|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 3 July 2006
doi: 10.1242/dev.02491
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.
* Author for correspondence (e-mail: spoethig{at}sas.upenn.edu)
Accepted 13 June 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: siRNA, RNAi, Heterochrony, Arabidopsis
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). This phenomenon is known as `heteroblasty' or `phase
change', and is brought about by the intersection of pathways controlling
developmental timing and leaf morphogenesis. In Arabidopsis,
heteroblasty can be observed in the gradual transition from leaves with long
petioles and round, smooth, flat blades to leaves with short petioles and
elliptical, serrate, epinastic blades
(Telfer et al., 1997). The
number of hydathodes per leaf also increases steadily throughout vegetative
growth (Hunter et al., 2003;
Tsukaya and Uchimiya, 1997),
whereas trichome distribution defines discrete phases, with leaves gaining
abaxial trichomes at the juvenile-to-adult transition, and losing adaxial
trichomes at the adult-to-reproductive transition
(Telfer et al., 1997).
Several genes responsible for the temporal control of this process have
recently been identified. These include the ARGONAUTE family member
ZIPPY (ZIP) (Hunter et
al., 2003), the RNA-dependent RNA polymerase RDR6, the
plant-specific gene SGS3
(Peragine et al., 2004), the
Dicer-like gene DCL4 (Gasciolli
et al., 2005; Xie et al.,
2005; Yoshikawa et al.,
2005), the exportin-5 homolog HASTY (HST)
(Bollman et al., 2003;
Park et al., 2005;
Telfer and Poethig, 1998), and
the zinc finger gene, SERRATE (SE)
(Clarke et al., 1999;
Prigge and Wagner, 2001).
Mutations in these genes cause the precocious expression of traits associated
with later stages of development; for example, mutations in ZIP, RDR6,
SGS3 and DCL4 cause premature leaf elongation, downward curling
of the leaf margin, serration, and abaxial trichome production
(Gasciolli et al., 2005;
Hunter et al., 2003;
Peragine et al., 2004;
Yoshikawa et al., 2005). All
of these genes have roles in the biogenesis of miRNAs or endogenous siRNAs
(Allen et al., 2005;
Dunoyer et al., 2005;
Park et al., 2005;
Peragine et al., 2004;
Vazquez et al., 2004;
Yoshikawa et al., 2005), which
strongly suggests that their mutant phenotype can be attributed to the
aberrant expression of genes normally repressed by these small RNAs.
Expression profiling of zip, rdr6 and sgs3 revealed several
genes whose transcripts accumulate in these mutants
(Peragine et al., 2004), but
which of these upregulated genes, if any, is responsible for their precocious
phenotype is still unknown.
We addressed this question using a genetic approach. Assuming that the
precocious phenotype of zip is indeed a result of upregulated gene
expression, we looked for loss-of-function mutations that suppress this
phenotype. Remarkably, this screen produced mutations in the two most-highly
upregulated genes in zip, rdr6 and sgs3: ETTIN
(ETT/ARF3) and AUXIN RESPONSE FACTOR 4 (ARF4). ETT and
ARF4 are closely related members of the auxin response factor family
of transcription factors (Remington et
al., 2004; Ulmasov et al.,
1999). The first ett mutants were isolated as plants with
severely malformed gynoecia: apical-basal defects caused the expansion of the
style and stipe at the expense of the ovary, and adaxialization led to the
peripheral expansion of central tissues, including the stigma and the
transmitting tract (Sessions and
Zambryski, 1995). The finding that ETT is expressed
abaxially in the developing gynoecium
(Sessions et al., 1997)
supported its role in establishing abaxial/peripheral tissues. Both
ETT and ARF4 have been shown to be expressed during
vegetative development: ETT mRNA is present in the shoot apical
meristem, leaf primordia, and the margins, vascular bundles and stipules of
mature leaves, whereas ARF4 mRNA is expressed in the abaxial domain
of leaf primordia and the phloem of mature leaves
(Pekker et al., 2005). Plants
doubly mutant for ett and arf4 resemble kan1;kan2
double mutants, suggesting that these genes are involved in the specification
of abaxial identity (Alvarez et al.,
2006; Pekker et al.,
2005). However, neither gene has been shown to have an independent
vegetative phenotype.
Here, we describe the effect of ett and arf4 on leaf
morphology, and their interaction with the ZIP-mediated timing
pathway. We show that upregulation of ETT and ARF4 is
largely responsible for the vegetative morphology of zip. We further
show that the zip phenotype can be attributed to a defect in the
production or stability of a trans-acting (ta) siRNA from the TAS3
locus (tasiR-ARF), which is known to direct the cleavage of the mRNA
of these genes (Allen et al.,
2005; Williams et al.,
2005). The abundance of tasiR-ARF, as well as the
ETT and ARF4 mRNAs, does not change significantly during
vegetative development. This result is consistent with the mutant phenotype of
zip (Hunter et al.,
2003), and suggests that tasiR-ARF regulation of
ETT and ARF4 transcript levels influences heteroblasty by
determining the sensitivity of leaf primordia to a temporal signal, rather
than by serving as a component of a developmental clock.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) was
obtained from Jason Reed (Chapel Hill, NC). ett-7 was a gift from
Patricia Zambryski (Berkeley, CA), and the zip (Ler) T-DNA
insertion line (CSH3629) was obtained from the Cold Spring Harbor Laboratory.
Plant growth, leaf shape analysis and trichome counts were carried out as
described by Peragine et al. (Peragine et
al., 2004) and camera lucida tracings were performed as described
by Kerstetter et al. (Kerstetter et al.,
2001). Leaf length was measured as the distance between the leaf
tip and the intersection point of lines drawn tangent to the leaf base. The
width of the leaf blade was measured at the midpoint of this line.
Mutant isolation and identification
zip-2 seeds were mutagenized in 0.4% EMS for 6-9 hours prior to
planting. Individually harvested M2 families (n=2400) were screened
for mutations that suppressed the leaf shape phenoype of zip-2.
ett-15 was mapped using the F2 plants from a cross to the zip
(Ler) T-DNA insertion line CSH3629, which we obtained from the Cold
Spring Harbor Laboratory. Five additional suppressors were classified
as ett or arf4 mutations by their failure to complement
ett-15 or arf4-2.
RNA analysis
Isolation of low molecular weight RNA, northern analysis, and
RLM-5'RACE were carried out as described previously
(Yoshikawa et al., 2005).
tasiR-ARF was identified on blots of low molecular weight RNA using
the locked nucleic-acid oligonucleotide probe:
5'-T(+G)GGG(+T)CTT(+A)CAA(+G)GTCA(+A)GAA-3'.
For RT-PCR, total RNA was isolated from leaves with Trizol reagent (Invitrogen) and PCR amplification was carried out with the following primers for the analysis of ARF3 and ARF4 expression:
ARF3F, 5'-TGGTCCCAAGAGAAGCAGG-3';
ARF3R, 5'-TCCACCATCCGAACAAGTG-3';
ARF4F, 5'-GCCGCTGAAGATTGTTTTGCTC-3';
ARF4R, 5'-AGTAGATGCCTCCTTGGTTGACC-3';
EIF4AF, 5'-GCGCATCCTCCAAGCTGGTGTCC-3'; and
EIF4AR, 5'-GGTGGAAGAAGCTGGAATATGTCAT-3'.
The following primers were used for the analysis of splicing defects in arf4 alelles:
Ex1F, 5'-GATGCTATGGTTTCATATTCGTCTCC-3';
Ex2R, 5'-TGTAGACCTCATCGGTGTCCTTATTAG-3';
Ex8F, 5'-AACTCTAAATGGAGGTGCTTGTTG-3';
Ex9R, 5'-GCCTTGGAGATGACTGAATGC-3';
Ex2F, 5'-CCAGTTGCTTGCTAATAAGGACAC-3';
Ex3R; 5'-CTTGACCTCTTTCCCCTCCC-3';
Ex5F; 5'-CTCGTCTCTGGTGATGCGG-3';
Ex6R; 5'-TCAGGAAGTCCATTTCTTGGC-3'.
ETT overexpression constructs
The ETT open reading frame (ORF) was amplified using primers
adding BspHI and BstEII sites to the 5' and 3'
end of this transcript, respectively (ARF3fBsp,
5'-ATCATGAGCGGTGGTTTAATCGATCTGAACG-3'; ARF3rBst,
5'-TGGTTACCCTAGAGAGCAATGTCTAGCAAC-3'). Addition of the 5'
BspHI site added a Ser residue after the initial Met, but this did
not affect the ability of the construct to rescue ett-15. The
resulting product was inserted into the T-easy vector (Promega) and used as a
template for site-directed mutagenesis using the following oligonucleotides,
as described previously (Wang and Malcolm,
1999).
mAf, 5'-CCAGAGGGTCCTGCAGGGACAGGAGATTTTTCCGGG-3';
mAr, 5'-CCCGGAAAAATCTCCTGTCCCTGCAGGACCCTCTGG-3';
mBf, 5'-CCATAAGGTCCTGCAGGGACAGGAGACAGTTCCCGCC-3';
mBr, 5'-GGCGGGAACTGTCTCCTGTCCCTGCAGGACCTTATGG-3'.
The mutated ETTIN ORFs were inserted behind the CaMV 35S promoter
in NcoI/BstEII digested pCambia3301, and transformed into
Col and ett-15 plants by the floral dip method
(Clough and Bent, 1998).
|
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) in the
closely related gene ARF4. One of these mutations (arf4-3)
is located in the middle of the first intron and two others (arf4-4
and arf4-5) are located at splice donor sites
(Fig. 1A). All three of these
mutations produce transcripts with unspliced introns
(Fig. 1B) that introduce stop
codons in the ARF4 ORF. arf4-6 is a missense mutation that converts
an arginine to a lysine (Fig.
1A,B). Most of the research presented here was conducted with
ett-7, a point mutation that generates a stop codon at the extreme
N-terminal end of ETT, and arf4-2, a T-DNA insertion near
the C-terminal end of ARF4 (Fig.
1A). These mutations were chosen because their phenotype is
slightly stronger than the suppressors isolated in our screens.
|
|
2.7 for leaf 7 and remained constant for several leaves
before declining. ett-7 and arf4-2 produced a significant
(n=10; P<0.01) decrease in the L:W ratio of leaves 1-8,
but did not affect the morphology of the last several leaves of the shoot.
Consistent with this effect, ett and arf4 delayed abaxial
trichome production in both the presence and the absence of zip-2
(Fig. 3C,D). On average,
zip-2 plants had 2.8±0.3 leaves without abaxial trichomes
(compared with 3.5±0.3 in Col). This number was increased to
6.6±0.5 in zip-2;ett-15, to 5.6±0.7 in
zip-2;ett-7, and to approximately 4 in zip-2;arf4-2,
zip-2;arf4-3, zip-2;arf4-4, zip-2;arf4-5 and zip-2;arf4-6. These
mutations produced a slightly greater delay in abaxial trichome production as
single mutants, but in most cases this effect was so small that it was not
statistically significant (Fig.
3C; data not shown). Interestingly, the trichome phenotype of
+/ett-7 was intermediate between that of homozygous mutant and
homozygous wild-type plants (Fig.
3D). Although ett mutations that truncate within the
DNA-binding domain appear to have dominant-negative activity
(Pekker et al., 2005), the
stop codon in ett-7 is located at the extreme N terminus of this
region, and should therefore eliminate most of this conserved domain. Whether
the semi-dominant phenotype of ett-7 results from haploinsufficiency
or dominant-negative activity, this result implies that abaxial trichome
production is sensitive to the dose of ETT. In summary, these results
indicate that ETT and ARF4 promote the expression of at least two juvenile
traits - `circular/elliptical' leaf morphology and the absence of abaxial
trichomes- and are required for the effect of zip-2 on these
traits.
|
). To
determine whether ett-7 also affects this trait, we introduced the
hydathode marker E340 (Hunter et al.,
2003) into an ett-7 background. ett-7 had no
effect on the number of hydathodes on leaves 1 through 7, indicating either
that upregulation of ETT is not responsible for this aspect of the
zip phentoype, or that hydathode production is regulated redundantly
by ETT and ARF4.
zip plants are often semi-sterile because stamen elongation is
delayed relative to the elongation of the pistil, and because the septum is
usually split near the tip of the carpels
(Hunter et al., 2003).
ett-7 and ett-15 also cause splitting of the septum, with
ett-7 having a much more severe phenotype than ett-15,
whereas arf4-2 has no observable carpel phenotype.
zip-2;ett-15 and zip-2;arf4-2 double mutants had a lower
frequency of septum splitting than did zip-2, suggesting that the
effect of zip-2 on septum development is attributable to the
increased expression of ETT and ARF4 in this mutant
(Fig. 3C). ett-15 had
a less significant effect than did arf4, probably because
ett-15 causes septum splitting in the absence of zip-2. The
fact that septum splitting is associated with both increased expression (in
zip-2) and decreased expression (in ett-7 and
ett-15) of ETT may reflect the marginal origin of the septum
(Liu et al., 2000): marginal
outgrowth typically only occurs at the boundary of two distinct cell types,
and loss of either cell fate can disrupt this process
(Bowman, 2000). Indeed, this
effect is consistent with the role of ETT and ARF4 in the
promotion of abaxial identity (Pekker et
al., 2005).
ETT has a non-redundant function in leaf polarity
In contrast to kan1 mutants, which have up-curled or unusually
flat leaves (Fig. 4A)
(Kerstetter et al., 2001),
ett and arf have little or no effect on leaf expansion as
single mutants and are therefore thought to have redundant functions in leaf
polarity (Pekker et al.,
2005). To further explore this theory, we examined the morphology
of the mesophyll cells in leaf 6 of wild type, kan1-12, ett-7, ett-15
and arf4-2 (Fig. 4B).
Adaxial (palisade) mesophyll cells of wild-type plants are round and tightly
packed, whereas abaxial (spongy) mesophyll cells are highly convoluted and
have abundant intercellular spaces. kan1-12 has a minor effect on
adaxial cell size, but simplifies the morphology of abaxial mesophyll cells
and reduces the amount of intercellular space in this tissue, causing it to
resemble adaxial mesophyll (Kerstetter et
al., 2001). arf4-2 and ett-15 did not have a
major effect on mesophyll morphology, but ett-7 produced a noticeable
reduction in the irregularity of spongy mesophyll cells. This observation
correlates with the observation that ett, but not arf4, can
correct the abaxialized phenotype of ANT::KAN2 plants
(Pekker et al., 2005). The
observation that ett-7 has a more dramatic effect on mesophyll cell
shape than ett-15 is consistent with the strength of their floral
phenotypes. These results demonstrate that ETT promotes the
differentiation of abaxial tissue in the leaf blade, and that this process is
more sensitive to the loss of ETT than to the loss of
ARF4.
zip upregulates ETT by blocking tasiR-ARF production
The ETT and ARF4 transcripts are targets of a ta-siRNA
from the TAS3 locus, tasiR-ARF, and accumulate in mutants
that block the production of this ta-siRNA
(Allen et al., 2005;
Peragine et al., 2004;
Williams et al., 2005). Both
of these transcripts are also upregulated in zip, and the isolation
of loss-of-function mutations of ETT and ARF4 as suppressors
of zip suggests that this increase plays a key role in the
zip phenotype. The basis for this increase is unclear, however,
because zip does not affect the accumulation of any of the siRNAs
that have been examined, including ta-siRNAs from the TAS1 and
TAS2 loci (Allen et al.,
2005; Peragine et al.,
2004; Vazquez et al.,
2004; Williams et al.,
2005; Yoshikawa et al.,
2005). Whether ZIP is required for the biogenesis of
tasiR-ARF is unknown.
To determine whether ZIP regulates ETT and ARF4
expression via the ta-siRNA pathway, we examined the effect of zip-2
on the expression of tasiR-ARF and mir390, a miRNA that is
involved in the biogenesis of tasiR-ARF
(Allen et al., 2005).
sgs3-11 and rdr6-11 had no effect on the level of
miR390, but the level of this miRNA was slightly increased in
zip-1. All three mutations dramatically reduced the level of
tasiR-ARF and produced a corresponding loss of tasiR-ARF-mediated
cleavage of ETT, as revealed by RLM-RACE
(Fig. 5A). As expected
(Yoshikawa et al., 2005),
siR1511 was absent in sgs3-11 and rdr6-11, but
present in zip-2. These results suggest that ZIP is required
for the biogenesis of siRNAs from TAS3, and also indicate that
ZIP either directly or indirectly represses the transcription or
biogenesis of the miRNA (miR390) that contributes to the generation
of these siRNAs. The observation that sgs3 and rdr6 do not
affect miR390 suggests that this effect is not mediated by a
ta-siRNA, as SGS3 and RDR6 appear to be generally required
for ta-siRNA biogenesis.
Based on its mutant phenotype, we originally suggested that zip
acts in a pathway that sets the threshold for the juvenile-to-adult
transition, rather that being a component of the developmental `clock' that
initiates this transition (Hunter et al.,
2003). This hypothesis was based on the observation that
zip affects the onset of the juvenile-to-adult transition (e.g.
abaxial trichome production, hydathode number) without affecting the number or
the character of transition leaves. This is in contrast to mutations, such as
hst, which accelerate both the onset of phase change and the rate at
which this process occurs (Telfer and
Poethig, 1998). To test this hypothesis, we examined the level of
tasiR-ARF, ETT and ARF4 at different times in shoot
development. Northern analysis revealed that the tasiR-ARF and
ARF3 transcripts are present at a relatively constant level during
the first three weeks of growth in plants grown in continuous light
(Fig. 5B). To obtain a more
accurate picture of the expression of ETT and ARF4, we
performed semi-quantitative RT-PCR on 3-mm and 6-mm long leaf primordia of
leaves 1 through 8 from zip and wild-type plants grown in either
short days (Fig. 5C) or
continuous light (Fig. 5D).
Both transcripts were more abundant in zip than in wild type, and
this difference was accentuated in short days. However, in both genotypes,
there was no apparent difference in the level of ETT or ARF4
mRNA in successive leaves. These results suggest that tasiR-ARF
constitutively represses ETT and ARF4, and they support the
hypothesis that ZIP sets the threshold at which leaves respond to a
temporal signal (via its effect on the level of ETT and
ARF4), rather than by regulating the production of this signal.
|
; Williams et al.,
2005) were mutated (Fig.
6A), under the regulation of the 35S promoter. The mutations that
were introduced in the tasiR-ARF target sites do not affect the amino
acid sequence of the protein, but are expected to eliminate or diminish the
ability of tasiR-ARF to repress ETT. 35S::ETT produced a
lower frequency of morphologically aberrant phenotypes than did
35S::ETTmAB, and also had a less significant effect on abaxial
trichome production. Twenty-seven percent of 35S::ETTmAB plants
produced abaxial trichomes at an abnormally early position (leaf 1-4), as
compared with 8% of plants expressing 35S::ETT
(Fig. 6B); furthermore, only
35S::ETTmAB produced plants with abaxial trichomes on leaf 1 or 2.
Many plants transformed with either 35S::ETT or 35S::ETTmAB
had abnormally late trichomes, presumably reflecting a co-suppression of
ETT by these overexpressed transgenes. RT-PCR analysis demonstrated
that the severity of the mutant phenotype in 35S::ETTmAB
transformants was correlated with the level of ETT expression, and
also revealed that many plants that appeared phenotypically normal had
equivalent, or higher, levels of ETT than did zip plants
(Fig. 6C). This latter result
suggests that ARF4, or another as yet unknown target of ZIP,
also contributes to the precocious phenotype of zip mutations.
About 16% of the 35S::ETTmAB primary transformants resembled
zip in that they had elongated, epinastic leaves and abaxial
trichomes on leaf 3 or 4 (Fig.
6D). These plants also resembled zip in having flowers
with short stamens and split replums (Fig.
6F). Plants with the highest levels of ETT had a more
severe phenotype that was strikingly similar to that of mutations in
asymmetric leaves 2 (as2)
(Iwakawa et al., 2002;
Ori et al., 2000;
Semiarti et al., 2001) and
blade-on-petiole1 (Ha et al.,
2003) (Fig. 6D-F).
These plants were very small, with tightly curled, deeply lobed leaves that
produced abaxial trichomes starting with leaf 1 or 2. Many leaves also had
small leaflets extending from the petiole; this phenotype was apparent on leaf
3 or 4 and became more severe on successive leaves. Petal elongation was
delayed in severely affected plants, and fully mature flowers had outwardly
curved, irregularly positioned petals (Fig.
6F). These flowers had short stamens that often failed to produce
pollen, but had relatively normal carpels with a low frequency of septum
splitting.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Here,
we show that the increase in ETT and ARF4 expression in
zip (Peragine et al.,
2004) is a consequence of a reduction in the level of a ta-siRNA
from the TAS3 locus, tasiR-ARF, and demonstrate that these
transcription factors are responsible for the precocious phenotype of
zip. Our results also indicate that tasiR-ARF may act as a
temporally constitutive repressor of ETT and ARF4, and that
the level of tasiR-ARF determines the sensitivity of leaf primordia
to the signal(s) responsible for the temporal variation in leaf
morphology.
|
; Sessions and Zambryski,
1995), and ett;arf4 double mutants have a vegetative
phenotype that is characteristic of adaxialized mutants, such as
kan1;kan2 or phb-D
(Alvarez et al., 2006;
Eshed et al., 2004;
McConnell and Barton, 1998;
Pekker et al., 2005). The
discoveries that loss-of-function mutations in ETT and ARF4
partially suppress the phenotype of zip, and that overexpression of
ETT in wild-type plants phenocopies zip, demonstrate that
overexpression of ETT and ARF4 is largely responsible for
the precocious phenotype of zip, and points to a relationship between
leaf polarity and heteroblasty.
Many, if not most, heteroblastic aspects of leaf morphology are polarized
along either an adaxial-abaxial or proximodistal axis. For example, the
increase in leaf epinasty that occurs during rosette development reflects an
increase in the growth rate of adaxial tissue relative to the growth rate of
abaxial tissue. Similarly, the differential distribution of trichomes on
rosette and inflorescence leaves is a consequence of an inherent difference in
the developmental potential of the adaxial and abaxial epidermis
(Telfer et al., 1997). It is
not surprising, therefore, that genes involved in the specification of leaf
polarity have a role in heteroblasty. But, while the ability of ett
and arf4 to suppress epinasty is readily explained by their
adaxialized phenotype, their effect on abaxial trichome production does not
have this explanation. Trichomes are absent or less abundant on the abaxial
than on the adaxial surface of rosette leaves, and mutations that cause
adaxialization, such as kan1-12
(Kerstetter et al., 2001) and
phb-D (McConnell and Barton,
1998), typically enhance trichome production on the abaxial leaf
surface. The delayed production of abaxial trichomes in ett and
arf4 is therefore inconsistent with an adaxialized phenotype. This
phenotype may reflect a separable function of these genes, as ett-7
and ett-15 have almost equivalent effects on trichome production
despite the much stronger effect of ett-7 on leaf and gynoecium
polarity. Similarly, arf4-2 delays abaxial trichome production and
affects leaf shape, but has no independent effect on leaf or carpel polarity.
Whether the effect of ett and arf4 on leaf shape is linked
to their function in leaf polarity or some other developmental pathway is
unclear. Whatever the case, the phenotype of these mutations individually and
in combination with zip suggests that ETT and ARF4
sit at a branch point in the mechanism of heteroblasty - linking a variety of
morphogenetic pathways to one or more temporal regulatory signals
(Fig. 7A).
|
). The
fact that the ETT transcript is not localized in the leaf
(Pekker et al., 2005), despite
its clear role in abaxial-adaxial polarity, suggests that ETT is
subject to additional levels of regulation during vegetative development as
well. In addition to regulation at the level of translation, it is likely that
ETT and ARF4 activities are regulated by their interaction with other
auxin-related proteins. The floral phenotype of ett can be partially
phenocopied by the application of polar auxin transport inhibitors, and
ETT is thought to establish boundaries between the stipe, ovary and
style in response to auxin levels
(Nemhauser et al., 2000). In
the absence of auxin, ARF proteins that activate transcription are inhibited
by binding to transcriptional repressors of the AUX/IAA family via domains III
and IV. Auxin promotes the degradation of AUX/IAA proteins, freeing the ARF
protein to promote transcription (Tiwari
et al., 2003). However, both ETT and ARF4 have both been shown to
act as transcriptional repressors, and ETT lacks domains III and IV and should
therefore be incapable of interacting with AUX/IAA proteins
(Tiwari et al., 2003;
Ulmasov et al., 1999). It will
be important to determine whether the activity of ARF4 is regulated by its
interaction with AUX/IAA proteins and if ARF4 has a role in the activity of
ETT.
Is AS2 a target of the zip pathway?
ETT and AS2 appear to play opposing roles in leaf
polarity, with as2 mutants having a weak abaxialized phenotype
(Lin et al., 2003;
Xu et al., 2003) and
ett mutants having a weak adaxialized phenotype. The possibility that
these genes may have closely related functions in leaf polarity is suggested
by our observation that plants with high levels of ectopic ETT
expression strongly resemble as2 mutant plants. This conclusion
receives additional support from the report that rdr6 (which
upregulates ETT) enhances the as2 phenotype
(Li et al., 2005), and the
observation that overexpressing AS2 produces a phenotype very similar
to that of ett;arf4 double mutants
(Lin et al., 2003;
Xu et al., 2003). All of these
observations are consistent with a model in which ETT and
AS2 act to mutually repress the activity of each other. This
repression does not appear to take place at a transcriptional level, however.
Although rdr6 upregulates ETT and enhances the as2
phenotype, it does not alter AS2 or AS1 expression, nor do
as1 and as2 alter the level of the RDR6 transcript
(Li et al., 2005). This would
suggest that AS2 is not a transcriptional target of ETT or
ARF4, and that the interaction of these genes either takes place
post-transcriptionally or at a convergent point in their pathways.
Alternatively, the phenotype of 35S::ETTmAB may reflect a role for
ETT in the repression of BLADE-ON-PETIOLE1 (BOP1),
as mutations in BOP1 resemble as2
(Ha et al., 2003).
The role of threshold genes in defining the juvenile-to-adult transition
Two classes of mutations have been identified in screens for mutants with
defects in the juvenile-to adult-transition: those that affect the time at
which adult traits are first produced without altering the length of the
transition zone or the total number of leaves produced by the shoot, and
mutations such as hasty (Telfer
and Poethig, 1998) and squint
(Berardini et al., 2001), which
accelerate the appearance of adult traits, nearly completely eliminate
transition leaves, and reduce leaf number. zip is an example of the
first class of mutations, and we hypothesized that ZIP acts to
establish a threshold for entry into the adult phase of development
(Hunter et al., 2003). Loss of
ZIP lowers this threshold, allowing the shoot to undergo phase change
prematurely (Fig. 7B). The
evidence that ZIP -and genes (RDR6, SGS3 and DCL4)
that have mutant phenotypes identical to zip - is required for the
production of tasiR-ARF suggests that this threshold is set by
ta-siRNA-mediated repression of ETT and ARF4. Loss of
tasiR-ARF regulation in zip, rdr6 and sgs3 mutants,
or as a result of introducing target site mutations in ETT
(35S::ETTmAB), lowers or bypasses the threshold for entry into the
adult phase. Although we did not determine the effect of repressing
ETT and ARF4 by overexpressing tasiR-ARF,
loss-of-function mutations of these genes produce the expected prolonged
juvenile phenotype, supporting the conclusion that the expression level of
these genes plays a crucial role in this transition. This conclusion receives
further support from the observation that ett-7 has a semi-dominant
(i.e. dose-dependent) effect on abaxial trichome production.
The independence of the threshold and clock mechanisms can be seen even in
plants with the most severe phenotypes, e.g. ett;arf4 double mutants
(Pekker et al., 2005) and
severely affected 35S::ETTmAB plants, which still show the gradual
changes in leaf morphology indicative of an intact developmental clock. This
result is consistent with the phenotype of zip, sgs3, rdr6 and
dcl4, and demonstrates that ETT and ARF4 transcript
levels are important for leaf identity and morphogenesis, but that subsequent
input from a temporal signal is needed to complete the regulation of
heteroblasty and phase change.
The function of the RNAi pathway in plants
In plants, RNAi has been studied primarily for its role in virus resistance
(Baulcombe, 2004). The evidence
that genes required for this process (SGS3, RDR6 and DCL4)
also affect vegetative phase change
(Gasciolli et al., 2005;
Peragine et al., 2004;
Xie et al., 2005;
Yoshikawa et al., 2005) and
salt sensitivity (Borsani et al.,
2005) reveal that this regulatory mechanism is involved in a wider
array of processes than has previously been recognized. However, mutations in
these genes cause relatively subtle phenotypes - particularly in comparison to
the highly pleiotropic phenotype of mutations in genes involved in miRNA
biogenesis (Schauer et al.,
2002). This may be a result of the redundancy in the pathways that
produce siRNAs; however, we think this is unlikely because plants doubly
mutant for DCL3 and DCL4 - the dicers that generate the vast
majority of endogenous siRNAs - resemble sgs3, rdr6 and dcl4
(Gasciolli et al., 2005).
Furthermore, although these plants show severe stochastic effects in advanced
generations - probably as a result of the reactivation of transcriptionally
silenced genes (Gasciolli et al.,
2005) - these epigenetic effects cannot be the basis for rapid,
reproducible developmental changes and physiological responses. This suggests
that RNAi is either employed sparingly to regulate endogenous gene expression,
or that only a few of its endogenous targets have important biological
functions.
Given that other ARF genes are targets of miRNAs, and that the biogenesis
of ta-siRNAs requires miRNA-directed cleavage, it is worth asking why
ETT and ARF4 are not direct targets of a miRNA. Is this
simply an accident of evolution, or is there something special about ta-siRNA
regulation that makes it particularly useful for some types of processes?
There is evidence that miRNAs act in a spatially restricted fashion (Alvarez
et al., 2006a; Parizotto et al.,
2004; Schwab et al.,
2006), whereas siRNAs are capable of moving long distances
(Dunoyer et al., 2005;
Himber et al., 2003;
Schwach et al., 2005).
Although this makes it tempting to think that ta-siRNAs may act as inducers of
phase change (the vegetative equivalent of a `florigen'), it is important to
remember that ta-siRNAs repress adult development, and do not accumulate (or
decline) over the course of development. The analogy may hold, however, in the
power of siRNAs to act non-autonomously to coordinate developmental
transitions that must take place simultaneously in a broad range of tissues.
Mobile siRNAs play an important role in repressing transgene expression
throughout a plant, and it is possible that they play a similar role in the
regulation of endogenous gene expression. This makes them excellent candidates
for establishing a uniform threshold for entry into the adult phase
(Fig. 7B). Finding the
additional targets of these ta-siRNAs, and understanding their interactions
with the developmental clock, are important problems for future research.
Note added in proof
While this paper was under review, evidence that tasiR-ARF is responsible
for the morphological phenotype of sgs3, rdr6 and dcl4 was
published by Adenot et al., Garcia et al. and Fahlgren et al.
(Adenot et al., 2006;
Garcia et al., 2006;
Fahlgren et al., 2006).
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adenot, X., Elmayan, T., Lauressergues, D., Boutet, S., Bouche, N., Gasciolli, V. and Vaucheret, H. (2006). DRB4-dependent TAS3 trans-acting siRNAs control leaf morphology through AGO7. Curr. Biol. 16,927 -932.[CrossRef][Medline]
Allen, E., Xie, Z., Gustafson, A. M. and Carrington, J. C. (2005). microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121,207 -221.[CrossRef][Medline]
Alonso, J. M., Stepanova, A. N., Leisse, T. J., Kim, C. J., Chen, H., Shinn, P., Stevenson, D. K., Zimmerman, J., Barajas, P., Cheuk, R. et al. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301,653 -657.
Alvarez, J. P., Pekker, I., Goldshmidt, A., Blum, E., Amsellem,
Z. and Eshed, Y. (2006). Endogenous and synthetic microRNAs
stimulate simultaneous, efficient, and localized regulation of multiple
targets in diverse Species. Plant Cell
18,1134
-1151.
Baulcombe, D. (2004). RNA silencing in plants. Nature 431,356 -363.[CrossRef][Medline]
Berardini, T. Z., Bollman, K., Sun, H. and Poethig, R. S.
(2001). Regulation of vegetative phase change in Arabidopsis
thaliana by cyclophilin 40. Science
291,2405
-2407.
Bollman, K. M., Aukerman, M. J., Park, M. Y., Hunter, C.,
Berardini, T. Z. and Poethig, R. S. (2003). HASTY, the
Arabidopsis ortholog of Exportin 5/MSN5, regulates phase change and
morphogenesis. Development
130,1493
-1504.
Borsani, O., Zhu, J., Verslues, P. E., Sunkar, R. and Zhu, J. K. (2005). Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123,1279 -1291.
Bowman, J. L. (2000). Axial patterning in leaves and other lateral organs. Curr. Opin. Genet. Dev. 10,399 -404.[CrossRef][Medline]
Clarke, J. H., Tack, D., Findlay, K., Van Montagu, M. and Van Lijsebettens, M. (1999). The SERRATE locus controls the formation of the early juvenile leaves and phase length in Arabidopsis. Plant J. 20,493 -501.[CrossRef][Medline]
Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,735 -743.[CrossRef][Medline]
Dunoyer, P., Himber, C. and Voinnet, O. (2005). DICER-LIKE 4 is required for RNA interference and produces the 21-nucleotide small interfering RNA component of the plant cell-to-cell silencing signal. Nat. Genet. 37,1356 -1360.[CrossRef][Medline]
Eshed, Y., Izhaki, A., Baum, S. F., Floyd, S. K. and Bowman, J.
L. (2004). Asymmetric leaf development and blade expansion in
Arabidopsis are mediated by KANADI and YABBY activities.
Development 131,2997
-3006.
Fahlgren, N., Montgomery, T. A., Howell, M. D., Allen, E., Dvorak, S. K., Alexander, A. L. and Carrington, J. C. (2006). Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Curr. Biol. 16,939 -944.[CrossRef][Medline]
Garcia, D., Collier, S. A., Byrne, M. E. and Martienssen, R. A. (2006). Specification of leaf polarity in Arabidopsis via the trans-acting siRNA pathway. Curr. Biol. 16,933 -938.[CrossRef][Medline]
Gasciolli, V., Mallory, A. C., Bartel, D. P. and Vaucheret, H. (2005). Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr. Biol. 15,1494 -1500.[CrossRef][Medline]
Ha, C. M., Kim, G. T., Kim, B. C., Jun, J. H., Soh, M. S., Ueno,
Y., Machida, Y., Tsukaya, H. and Nam, H. G. (2003). The
BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the
modulation of meristematic activity in Arabidopsis.
Development 130,161
-172.
Himber, C., Dunoyer, P., Moissiard, G., Ritzenthaler, C. and Voinnet, O. (2003). Transitivity-dependent and -independent cell-to-cell movement of RNA silencing. EMBO J. 22,4523 -4533.[CrossRef][Medline]
Hunter, C., Sun, H. and Poethig, R. S. (2003). The Arabidopsis heterochronic gene ZIPPY is an ARGONAUTE family member. Curr. Biol. 13,1734 -1739.[CrossRef][Medline]
Iwakawa, H., Ueno, Y., Semiarti, E., Onouchi, H., Kojima, S.,
Tsukaya, H., Hasebe, M., Soma, T., Ikezaki, M., Machida, C. et al.
(2002). The ASYMMETRIC LEAVES2 gene of Arabidopsis
thaliana, required for formation of a symmetric flat leaf lamina, encodes
a member of a novel family of proteins characterized by cysteine repeats and a
leucine zipper. Plant Cell Physiol.
43,467
-478.
Kerstetter, R. A. and Poethig, R. S. (1998). The specification of leaf identity during shoot development. Annu. Rev. Cell Dev. Biol. 14,373 -398.[CrossRef][Medline]
Kerstetter, R. A., Bollman, K., Taylor, R. A., Bomblies, K. and Poethig, R. S. (2001). KANADI regulates organ polarity in Arabidopsis. Nature 411,706 -709.
Li, H., Xu, L., Wang, H., Yuan, Z., Cao, X., Yang, Z., Zhang,
D., Xu, Y. and Huang, H. (2005). The Putative RNA-dependent
RNA polymerase RDR6 acts synergistically with ASYMMETRIC
LEAVES1 and 2 to repress BREVIPEDICELLUS and
microRNA165/166 in Arabidopsis leaf development. Plant
Cell 17,2157
-2171.
Lin, W. C., Shuai, B. and Springer, P. S.
(2003). The Arabidopsis LATERAL ORGAN BOUNDARIES-domain
gene ASYMMETRIC LEAVES2 functions in the repression of KNOX
gene expression and in adaxial-abaxial patterning. Plant
Cell 15,2241
-2252.
Liu, Z., Franks, R. G. and Klink, V. P. (2000).
Regulation of gynoecium marginal tissue formation by LEUNIG and
AINTEGUMENTA. Plant Cell
12,1879
-1892.
McConnell, J. R. and Barton, M. K. (1998). Leaf polarity and meristem formation in Arabidopsis. Development 125,2935 -2942.[Abstract]
Nemhauser, J. L., Feldman, L. J. and Zambryski, P. C. (2000). Auxin and ETTIN in Arabidopsis gynoecium morphogenesis. Development 127,3877 -3888.[Abstract]
Nishimura, T., Wada, T. and Okada, K. (2004). A key factor of translation reinitiation, ribosomal protein L24, is involved in gynoecium development in Arabidopsis. Biochem. Soc. Trans. 32,611 -613.[Medline]
Ori, N., Eshed, Y., Chuck, G., Bowman, J. L. and Hake, S. (2000). Mechanisms that control KNOX gene expression in the Arabidopsis shoot. Development 127,5523 -5532.[Abstract]
Parizotto, E. A., Dunoyer, P., Rahm, N., Himber, C. and Voinnet,
O. (2004). In vivo investigation of the
transcription, processing, endonucleolytic activity, and functional relevance
of the spatial distribution of a plant miRNA. Genes
Dev. 18,2237
-2242.
Park, M. Y., Wu, G., Gonzalez-Sulser, A., Vaucheret, H. and
Poethig, R. S. (2005). Nuclear processing and export of
microRNAs in Arabidopsis. Proc. Natl. Acad. Sci.
USA 102,3691
-3696.
Pekker, I., Alvarez, J. P. and Eshed, Y.
(2005). Auxin response factors mediate Arabidopsis organ
asymmetry via modulation of KANADI activity. Plant
Cell 17,2899
-2910.
Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L. and
Poethig, R. S. (2004). SGS3 and
SGS2/SDE1/RDR6 are required for juvenile development and the
production of trans-acting siRNAs in Arabidopsis. Genes
Dev. 18,2368
-2379.
Prigge, M. J. and Wagner, D. R. (2001). The
Arabidopsis SERRATE gene encodes a zinc-finger protein required for
normal shoot development. Plant Cell
13,1263
-1279.
Remington, D. L., Vision, T. J., Guilfoyle, T. J. and Reed, J.
W. (2004). Contrasting modes of diversification in the
Aux/IAA and ARF gene families. Plant
Physiol. 135,1738
-1752.
Schauer, S. E., Jacobsen, S. E., Meinke, D. W. and Ray, A. (2002). DICER-LIKE1: blind men and elephants in Arabidopsis development. Trends Plant Sci. 7, 487-491.[CrossRef][Medline]
Schwab, R., Ossowski, S., Riester, M., Warthmann, N. and Weigel,
D. (2006). Highly specific gene silencing by artificial
microRNAs in Arabidopsis. Plant Cell
18,1121
-1133.
Schwach, F., Vaistij, F. E., Jones, L. and Baulcombe, D. C.
(2005). An RNA-dependent RNA polymerase prevents meristem
invasion by potato virus X and is required for the activity but not the
production of a systemic silencing signal. Plant
Physiol. 138,1842
-1852.
Semiarti, E., Ueno, Y., Tsukaya, H., Iwakawa, H., Machida, C. and Machida, Y. (2001). The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates formation of a symmetric lamina, establishment of venation and repression of meristem-related homeobox genes in leaves. Development 128,1771 -1783.[Abstract]
Sessions, A., Nemhauser, J. L., McColl, A., Roe, J. L., Feldmann, K. A. and Zambryski, P. C. (1997). ETTIN patterns the Arabidopsis floral meristem and reproductive organs. Development 124,4481 -4491.[Abstract]
Sessions, R. A. and Zambryski, P. C. (1995). Arabidopsis gynoecium structure in the wild and in ettin mutants. Development 121,1519 -1532.[Abstract]
Telfer, A. and Poethig, R. S. (1998). HASTY: a gene that regulates the timing of shoot maturation in Arabidopsis thaliana. Development 125,1889 -1898.[Abstract]
Telfer, A., Bollman, K. M. and Poethig, R. S. (1997). Phase change and the regulation of trichome distribution in Arabidopsis thaliana. Development 124,645 -654.[Abstract]
Tiwari, S. B., Hagen, G. and Guilfoyle, T.
(2003). The roles of auxin response factor domains in
auxin-responsive transcription. Plant Cell
15,533
-543.
Tsukaya, H. and Uchimiya, H. (1997). Genetic analyses of the formation of the serrated margin of leaf blades in Arabidopsis: combination of a mutational analysis of leaf morphogenesis with the characterization of a specific marker gene expressed in hydathodes and stipules. Mol. Gen. Genet. 256,231 -238.[CrossRef][Medline]
Ulmasov, T., Hagen, G. and Guilfoyle, T. J. (1999). Dimerization and DNA binding of auxin response factors. Plant J. 19,309 -319.[CrossRef][Medline]
Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli, V., Mallory, A. C., Hilbert, J. L., Bartel, D. P. and Crete, P. (2004). Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol. Cell 16, 69-79.[CrossRef][Medline]
Wang, W. and Malcolm, B. A. (1999). Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange site-directed mutagenesis. Biotechniques 26,680 -682.[Medline]
Williams, L., Carles, C. C., Osmont, K. S. and Fletcher, J.
C. (2005). A database analysis method identifies an
endogenous trans-acting short-interfering RNA that targets the Arabidopsis
ARF2, ARF3, and ARF4 genes. Proc. Natl. Acad. Sci.
USA 102,9703
-9708.
Xie, Z., Allen, E., Wilken, A. and Carrington, J. C.
(2005). DICER-LIKE 4 functions in trans-acting small
interfering RNA biogenesis and vegetative phase change in Arabidopsis
thaliana. Proc. Natl. Acad. Sci. USA
102,12984
-12989.
Xu, L., Xu, Y., Dong, A., Sun, Y., Pi, L. and Huang, H.
(2003). Novel as1 and as2 defects in leaf
adaxial-abaxial polarity reveal the requirement for ASYMMETRIC
LEAVES1 and 2 and ERECTA functions in specifying leaf
adaxial identity. Development
130,4097
-4107.
Yoshikawa, M., Peragine, A., Park, M. Y. and Poethig, R. S.
(2005). A pathway for the biogenesis of trans-acting siRNAs in
Arabidopsis. Genes Dev.
19,2164
-2175.
This article has been cited by other articles:
|
|
 |
|
  J.-W. Wang, R. Schwab, B. Czech, E. Mica, and D. Weigel Dual Effects of miR156-Targeted SPL Genes and CYP78A5/KLUH on Plastochron Length and Organ Size in Arabidopsis thaliana PLANT CELL, May 1, 2008; 20(5): 1231 - 1243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. A. Farazi, S. A. Juranek, and T. Tuschl The growing catalog of small RNAs and their association with distinct Argonaute/Piwi family members Development, April 1, 2008; 135(7): 1201 - 1214. [Abstract] [Full Text] [PDF]
|
|
|
|
18, ±s.e.m.). The
number of cauline leaves is indicated by the white bar. The numbers above each
bar represent the percentage of flowers with a split septum, based on an
analysis of the first 10 flowers of five plants of each genotype.
ett-15 and arf4 partially suppress the septum splitting
observed in zip. Other arf4 mutations also delay abaxial
trichome production in a zip-2 background. (D) The number of
leaves without abaxial trichomes among plants segregating ett-7 and
zip-2. ett-7 has a semi-dominant effect on abaxial trichome
production.
