotide miR166 was increased compared with wild type. In seedlings, measurement of the level of expression shows that jba-1D plants homozygous for the T-DNA insertion generated a higher level of miR166 transcripts than hemizygous jba-1D/+ plants, accounting for the dose dependence of the jba phenotypes. miR166 is also expressed at much higher levels in both wild-type and jba-1D inflorescences than in seedlings (Fig. 5C).
Embryo expression of miR166 and an HD-ZIP target gene
To investigate the role played by miR166g in regulating
Arabidopsis morphogenesis, we determined the tissue distribution of
the mature miR166 sequence in wild-type and jba-1D/+ plants
using in situ hybridization. Because the miR166g-mediated
jba-1D phenotype was detectable in mature embryos, we focused our
analysis on the embryonic stage of development. The miR166 expression
pattern was identical in developing wild-type
(Fig. 6A-E) and
jba-1D/+ (Fig. 6G-K)
embryos, confirming that the 35S enhancer elements present in jba-1D
up-regulate the affected gene in its normal expression domain rather than
inducing ectopic expression. The first stage at which we could reliably detect
miR166 transcripts was the late globular stage, where initial
expression is restricted to the periphery of the hypocotyl and the tips of the
initiating cotyledons (Fig.
6A,G). As the cotyledons emerge, expression expands to include the
abaxial region and the distal tip (Fig.
6B,C,H,I). At the late torpedo stage, miR166 was detected
in the peripheral cells of the hypocotyl and along the adaxial and abaxial
margins of the cotyledons (Fig.
6D,J). Weaker, if any, expression was detected in the central
portion of the cotyledons corresponding to the developing vasculature. In
mature embryos, the expression of miR166 changed dramatically in both
wild-type and jba-1D/+ backgrounds. At this stage miR166
accumulated in the SAM, the adaxial region of the cotyledons, and the
provascular tissues of the embryos (Fig.
6E,K). These results demonstrate that miR166 has a
dynamic expression pattern during Arabidopsis embryogenesis.
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Finally, to analyze whether the increase in REV transcript levels and the decrease in CNA and PHB transcript levels in jba-1D plants is due to alteration in their tissue distribution, we performed an in situ hybridization analysis on seedlings using REV, CNA and PHB probes. The REV expression pattern in wild-type (Fig. 8A) and jba-1D (Fig. 8B) seedlings was similar. In both backgrounds, the REV mRNA accumulated in the adaxial regions of the leaf primordia, in the vascular tissues and in the central zone of the SAM. These results show that the tissue distribution of REV mRNA is not altered in jba-1D seedlings. The CNA expression in wild-type seedlings was restricted to the adaxial region of developing leaves, to the vascular tissues and to the central, interior region of the SAM (Fig. 8C). In jba-1D seedlings, the CNA expression pattern changed significantly (Fig. 8D). CNA transcripts accumulated at a very low level in the adaxial region of initiating leaf primordia and in the adaxial region of developing leaves (data not shown), but no signal was detected in the vascular tissues or in the center of the SAM. The PHB expression pattern was also altered in jba-1D seedlings. Wild-type seedlings express PHB in the adaxial region of the leaf primordia, in the vascular tissues and in the SAM, mostly in the overlying layers (Fig. 8E). In jba-1D seedlings, PHB was expressed at only a very low level in the adaxial region of initiating leaf primordia on the flanks of the SAM and in the vascular tissues, but no transcripts are detected in the SAM itself. These results are consistent with the RT-PCR data and demonstrate that over-expression of miR166g has differential effects on the transcript levels and tissue distributions of its AtHD-ZIP target genes.
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Discussion |
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Over-expression of miR166g causes severe developmental defects
Several independent lines of evidence demonstrate that the jba-1D
phenotypes are caused by over-expression of miR166g. First,
miR166 transcripts are elevated in jba-1D seedlings and
inflorescences, and this increase in transcript level occurs in a
dose-dependent manner that corresponds with the increase in phenotype
severity. Second, a 543 bp genomic region from the 35S enhancers through the
MIR166g locus, but lacking coding sequences for any of the
surrounding genes, is sufficient to recapitulate all aspects of the
jba phenotype when introduced into wild-type Col plants. Third, DCL1
function is required to obtain the jba phenotypes, indicating that
the effects of the jba-1D mutation require miRNA activity. Finally,
mRNA expression levels of the targets of miR166g, the five members of
the class III HD-ZIP family of transcription factors, are altered in
jba-1D plants.
Over-expression of miR166g in jba-1D plants causes
specific developmental defects. The earliest detectable phenotype is an
increase in jba-1D mature embryo SAM size, indicating that the
regulatory activity of miR166 is already required during
embryogenesis. The jba-1D SAM continues to enlarge throughout the
vegetative and inflorescence phases, culminating in the splitting of the
primary shoot apex into multiple independent SAMs and the fasciation of the
inflorescence meristems. Meristem enlargement in jba-1D plants occurs
through the coordinate expansion of CLV3-expressing cells in the
overlying layers of the meristem and WUS-expressing cells in the
interior, and requires wild-type WUS activity. The fasciated
meristems of clv mutants also display coordinate expansion of
CLV3-expressing and WUS-expressing cells, and are
WUS dependent (Brand et al.,
2000; Schoof et al.,
2000
). However, the fasciated meristems of clv plants and
those of jba-1D plants are generated by different molecular
mechanisms. The CLV pathway restricts the size of the WUS-expressing
cell population by preventing its expansion laterally and upward into the L2
cell layer (Brand et al.,
2000
; Schoof et al.,
2000
). In contrast, miR166 appears to regulate meristem
size by indirectly controlling the amount of WUS transcription within
the organizing center itself. Thus miR166 and the CLV
pathway function in parallel to regulate the level of WUS
transcription and the number of WUS-expressing cells in vegetative
and inflorescence meristems, respectively.
The jba-1D mutation also causes the adaxialization of rosette
leaves and vascular bundles in the stem, and a reduction in gynoecium tissue.
Given that PHB and PHV confer adaxial identity
(Emery et al., 2003;
McConnell and Barton, 1998
),
the adaxialization of jba-1D leaves seems counterintuitive. However,
experiments have shown that a SAM-derived signal(s) is important for
specifying adaxial leaf identity (Snow and
Snow, 1959
; Sussex,
1954
; Waites and Hudson,
1995
), and thus initiating jba-1D leaves may receive an
excess of adaxializing signal(s) emanating from multiple SAMs. Increased
accumulation of transcripts from REV, which plays a redundant role
with PHB and PHV in conferring adaxial fate
(Emery et al., 2003
), may also
contribute to conditioning this phenotype, as increased meristem size per se
is not sufficient to cause leaf adaxialization
(Clark et al., 1993
).
Similarly, the decrease in PHB, PHV and CNA transcript
levels observed in jba-1D plants is unlikely to be the sole cause of
the reduced gynoecium phenotype, as phb phv cna mutants form extra
carpels instead of fewer carpels (Prigge
et al., 2005
). jba-1D floral meristems are the same size
as wild-type meristems, and thus the gynoecium defect is probably not due to
premature floral meristem termination. jba-1D gynoecia consist of
fewer cell types than normal and occasionally lack vasculature (data not
shown), suggesting instead a possible defect in gynoecium patterning and/or
polarity. Further work will be required to unravel the precise roles of
miR166 and the class III HD-ZIP genes in gynoecium
development.
miR166 expression and regulation of the class III HD-ZIP genes
Our experiments show that over-expression of miR166g in
activation-tagged jba-1D plants causes significant changes in the
mRNA accumulation of their target class III HD-ZIP family members. We
find that the overall transcription levels of the HD-ZIP genes are
affected in jba-1D seedlings and inflorescences, but that the five
target genes do not respond to miR166g over-expression in the same
way. ATHB8 transcript levels are unaffected by miR166g
over-expression, suggesting that ATHB8 may be targeted by other
members of the miR165/166 group, such as miR166a
(Kim et al., 2005). PHB,
PHV and CNA are all down-regulated in jba-1D seedlings
and inflorescence meristems, while REV transcription is elevated.
Since REV shows a similar expression pattern in wild-type and
jba1-D seedlings (Fig.
8) and inflorescence meristems (data not shown), an explanation
for the unexpected up-regulation of REV may be that REV is a
target of negative regulation by PHB, PHV and/or CNA. This
theory is consistent with previous observations that CNA and
ATHB8 partially suppress the rev and rev phv
lateral and floral meristem defects, indicating an antagonistic relationship
between them (Prigge et al.,
2005
).
Interestingly, we found that miR166g has a dynamic expression
pattern in developing wild-type and jba-1D embryos. During the early
stages of embryogenesis, miR166 accumulates predominantly in the
peripheral regions of the hypocotyls and throughout the developing cotyledons.
The hypocotyl expression pattern of miR166 is reciprocal to those of
its five target genes, which are all expressed in overlapping patterns but are
largely restricted to the central cells
(Emery et al., 2003;
Prigge et al., 2005
). These
data suggest that during early embryogenesis miR166 acts to clear the
transcripts of its target gene(s) from the periphery of the developing
hypocotyl. In contrast, the expression patterns of miR166 and
REV, PHB, PHV and CNA overlap in the developing cotyledons,
indicating that the presence of the miRNA does not cause complete turnover of
its target transcripts in these tissues. In mature embryos the miR166
expression pattern alters dramatically, becoming confined to the SAM, the
adaxial side of the cotyledons, and the vasculature. At this stage of
development the expression pattern of miR166 is coincident with that
of its targets [REV in the SAM; REV, PHB and PHV in
the cotyledons and all five genes in the vasculature
(Prigge et al., 2005
)]
suggesting that a major effect of miR166 expression at this stage may
be to modulate the mRNA transcript levels of the class III HD-ZIP
genes. The Arabidopsis class III HD-ZIP genes may thus
represent examples of so-called miRNA `tuning targets', messages for which
miRNA regulation adjusts the protein output in a fashion that permits
customized expression in different cells types yet a more uniform expression
level within each cell type (Bartel and
Chen, 2004
). Alternatively, or additionally, the overlap of the
miRNA and target mRNA expression patterns may suggest that the HD-ZIP
genes are at this stage of development either targeted for translation
repression or for methylation of their coding regions, as has been previously
shown for the PHB and PHV loci
(Bao et al., 2004
).
Regulation of shoot apical meristem activity by class III HD-ZIP genes
The most dramatic phenotype caused by over-expression of miR166g
is the extensive SAM enlargement and stem fasciation. Previous work has
implicated members of the class III HD-ZIP family in meristem regulation.
rev phb phv mutants lack a functional embryonic SAM
(Emery et al., 2003),
indicating that REV plays a redundant role with PHB and
PHV in SAM establishment. rev mutants have reduced lateral
and floral meristem activity (Otsuga et
al., 2001
; Talbert et al.,
1995
), indicating that REV also promotes post-embryonic
meristem initiation and function. REV gain-of-function mutations
avb1 and rev-10D lead to SAM enlargement, defective leaf
polarity and altered vascular patterning
(Emery et al., 2003
;
Zhong and Ye, 2004
). These
phenotypes resemble those observed in jba-1D plants, which accumulate
higher than normal levels of REV transcripts. However, eliminating
REV activity in a jba-1D background did not significantly
attenuate the jba shoot meristem phenotypes. This result indicates
that REV plays at most a minor role in conditioning the SAM
enlargement and stem fasciation defects caused by over-expression of
miR166g.
The shoot apical meristem phenotypes of jba-1D plants are also
very similar to those of phb phv cna plants
(Prigge et al., 2005). Like
jba-1D mutants, phb phv cna mutants produce enlarged SAMs,
fasciated meristems and internal vascular bundles in the stem. The redundant
role of PHB, PHV and CNA in meristem regulation is
independent of REV (Prigge et
al., 2005
), as is the role of miR166g (see Fig. S2 in
supplementary material). Although cna mutations have no discernable
effect on their own they enhance the stem cell accumulation defects of
clv null mutant meristems, suggesting that CNA acts in parallel with
the CLV pathway to regulate shoot apical meristem size
(Green et al., 2005
).
Our data confirm that proper regulation of class III HD-ZIP gene
activity is a critical feature of normal shoot apical meristem function. We
propose that in wild-type plants, PHB, PHV and CNA restrict
SAM activity by down-regulating WUS transcription. During the late
globular and heart stages of embryogenesis, PHB, PHV and CNA
are all expressed in apical cells of the presumptive SAM
(Prigge et al., 2005), in a
pattern coincident with that of WUS
(Mayer et al., 1998
). After
germination, PHB and CNA continue to be expressed in the
meristem (Fig. 8)
(McConnell et al., 2001
), the
CNA expression domain remaining coincident with that of WUS.
Thus the transient expression of PHB, PHV and CNA early in
embryogenesis, and the persistent expression of PHB and CNA
in post-embryonic development, may be required to modulate the level of
WUS transcription, leading to the maintenance of a stem cell
population of the appropriate size. In jba-1D SAMs, over-expression
of miR166g would cause a reduction in the level of PHB, PHV
and CNA mRNAs and a resultant elevation of WUS
transcription. Abnormally high levels of WUS activity could then
promote excess stem cell accumulation and the eventual establishment of new
stem cell foci, leading to SAM enlargement and ultimately to the splitting and
fasciation of the jba-1D shoot apical meristem. We also find that
this activity of PHB, PHV and CNA in regulating SAM size is
largely independent of REV, consistent with data from analysis of
HD-ZIP triple and quadruple mutant plants
(Prigge et al., 2005
).
Nine MIR165/166 loci are present in the Arabidopsis genome, each of which has the potential to regulate any or all of its five class III HD-ZIP gene targets either by clearing the transcripts from specific cells or by regulating the level of transcript accumulation. To account for the observed expression of the HD-ZIP genes in combinatorial tissue- and stage-specific patterns, it seems reasonable to expect that the nine MIR loci will also have dynamic and differential transcription profiles. Further examination of the regulatory interactions between the various MIR165/166 family members, their class III HD-ZIP target genes, and meristem maintenance factors will reveal additional insights into the complex interplay between polar lateral organs and the shoot apical meristem from which they derive.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/16/3657/DC1
* Present address: Department of Plant Sciences, Oxford University, Oxford
OX1 3RB, UK
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