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First published online 16 August 2006
doi: 10.1242/dev.02521
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Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.
* Author for correspondence (e-mail: spoethig{at}sas.upenn.edu)
Accepted 6 July 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: miRNA, Heterochrony, Phase change
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
let-7 (Reinhart et al.,
2000), are expressed late in larval development and promote adult
development by repressing the expression of genes required for the expression
of juvenile traits (Ambros,
2000; Pasquinelli and Ruvkun,
2002). Recent studies indicate that miRNAs and other endogenous
small RNAs also regulate developmental transitions in plants. In plants, the
shoot apex progresses through juvenile and adult phases of vegetative
development before undergoing a transition to reproductive development
(Baurle and Dean, 2006;
Poethig, 1990). Several miRNAs
have been shown to affect flowering time when over-expressed in
Arabidopsis (Achard et al.,
2004; Chen, 2004;
Schwab et al., 2005), and the
temporal expression pattern of one of these, miR172, suggests that it
may promote both vegetative phase change and floral induction
(Aukerman and Sakai, 2003).
miR172 exhibits a similar temporal expression pattern in maize, where
it targets Glossy15(Gl15), a gene required for the
expression of juvenile epidermal traits
(Evans et al., 1994;
Lauter et al., 2005;
Moose and Sisco, 1994).
Additional evidence for the involvement of small RNAs in vegetative phase
change is provided by the observation that mutations in the genes required for
the biogenesis of miRNAs (Clarke et al.,
1999; Grigg et al.,
2005; Park et al.,
2005; Ray et al.,
1996; Telfer and Poethig,
1998), or for post-transcriptional gene silencing (PTGS, also
known as RNAi) (Morel et al.,
2002; Peragine et al.,
2004), have a precocious vegetative phenotype. However, there is
still no conclusive evidence that temporal variation in miRNAs or endogenous
siRNAs contribute to vegetative phase change or floral induction.
To identify genes directly involved in vegetative phase change, we used
microarray analysis to search for genes whose mRNA is elevated in zip-1,
rdr6-11 and sgs3-11
(Peragine et al., 2004). These
mutations have identical phenotypes, and we reasoned that the genes
responsible for this phenotype would be expressed in a similar way in all
three mutants. Three such genes were identified: ETTIN/ARF3, ARF4 and
SPL3. This paper concerns the regulation and function of
SPL3 and the closely related genes, SPL4 and SPL5.
These genes are members of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE
(SPL) gene family, members of which share a DNA-binding domain (the
SPB domain) first identified in proteins that bind to sequence motif present
in the promoter of the SQUAMOSA gene in Antirrhinum majus
(Cardon et al., 1999;
Cardon et al., 1997;
Klein et al., 1996). Members
of this plant-specific structurally diverse family are found in non-vascular
(Arazi et al., 2005) and
vascular plants, and are required for such processes as ligule
(Moreno et al., 1997) and
glume (Wang et al., 2005b)
development in maize, and leaf (Stone et
al., 2005) and flower (Klein
et al., 1996; Unte et al.,
2003) development in Arabidopsis and
Antirrhinum. Cardon and colleagues
(Cardon et al., 1997)
originally proposed a role for SPL3 in floral induction based on the
observations that SPL3 mRNA increases during the transition to
flowering in plants grown in long or short days, and that overexpression of
SPL3 produces early flowering. Consistent with this hypothesis,
SPL3 mRNA rapidly increases in plants exposed to a photoinductive
stimulus (Schmid et al.,
2003). SPL4 and SPL5 are closely related to
SPL3, and have a similar temporal expression pattern
(Cardon et al., 1999). The
function of these genes is unknown, however, as plants overexpressing
SPL4 and SPL5 have no apparent phenotype
(Cardon et al., 1999).
SPL genes were among the first miRNA-regulated genes to be identified
in Arabidopsis (Rhoades et al.,
2002). Ten out of the 16 SPL genes in
Arabidopsis - including SPL3, SPL4 and SPL5 - have
target sites for miR156. All of these genes are specifically
downregulated in plants that constitutively overexpress miR156b
(Schwab et al., 2005), but
only two of these transcripts have been shown to be cleaved by this miRNA
(Chen et al., 2004;
Kasschau et al., 2003).
Here, we show that SPL3, SPL4 and SPL5 have overlapping functions in the regulation of vegetative phase change and floral induction in Arabidopsis and demonstrate that miR156 is responsible for the temporal change in SPL3 expression during vegetative development. We also show that ZIP and RDR6 either directly or indirectly repress the expression of SPL3 during vegetative development, but do not contribute significantly to the temporal expression pattern of this gene.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RNA blots
Total RNA was isolated using Trizol (Invitrogen) from shoot apices of
plants grown under short-day conditions (10 hours light:14 hours dark) at
22°C. Whole seedlings (including cotyledons) were used for 8- to
14-day-old seedlings, the cotyledons and leaves 1 and 2 were removed from
plants harvested between 18-21 days after planting (dap), and the cotyledons
and leaves 1-4 were removed from plants harvested between 23-28 dap. The
number of leaves present on the shoot apex was determined by comparison with a
growth curve generated by dissecting shoot apices grown under the same SD
conditions. To measure SPL3 mRNA, 20 µg of total RNA was run on
1.2% agarose gels, transferred to Hybond N+ nylon membranes
(Amersham Pharmacia), and crosslinked under UV light. Hybridizations were
performed at 68°C in PerfectHyb plus buffer (Sigma). The SPL3
probe was PCR amplified using the primers 5 ' ACGAGAGAAGGCGGAAAAGCACAA 3
' and 5 ' CGGGATCCCTAAGTCTCAATGCATTTAT 3 ' from a
SPL3 cDNA clone and was labeled with 32P-dCTP using
Prime-II Random Primer Labeling Kit (Stratagene). Blots were hybridized for 8
hours at 68°C, washed once in 2 'SSC and 0.1% SDS solution for 5
minutes at room temperature, twice in 0.5xSSC and 0.1% SDS for 20
minutes at 68°C, and once in 0.1xSSC and 0.1% SDS for 20 minutes at
68°C, and were scanned with a Storm 860 (Molecular Dynamics). To measure
miR156, 50 µg total RNA was separated on 8 M urea/15% denaturing
polyacrylamide gels and electrically transferred to Hybond N+ nylon
membranes. Blots were hybridized with a miR156-complementary
oligonucleotide labeled with 32P-ATP (New England Biolabs) at
40°C in ULTRAhyb-oligo hybridization buffer (Ambion, Austin, TX). Blots
were washed twice at 40°C in 2xSSC and 0.5% SDS for 30 minutes
before scanning.
5' and 3' RACE
Total RNA was isolated from leaf or floral tissue as described above. 5
'RACE and 3 'RACE were carried out using the FirstChoice RLM-RACE
Kit (Ambion, Austin, TX). For standard 5 ' RACE, 5 µg of total RNA
was ligated to the RNA adapter after treatment with calf intestinal
phosphatase and tobacco acid pyrophosphatase. For 5 'RLM-RACE, RNA was
ligated to the RNA oligo adapter without pre-treatment. cDNA was synthesized
using the 3 'RACE oligo d(T) adapter supplied by the manufacturer.
Nested PCR was carried out using the nested adapter primer, and primers
specific for SPL3, SPL4 and SPL5. RACE products were gel
purified and cloned into pGEM T easy vector (Promega) for sequencing.
Transgenic plants
SPL3 (ORF plus 3 'UTR) and a sequence lacking the 3 '
UTR (SPL3
) were PCR-amplified with pfu TURBO (Stratagene) using
cDNA as template. SPL3m was generated by introducing 7 mutations into
the predicted miR156 binding site using recombinant PCR.
GUS-Plus was amplified from the pCAMBIA1305.1 vector and
fused in frame to the 5 ' end of these genes to generate GUS-tagged
proteins. All of these constructs were cloned downstream of the CaMV 35S
promoter in pEZR-CL. Constructs overexpressing putative miR156
precursors were generated by cloning 0.3-0.8 kb of intergenic genomic sequence
containing the precursor in this vector. Plants were transformed using the
floral dip method (Bechtold et al.,
1993).
GUS activity assays
Visual (i.e. non-quantitative) analyses of GUS expression were conducted by
staining plants according to the method of Senecoff et al.
(Senecoff et al., 1996). The
effect of various mutations on the expression of 35S::GUS-SPL3
constructs was determined as follows. T1 seeds from plants treated with
Agrobacterium were grown on 1/2 MS medium supplemented with 50 mg/l
kanamycin to identify transgenic plants. Resistant plants were transferred to
soil in 96-well flats and grown under continuous light at 22°C. Leaves 3
and 4 were harvested when plants had about five fully expanded leaves, and
stored at -80°C for subsequent analysis. Upon flowering, adult rosette or
cauline leaves were visually assayed for GUS activity to identify plants
containing an active transgene. Quantitative GUS assays were carried out on
stored leaves from these plants. Leaves were ground to a fine powder in liquid
nitrogen in a microfuge tube, and suspended in a buffer containing 50 mM
sodium phosphate (pH 7.0), 10 mM ß-mercaptoethanol, 10 mM EDTA, 0.1%
sodium lauryl sarcosine and 0.1% Triton X-100. After centrifugation, 10 µl
of the supernatant was added to preheated GUS assay buffer (2 mM
4-Methylumbelliferyl ß-D-Glucuronide in GUS extraction buffer) at
37°C and incubated for 30 minutes. The reaction was stopped by addition of
900 µl of 0.2 M Na2CO3, and fluorescence was measured
with LS-50B luminescence spectrometer (PerkinElmer) with emission and
excitation filters set at 455 nm and 365 nm, respectively. Total protein was
determined using the Bradford Assay (BioRad).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), indicate that SPL3 (At2g33810) has a complex
transcription pattern. To determine the structure of this locus, we performed
5 ' and 3 ' RACE in both sense and antisense orientations. These
experiments revealed that the SPL3 sense transcript (ST) has a single
5 ' end and multiple poly(A) sites in its 3 ' UTR
(Fig. 1A). The miR156
target site is located in the 3 ' UTR, 50 nucleotides from the stop
codon. We also identified several 
950 nucleotide antisense transcripts
(AST), which completely encompass the ST and have an intron located in nearly
the same position as the intron in the ST
(Fig. 1A). Four transcription
start sites and 5 poly(A) sites were identified in these AS transcripts. SPL4 (At1g53160) and SPL5 (At3g15270) encode, respectively, 174 amino acid and 181 amino acid proteins that are 65% identical (76% similar) to the 131 amino acid protein encoded by SPL3. The structure of these genes is also very similar to SPL3 (Fig. 1A). These genes also possess a miR156 target site located within their 3 ' UTR and 5 'RLM-RACE demonstrated that SPL3/4/5 are all cleaved in the middle of this target site (Fig. 1B).
The regulation and function ofSPL3
In order to study the role of miR156 in the regulation of
SPL3, we produced transgenic plants expressing
miR156-sensitive and miR156-resistant forms of this gene
under the regulation of the constitutive CaMV 35S promoter. We chose to use
the 35S promoter rather than the endogenous SPL3 promoter in order to
eliminate effects on SPL3 expression due to transcriptional
cross-regulation. The constructs generated for this experiment express an
SPL3 transcript with a normal 3 ' UTR
(35S::SPL3), a transcript with seven mutations in the
miR156 target site (35S::SPL3m), and a transcript lacking
the 3 ' UTR (35S::SPL3)
(Fig. 2A). Constructs in which
the GUS Plus sequence was fused in frame to the 5 ' end of
these sequences were also generated in order to evaluate the expression of the
SPL3 protein. Plants expressing GUS-tagged versions of SPL3 resembled plants
expressing untagged proteins, although their phenotype was slightly weaker.
Homozygous stocks containing a single insert were established from transgenic
lines, and these stocks were subjected to Northern analysis to ensure that the
transgene was overexpressed.
GUS expression was absent in the progeny of a cross between transgenic plants carrying 35S::GUS-SPL3 and plants carrying 35S::miR156a (Fig. 2B). Progeny from a cross between 35S::GUS-SPL3m and 35S::miR156a had high levels of GUS activity, and there was no significant difference between the amount of GUS activity in these plants (35,821±2815 pmol 4-MU/minute µg protein) and the progeny of a control cross between 35S::GUS-SPL3m and plants carrying an empty vector (33,599±2109 pmol 4-MU/minute µg protein). These results demonstrate that the SPL3 mRNA is a direct target of miR156 and show that the mutations we introduced in the miR156 target site completely block the activity of this miRNA.
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), flowered early, had a significantly reduced number
of juvenile, adult and cauline leaves (Fig.
2C-E). The first two leaves of transgenic plants had a shorter
petiole and a more acute leaf base than wild-type leaves
(Fig. 2E); these features are
typical of adult leaves. These results indicate that SPL3 promotes
both vegetative and reproductive phase change and is normally repressed by
miR156.
Plants constitutively expressing the a, b, c, d, e and f isoforms of
miR156 were generated by transforming wild-type plants with
constructs containing 0.3-0.8 kb of intergenic genomic sequence under the
regulation of the 35S promoter (Fig.
3A). All of these transgenic lines had a similar phenotype
(Fig. 3B), and resembled the
35S::156b transgenics described by Schwab et al.
(Schwab et al., 2005). Thus,
all these loci are capable of producing miR156, although whether they
are all transcribed in vivo is unknown. A detailed phenotypic analysis was
performed on plants overexpressing miR156a. In continuous light,
transgenic plants had a significantly larger number of leaves without abaxial
trichomes (n=24; P<0.01 for all traits)
(Fig. 3C). Furthermore, most of
the leaves produced by these plants had a round lamina, long petiole and were
relatively small in size - all characteristics of juvenile leaves. Transgenic
plants also flowered significantly later than vector controls with many
additional leaves (Fig. 3C). To
determine if downregulation of SPL3 is responsible for this
phenotype, we examined the phenotype of F2 progeny from plants heterozygous
for 35S::miR156a and the miR156-insensitive transgene,
35S::SPL3m. Plants carrying both of these transgenes produced
significantly fewer leaves without abaxial trichomes than plants expressing
35S::miR156a (Fig.
3D). Plants with both transgenes resembled 35S::SPL3m in
producing leaves with short petioles, but the shape of the lamina in double
transgenics was more similar to that of 35S::miR156a than
35S::SPL3m (Fig. 3E).
We conclude that downregulation of SPL3 accounts for the effect of
35S::miR156a on abaxial trichome production, petiole length and
flowering time, but that the effect of this miRNA on lamina shape is mediated
by a different gene or genes. In this respect, SPL3 resembles
Gl15 in maize, which regulates phase-specific patterns of leaf
epidermal differentiation and flowering time, but does not affect leaf shape
(Evans et al., 1994;
Lauter et al., 2005;
Moose and Sisco, 1994).
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, 35S::SPL5 and
35S::SPL5 constructs analogous in structure to the similarly
named SPL3 constructs (Fig.
2A). 35S::SPL4 and 35S::SPL5 reduced the number
of adult leaves (n=50; P<0.01), but had no significant
effect on the number of leaves without abaxial trichomes or on flowering time
(Fig. 4B-D). By contrast,
35S::SPL4 and 35S::SPL5 significantly
accelerated abaxial trichome production and floral induction (n=48;
P<0.01). These results indicate that SPL4 and
SPL5 have the same function as SPL3, and that they are
normally repressed by miR156.
Temporal regulation of SPL3 by miR156
We studied the level of SPL3 and miR156 RNA in plants
grown in short days (SD; 10 hours light: 14 hours dark) in order to
distinguish changes in the expression of these genes associated with
vegetative phase change from those associated with floral induction
(Schmid et al., 2003). Under
our SD conditions, the first leaf with abaxial trichomes (leaf 8 or 9) is
initiated 15-16 dap and flowering occurs approximately 60 dap, after the shoot
has produced about 45 leaves (Telfer et
al., 1997; Bollman et al.,
2003). Thus, changes in gene expression observed during the first
30 days of growth in SD are associated with vegetative phase change, not
floral induction.
The expression of SPL3 and miR156 was determined by
northern analysis of RNA from shoot apices harvested from juvenile (12 dap; 4
leaves), transition (19 dap; 10 leaves) and adult (26 dap; 16 leaves) plants
growing in SD. SPL3 mRNA was nearly undetectable at 12 dap, and was
present in increasing amounts in the 19 and 26 dap samples. A probe
complementary to miR156 detected both 20 and 21 nucleotide miRNAs;
the 20 nucleotide miRNA is derived from one or more of the miR156a-f
loci, whereas the 21 nucleotide miRNA is probably derived from
miR157a-d, which encode a miRNA that is very similar to
miR156 (Reinhart et al.,
2002; Xie et al.,
2005). The 21 nucleotide miRNA was expressed uniformly, whereas
the 20 nucleotide miRNA was expressed at a high level at 12 dap and at
uniformly low level thereafter (Fig.
5A). Thus, SPL3 and miR156 are expressed in a
complementary fashion early in vegetative development.
To determine if miR156 contributes to the temporal expression pattern of SPL3, we examined the expression of miR156-sensitive (35S::GUS-SPL3) and miR156-insensitive (35S::GUS-SPL3m) reporter genes under the regulation of the constitutive CaMV 35S promoter. All of the leaves of plants homozygous for 35S::SPL3m had high levels of GUS activity and there was relatively little variation in activity in plants harvested at different times (Fig. 5B,C). By contrast, plants transformed with 35S::GUS-SPL3 expressed GUS only in apical leaves. Lines with strong activity displayed GUS expression earlier in shoot development than did lines with lower activity, and often became completely silenced in subsequent generations. The line illustrated in Fig. 5 displays the pattern of GUS activity characteristic of the most stable lines we obtained. GUS expression in this line was observed starting with leaf 7 or 8 (Fig. 5B), and increased gradually during shoot development (Fig. 5C). Consistent with this expression pattern, miR156 was more abundant at 8 dap than at the later time points (Fig. 5D). These results demonstrate that temporal variation in the expression 35S::GUS-SPL3 is regulated post-transcriptionally by miR156.
ZIP and RDR6 regulate the amplitude of SPL3 expression, but not its temporal expression
SPL3 initially came to our attention because it is one of a
relatively small number of genes that is over-expressed in zip, rdr6
and sgs3 mutants. Two other genes that have this expression pattern,
ETTIN/ARF3 and ARF4, are targets of trans-acting
siRNAs (ta-siRNAs) (Allen et al.,
2005; Williams et al.,
2005). ta-siRNAs are produced by a pathway involving SGS3,
RDR6 and DCL4 from transcripts that are cleaved by a miRNA
(Dunoyer et al., 2005;
Gasciolli et al., 2005;
Yoshikawa et al., 2005). The
observation that SPL3 mRNA is elevated in these three mutants
therefore suggests that SPL3 is either directly or indirectly
regulated by this silencing pathway.
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) and has a
precocious vegetative phenotype (Telfer
and Poethig, 1998). miR156 was present at a reduced but
readily detectable level in hst-6 at 14 dap, and was nearly
undetectable in this mutant at 21 dap. Consistent with its effect on
miR156, SPL3 was only slightly elevated in hst-6 at 14 dap,
but was two- to threefold higher than normal at later times in development
(Fig. 6A,B). This result
supports the conclusion that temporal variation in miR156 is
responsible for changes in the abundance of SPL3 mRNA during
vegetative development,
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; Qi et al.,
2005). To determine if ZIP is required for
miR156-directed repression of SPL3, we transformed wild-type
and zip-1 plants with 35S::GUS-SPL3 and
35S::GUS-SPL3m. Quantitative GUS assays were performed on leaf 3 and
4 from 30 or more primary transformants in which these transgenes were
expressed in the inflorescence (see Materials and methods); this was carried
out to ensure that the transgenes were capable of being expressed.
35S::GUS-SPL3 was expressed at significantly lower levels
than 35S::GUS-SPL3m in the juvenile leaves of both wild-type and
zip-1 plants, and there was no significant difference in amount of
GUS activity in these genotypes (Fig.
6C). This result, and observation that zip-1 does not
affect the abundance of miR156
(Fig. 6A), indicate that the
increase in SPL3 mRNA in this mutant is not mediated by a change in
the expression of miR156 or by a defect in miR156-directed
repression of SPL3.
The effect of sgs3-11 and rdr6-11 on the level of
SPL3 mRNA suggests that this gene may be a direct target of RNAi. To
test this hypothesis, we searched for siRNAs derived from SPL3 by
hybridizing blots of low molecular weight from wild-type and mutant plants
with a probe that spans the SPL3 ORF and 3 'UTR, as well as
with oligonucleotide probes to sequences 5 ' and 3 ' of the
miR156 target site. No siRNAs were detected with any of these probes.
Furthermore, a search of the MPSS database
(mpss.udel.edu/at/)
revealed that no small RNAs have been cloned from SPL3. To determine
if SGS3 and RDR6 play a general role in the expression of
genes targeted by miR156, we examined the level of SPL4,
SPL9 and SPL15 mRNA in sgs3-11 and rdr6-11
mutants; SPL9 and SPL15 have a miR156 target site
in their ORF and are downregulated in plants constitutively expressing
miR156b (Schwab et al.,
2005). SPL4 mRNA was elevated in both mutants, but we
observed little or no change in SPL9 or SPL15
(Fig. 6D). Semi-quantitative
RT-PCR analysis of the expression of the SPL3 antisense transcript in
zip, rdr6-11 and sgs3-11 demonstrated that these mutations
also have no effect on this transcript
(Fig. 6E). These results
suggest that the increase in SPL3 mRNA in sgs3 and
rdr6 is an indirect effect, and does not reflect the involvement of
SGS3 and RDR6 in miR156-initiated transitive
silencing of this gene.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kidner and
Martienssen, 2005). Here, we show that miR156 regulates
the expression of three genes that promote vegetative phase change in
Arabidopsis - SPL3, SPL4 and SPL5 - and that
temporal variation in the level of this miRNA contributes significantly to the
temporal change in SPL3 activity during vegetative development.
The function of SPL3, SPL4 and SPL5
SPL3 was originally identified because of its homology to SBP
genes in Antirrhinum and was reported to cause early flowering when
overexpressed (Cardon et al.,
1997). We confirmed this observation, but found that
overexpression of SPL3 only has a major effect on flowering time if
the miR156 target site in the 3 ' UTR is mutated or deleted.
The results obtained by Cardon et al.
(Cardon et al., 1997) are
probably attributable to the fact that the construct used in that study had a
truncated miR156 target site. Overexpression of SPL3 also
accelerated two traits characteristic of adult leaves - abaxial trichome
production and short petioles - but had relatively little effect on other leaf
traits. This result demonstrates that SPL3 regulates a subset of
traits associated with the adult phase, but is not entirely responsible for
vegetative phase change. Plants overexpressing miR156-sensitive and
miR156-insensitive versions of the SPL4 and SPL5
mRNAs have essentially the same phenotype as plants expressing the
corresponding SPL3 constructs, indicating that these genes have
similar, if not the same functions as SPL3. Whether SPL4 and
SPL5 have unique functions is still unknown because loss-of-function
mutations in these genes have not yet been identified. Other genes that affect
both vegetative phase change and floral induction include genes involved in
gibberellin biosynthesis/signaling, as well as genes involved in the
autonomous floral induction pathway (Scott
et al., 1999; Silverstone et
al., 1997; Telfer et al.,
1997). It will be important to determine if SPL3/4/5
reside in one or the other of these pathways. The observation that
35S::SPL3m is incapable of rescuing the effect of
35S::miR156a on lamina shape suggests that other targets of this
miRNA also play a role in vegetative phase change.
Temporal regulation of SPL3 by miR156
The level of SPL3 mRNA increases early in vegetative development
(this study), and during the transition to flowering
(Cardon et al., 1999;
Cardon et al., 1997;
Schmid et al., 2003). Because
of our interest in vegetative phase change, we focused on the regulation of
SPL3 early in vegetative development, when the expression of this
gene and miR156 change in a complementary fashion. We found that the
constitutively transcribed miR156-sensitive reporter gene
35S::GUS-SPL3 displayed GUS activity only in apical leaves, whereas
the miR156-insensitive 35S::GUS-SPL3m transgene displayed
constitutive GUS activity. This result is consistent with the capacity of
these transgenes to affect plant morphology, and demonstrates that
SPL3 is repressed post-transcriptionally by miR156 early in
shoot development. Additional support for this conclusion was provided by the
observation that the temporal expression pattern of the endogenous
SPL3 transcript is disrupted by hst-6, a mutation that
strongly reduces the accumulation of miR156. Interestingly, the
amount of miR156 present in young plants appears far in excess of the
amount actually necessary to block SPL3 activity. This conclusion is
supported by the observation that 35S::SPL3 was unable to overcome
the effect of endogenous miR156, and the observation that
hst-6 had relatively little effect on SPL3 mRNA levels early
in shoot development, when hst-6 still has a significant amount of
miR156.
It should be emphasized that our results do not eliminate the possibility
that transcriptional regulation also plays a role in the temporal expression
of SPL3. SPL3/4/5 are transcription factors and may crossregulate
each other's expression, as has been observed in the case of the AP2
family of miR172-regulated transcription factors
(Chen, 2004;
Schmid et al., 2003). Indeed,
the dramatic increase in the expression of SPL3/4/5 during the floral
transition is probably regulated at a transcriptional level rather than by
miR156 because miR156 does not decrease markedly during this
transition (G.W. and R.S.P., unpublished).
How is the expression of miR156 regulated early in vegetative
development? miR172 is expressed in a pattern complementary to that
of miR156, increasing with time during shoot development
(Aukerman and Sakai, 2003).
Furthermore, the phenotype of plants overexpressing miR172 is nearly
the exact opposite of plants overexpressing miR156, and strongly
resembles the phenotype of plants over-expressing SPL3m
(Aukerman and Sakai, 2003;
Chen, 2004). The targets of
miR172 include the AP2-like transcription factors, TOE1,
TOE2, SMZ and SNZ in Arabidopsis
(Aukerman and Sakai, 2003;
Chen, 2004;
Schmid et al., 2003), and
Gl15 in maize (Lauter et al.,
2005). These genes act as floral repressors and, in the case of
Gl15, also promote the expression of juvenile vegetative traits. The
complementary relationship between the expression pattern and functions of
miR172 and miR156 is striking and raises the possibility
that these two miRNAs and their targets act in sequence in the same regulatory
pathway.
The function of RNAi in the regulation of SPL3
Transcripts that contain a miRNA target site are frequently silenced in an
RDR6-dependent fashion when they are expressed as transgenes, and it
has been reported that miRNA-directed cleavage sensitizes transcripts to
transitive silencing (Parizotto et al.,
2004). Consistent with this report, we found that all of our
constructs with a functional miR156 target site were significantly
more susceptible to silencing than constructs that lacked this site. This made
it difficult to generate reporter lines and to maintain these lines over many
generations. It did not interfere with our ability to identify reliable
reporter lines, however, because transgenes undergoing post-transcriptional
silencing have a different expression pattern than SPL3. Whereas the
expression of SPL3 increases with time, transgenes undergoing
silencing are either permanently silenced very early in shoot development, or
display progressively lower levels of expression during shoot growth
(de Carvalho et al., 1992;
Glazov et al., 2003;
Palauqui et al., 1996;
Vaucheret et al., 2004).
Although it is clear that transgenes expressing transcripts with miRNA-target
sites are often subject to RNAi, whether this is also true for endogenous
transcripts is less certain. miRNA-directed transitive silencing is well
documented in the case of transcripts that produce trans-acting
siRNAs (Allen et al., 2005;
Yoshikawa et al., 2005).
However, most protein-coding transcripts that are cleaved by miRNAs -
including the miR156-regulated transcripts SPL9 and
SPL15 - are not affected by sgs3 or rdr6, and are
therefore unlikely to be targets of RNAi
(Allen et al., 2005;
Peragine et al., 2004). The
observation that SPL3 and SPL4 are elevated in these mutants
suggests that these genes either have features that make them unusually
susceptible to RNAi (e.g. the presence of the miR156 target site in
their 3 ' UTR), or that they are regulated indirectly by this mechanism
- for example, by a transcription factor that itself is a target of RNAi. We
have no conclusive evidence for either possibility. However, the observation
that zip-1, rdr6-11 and sgs3-11 have nearly identical
effects on SPL3 expression (Fig.
6A) (Peragine et al.,
2004) suggests that these genes regulate SPL3 by the same
mechanism. Because ZIP is not generally required for RNAi
(Hunter et al., 2003) or for
the miR156-dependent suppression of 35S::GUS-SPL3
(Fig. 6C), this observation may
indicate that these genes regulate SPL3 expression indirectly.
A large fraction of the genes in Arabidopsis have antisense
transcripts (Borsani et al.,
2005; Jen et al.,
2005; Wang et al.,
2005a; Yamada et al.,
2003). In most cases, these transcript pairs represent
neighboring, divergently transcribed protein-coding genes that overlap for a
region of their 3 ' ends. By contrast, the SPL3 antisense
transcript is nearly the same size as the sense transcript, it completely
encompasses the sense transcript and it does have significant coding
potential, suggesting that this transcript is dependent on sequences that are
important for the expression of the sense transcript. Whether this transcript
plays a role in the expression of the sense transcript remains to be
determined.
The results of this and other (Schmid
et al., 2003; Schwab et al.,
2005) studies indicate that SPL3 and the related genes,
SPL4 and SPL5, are under complex transcriptional and
post-transcriptional regulation. We are particularly intrigued by these three
genes because they establish a link between miRNAs and vegetative phase change
in Arabidopsis, and reveal that temporal variation in miRNA
expression plays a regulatory role in developmental timing in plants. Further
studies will be necessary to determine the precise mechanism by which
SPL3/4/5 expression is controlled during vegetative and reproductive
phase change, and the regulatory pathways in which these genes operate.
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ACKNOWLEDGMENTS |
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