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First published online August 7, 2009
doi: 10.1242/10.1242/dev.031658
urczak
Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB, Jordi Girona, 18-26, 08034 Barcelona, Spain.
Author for correspondence
(paula.suarez{at}cid.csic.es)
Accepted 26 June 2009
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Developmental timing, Flowering, miR172, Photoperiod, Phytochrome B, Potato, Tuberization
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; González-Schain
and Suárez-López, 2008). Very few genes involved in
the photoperiodic control of tuber induction have been identified and their
possible functions in the timing of tuberization have hardly been
explored.
The photoreceptor phytochrome B (PHYB) plays an important role in the
response of tuberization to day length. PHYB is required to inhibit
tuberization under LD conditions, as PHYB antisense (anti-PHYB)
plants tuberize under LDs (Jackson et al.,
1996). This effect is transmitted through grafts, which has been
interpreted as evidence that PHYB induces the generation of a mobile inhibitor
of tuberization (Jackson et al.,
1998) that must be produced in the leaves in response to LDs.
However, the mechanism by which PHYB controls photoperiodic tuberization is
unknown. Although it has been shown that PHYB affects the mRNA levels of a
gibberellin-20-oxidase (GA20ox1), a key enzyme in gibberellin biosynthesis
that affects tuberization, the alteration of GA20ox1 transcript and
GA1 levels in anti-PHYB plants is not consistent with
their tuberization phenotype (Carrera et
al., 2000; Jackson et al.,
2000;
Martínez-García et al.,
2002). Therefore, how PHYB negatively regulates tuberization has
not been elucidated.
A homeodomain protein, BEL5, and a sucrose transporter, SUT4, are also
involved in the control of tuberization by the photoperiod
(Chen et al., 2003;
Banerjee et al., 2006b;
Chincinska et al., 2008). BEL5
overexpression promotes tuberization and overrides the inhibition caused by
LDs (Chen et al., 2003;
Banerjee et al., 2006b). A
recent report suggests that BEL5 mRNA is graft transmissible and its
movement to stolons correlates with tuber induction
(Banerjee et al., 2006b). By
contrast, SUT4 is a tuberization inhibitor, as SUT4 RNAi plants
tuberize under non-inductive LDs
(Chincinska et al., 2008). In
addition, SUT4 RNAi plants flower earlier than the wild type
(Chincinska et al., 2008),
revealing another link in the regulation of flowering and tuber induction.
Despite the involvement of PHYB, BEL5 and SUT4 in photoperiodic
tuberization, it is not known whether there are genetic or molecular
interactions between them. Recently, several studies have demonstrated that
microRNA 172 (miR172) plays a role in the timing of
flowering and vegetative phase change
(Aukerman and Sakai, 2003;
Chen, 2004;
Lauter et al., 2005) and is
involved in a photoperiodic pathway controlling flowering in Arabidopsis
thaliana (Jung et al.,
2007). All the genes targeted by miR172 encode members of
a subset of the APETALA2 (AP2)-like transcription factor family
(Aukerman and Sakai, 2003;
Schmid et al., 2003;
Chen, 2004;
Lauter et al., 2005;
Chuck et al., 2007). Like most
plant microRNAs (miRNAs), miR172 can both repress translation and
induce degradation of its target mRNAs
(Aukerman and Sakai, 2003;
Kasschau et al., 2003;
Chen, 2004;
Lauter et al., 2005;
Schwab et al., 2005;
Chuck et al., 2007;
Brodersen et al., 2008;
Voinnet, 2009).
The miR172 sequence had been previously found in a potato EST
(Aukerman and Sakai, 2003),
suggesting that this miRNA is produced in potato. Given the similarities in
the control of flowering and tuberization
(Suárez-López,
2005;
Rodríguez-Falcón et al.,
2006), we tested the hypothesis that miR172 can regulate
flowering and tuber induction in this plant species. Our results show that
miR172 affects the induction of tuberization, probably upstream of
BEL5 and, like in Arabidopsis, is involved in the temporal control of
flowering. The study of miR172 abundance and location strongly
suggests that miR172 acts downstream of PHYB to regulate tuber
induction and that this miRNA is a component or a regulator of long-distance
signals. Furthermore, the results suggest that PHYB acts upstream of BEL5. The
cloning and expression analysis of a potato AP2-like gene harboring a
miR172 target site indicates that this miRNA negatively regulates its
target transcripts in potato. Finally, we propose a model for a pathway
regulating potato tuberization.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). Plants were propagated from single-node stem cuttings on
Murashige and Skoog (MS) medium containing 20 g l-1 sucrose and 2 g
l-1 Gelrite (Duchefa Biochemie, Haarlem, The Netherlands) at
23°C under LD conditions (16 hours light and 8 hours darkness).
Two-week-old plants were planted in soil composed of substrate type 3
(Plantaflor) and sand (3:1) and grown in the greenhouse at 23°C under LD
conditions. During autumn and winter, the light period was extended to 16
hours with high-pressure sodium vapor lamps (SON-T Agro 400 W, Philips
Ibérica, Madrid, Spain). Whenever natural light intensity fell below 65
µmol m-2 second-1 during the light period, light
intensity was supplemented with the same lamps. Plants were watered daily with
modified Hoagland's solution (Johnson et
al., 1957) diluted 1/60.
Prediction of miR172 precursor secondary structure
The first 500 nucleotides of the reverse complement of a putative potato
miR172 precursor (accession number BQ114970) were used to predict its
secondary structure using Mfold
(http://www.bioinfo.rpi.edu/applications/mfold)
(Zuker, 2003). Nine different
structures were predicted, with 
G values ranging from -110.77 to -99.93
kcal mol-1. The structure with the lowest G value was chosen
as representative and is shown in Fig. S1 in the supplementary material.
Analysis of miR172 levels
Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA,
USA) according to the manufacturer's instructions with the following
modification: the isopropanol precipitation was done with a high-salt solution
(1.2 M NaCl and 0.8 M sodium citrate) to remove polysaccharide contaminations.
Alternatively, total RNA was isolated using the Real ARNzol Spin Kit (+PVP)
(Durviz, Valencia, Spain).
For RNA blots, 30 µg of total RNA were fractionated on denaturing 17.5%
polyacrylamide gels containing 7 M urea, which were then electroblotted to a
charged nylon membrane (Zeta probe, Bio-Rad, Hercules, CA, USA). Blots were
probed with 32P-labeled DNA oligonucleotides, complementary to
either miR172 (5'-ATGCAGCATCATCAAGATTCT-3') or U6 small
nuclear RNA (5'-GCAGGGGCCATGCTAATCTTCTCTGTATCGT-3'). Probes (10
pmol) were end-labeled with [
-32P]ATP using T4
polynucleotide kinase (Fermentas, Vilnius, Lithuania). Membranes were
prehybridized and hybridized in Perfect-Hyb Plus buffer (Sigma-Aldrich, St
Louis, MO, USA) at 37°C for miR172 and 50°C for U6 snRNA, and
then washed twice at 50°C with 2x SSC and 0.2% SDS for 15
minutes.
Mature miR172 was quantified by reverse transcription quantitative
real-time PCR (RT-qPCR) according to a previously described protocol
(Chen et al., 2005). Reverse
transcription reactions contained 1-2 µg RNA, 50 nM stem-loop RT primer
(5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATGCAG-3'),
1x RT buffer (Invitrogen), 0.25 mM dNTPs, 200 U Superscript III reverse
transcriptase (Invitrogen) and 2 U µl-1 RNaseOUT (Invitrogen).
The 20 µl reactions were incubated for 30 minutes at 16°C, 30 minutes
at 50°C and 5 minutes at 85°C. qPCR was performed on a LightCycler 480
Real-Time PCR System (Roche Diagnostics, Mannheim, Germany). The 20 µl PCR
included 0.2 µl cDNA, 1x LightCycler 480 SYBR Green I Master Mix
(Roche), 0.3 µM forward primer (5'-CGGCGGTAGAATCTTGATGATG-3')
and 0.3 µM reverse primer (5'-GTGCAGGGTCCGAGGT-3'). The
reactions were incubated at 95°C for 10 minutes, followed by 40 cycles of
95°C for 10 seconds, 60°C for 30 seconds and 72°C for 30 seconds.
For normalization, 5S rRNA was reverse transcribed (primer
5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGGAT-3') and
amplified (forward primer 5'-GGATGCGATCATACCAGCACT-3' and the same
reverse primer as for miR172) in the same conditions as for
miR172. All reactions were run in triplicate. The specificity of PCR
was checked with dissociation curve analysis. Data from RT-qPCR were analyzed
using the 2-Ct method
(Livak and Schmittgen,
2001).
In situ hybridization
Plant material was fixed for 8 hours at 4°C in 4% paraformaldehyde and
0.1 M phosphate buffer pH 7, dehydrated through a graded series of ethanol and
butanol dilutions, embedded in Paraplast Plus (Paraplast X-Tra, Oxford
Labware, St Louis, MO, USA) and sectioned to 7 µm with a microtome. Tissue
sections were deparaffinized with Histoclear, rehydrated through an ethanol
series, and then pre-treated with proteinase K (0.1 U ml-1) in
Tris-HCl pH 7.5 at 37°C for 15 minutes. Digestion was stopped by washing
with 0.2x PBS containing 0.04 M glycine and then twice with PBS for 2
minutes. After dehydrating in ethanol baths, hybridization was performed at
37°C overnight with 0.02 µM digoxigenin-labeled miRCURY LNA probe
complementary to miR172 (Exiqon, Vedback, Denmark) in hybridization
solution (50% formamide, 2x SSC, 4x Denhardt's solution, 20%
dextran, 2 mg ml-1 tRNA). An LNA oligonucleotide complementary to
mouse miR124 (5'-TTAAGGCACGCGGTGAATGCCA-3'), with no
predicted target sequences in plants, was used as negative control. After
hybridization, slides were washed in 2x SSC at 37°C for 45 minutes
and in 1x SSC for 15 minutes. For signal detection, samples were
incubated in 10% blocking reagent (Roche, Mannheim, Germany) in PBS for 1 hour
and afterwards for 30 minutes in blocking reagent containing anti-DIG alkaline
phosphatase-conjugated Fab fragment antibody (Roche) diluted to 1:500. After
three washes for 10 minutes in PBS, tissues were equilibrated in detection
buffer (100 mM Tris-HCl pH 9.5, 100 mM NaCl, 50 mM MgCl2) for 15
minutes prior to incubating in the same buffer supplemented with 0.2 mM NBT
and 0.2 mM BCIP substrates until a signal was visible.
Generation of plants overexpressing miR172
For miR172 overexpression, a 133 bp fragment of a putative
miR172 precursor from potato was PCR-amplified using EST600546
(GenBank accession number BQ114970) as a template and primers miR172
forward 5'-GGTCTAGACATACAGTTGTTGCTTGCTA-3' and miR172
reverse 5'-GGGTCGACATCAAGTCATCAATTTGCCA-3'. The PCR product was
digested with XbaI and SalI and cloned in sense orientation
in the XbaI and SalI sites of pBIB35S-Hyg to generate
pBIB-35S::miR172. The pBIB35S-Hyg vector was created by replacing the
EcoRI/HindIII cassette of pBIB-HYG
(Becker, 1990) with the
EcoRI/HindIII cassette of pBINAR
(Höfgen and Willmitzer,
1990). pBIB-35S::miR172 was electroporated into
Agrobacterium tumefaciens strain C58 GV2260, which was used to
transform S. tuberosum subspecies andigena essentially as
described (Banerjee et al.,
2006a). Hygromycin-B-resistant shoots were regenerated and then
propagated from single-node stem cuttings on MS medium containing 20 g
l-1 sucrose, 15 mg l-1 hygromycin B, 250 mg
l-1 cefotaxime and 2 g l-1 Gelrite (Duchefa Biochemie).
Plants were propagated for at least two rounds on selective medium before
being propagated in the absence of the antibiotic. Twenty-three putative
transgenic lines were planted in soil and the level of miR172 was
assessed by RNA blot. Seven 35S::miR172 lines and one line carrying
an empty vector were selected for further analysis.
Analysis of flowering and tuberization
To analyze flowering time, plants were grown either in the LD greenhouse or
in controlled environment chambers with the following photoperiods: SD (8
hours light and 16 hours darkness); and SD supplemented with a 30 minutes
white-light night break given 8 hours after the start of the dark period
(SD+NB). Lighting was provided by high-pressure sodium vapor lamps (SON-T Agro
400 W). The shoot apex was carefully checked for visible signs of flowering
every 2 or 3 days. Flowering time was measured as the number of days from
planting in soil to the appearance of the floral bud. Leaf number was also
recorded at the time the floral bud was visible.
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Grafting experiments
Five-week-old LD-grown 35S::miR172 (line 8) and wild-type plants
were used. V-shape grafts were made, bound with paper surgical tape, and
immediately covered up with a transparent plastic bag to preserve humidity.
Grafts were maintained in the LD greenhouse. Humidity was slowly released
after 48 hours. Ten days after grafting, when graft unions had healed, the
stock leaves were removed and plants were transferred to SD+NB conditions.
Twenty grafts of each of the following types were analyzed:
35S::miR172 scions onto wild-type stocks, wild-type scions onto
35S::miR172 stocks, wild-type scions onto wild-type stocks and
35S::miR172 scions onto 35S::miR172 stocks. Ungrafted plants
were also used as controls. Tuberization time was determined as described
above.
Identification of RAP1, a putative miR172 target gene
In order to isolate potential target genes of miR172, total RNA
was extracted from potato tissues with low miR172 levels. RNA was
reverse transcribed using the RNA-ligase-mediated method from the Gene Racer
Kit (Invitrogen). First, a forward primer corresponding to the miR172
sequence (5'-AGAATCTTGATGATGCTGCAT-3') was used in a 3' RACE
reaction to amplify the 3' part of cDNAs representing potential targets
of miR172. A PCR product was gel-excised, cloned and sequenced. Then,
a reverse primer (5'-GTCAAGAGTTGTCGAAGCAA-3') designed on the
obtained sequence was used for 5' RACE to obtain the full-length
sequence of the cDNA. This sequence was named RAP1 and has been
submitted to the EMBL Nucleotide Sequence Database under the accession number
FM246879.
Reverse transcription quantitative real-time PCR analyses
RAP1 and BEL5 mRNA accumulation was analyzed by RT-qPCR
in wild-type, anti-PHYB and 35S::miR172 plants grown under
different photoperiods. Total RNA was purified from frozen tissues by TRIzol
extraction, as described above. cDNA synthesis was performed in a 20 µl
volume, containing 2 µg RNA, 25 µg ml-1
oligo(dT)12-18 (Invitrogen), 0.5 mM dNTP Mix (Invitrogen), 10 mM
DTT (Invitrogen), 40 U RNase Inhibitor (RNA Guard, Pharmacia) and 200 U M-MLV
Reverse Transcriptase (Invitrogen). qPCR was performed on an ABI Prism 7000
Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with
gene-specific primer pairs. The reactions, performed in triplicate, contained
0.2 µl of cDNA, 1x SYBR Green PCR Master Mix (Applied Biosystems),
0.3 µM forward primer and 0.3 µM reverse primer, and were incubated at
95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and
60°C for 1 minute. The specificity of PCR was checked with dissociation
curves and quantification was standardized to ACTIN (ACT)
mRNA levels. Data from RT-qPCR were analyzed using the
2-Ct method
(Livak and Schmittgen, 2001).
Primers used for PCR were as follows: ACT forward,
5'-CCTTGTATGCTAGTGGTCG-3'; ACT reverse,
5'-GCTCATAGTCAAGAGCCAC-3'; BEL5 forward,
5'-TTTAATGGCAGCATACGCGA-3'; BEL5 reverse,
5'-CTGGCATGGCTAGGTTTTCAG-3'; RAP1 forward,
5'-AGGGAACAGCATTAGGGAAGGGT-3'; RAP1 reverse,
5'-AGTCAAGAGTTGTCGAAGCAATGTA-3'.
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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). A prediction of the secondary structure of this putative
precursor using Mfold showed that the miR172 sequence is present,
paired to the miR172* sequence, in an arm of the stem of a
stem-loop structure (see Fig. S1 in the supplementary material), as expected
for a miRNA precursor. This suggests that miR172 can be produced in
potato.
Analysis of miR172 levels in potato plants
The relative abundance of miR172 was analyzed by RT-qPCR and RNA
blot, using a miR172 antisense probe, in potato plants grown under
LD, SD+NB and SD conditions. As shown in
Fig. 1 and Fig. S2 in the
supplementary material, miR172 is present in all organs studied
(leaves, stems and stolons), with a higher accumulation in stems under all
photoperiods. miR172 levels were higher in all organs under
tuber-inducing conditions (SDs) than under non-inducing LDs
(Fig. 1A), whereas under
moderately inductive SD+NB conditions, levels were lower than under LDs
(Fig. 1B). Both under SD and
SD+NB conditions, an increase in miR172 levels correlated with tuber
initiation, as its abundance was higher in swollen than unswollen stolons
(Fig. 1; see Fig. S2 in the
supplementary material).
To examine the spatial distribution of miR172, we performed in situ hybridization on leaves, stems, stolons and swollen stolons. In leaves, miR172 was mainly detected in the epidermis and vascular cells (Fig. 2A). By contrast, in the stem, a signal corresponding to miR172 was detected only in the internal and external phloem (Fig. 2B). In stolons collected under LDs, miR172 was present mainly in vascular cells and in the apical meristem (Fig. 2C). Under SDs, however, miR172 was detected in all cell types of unswollen stolons (Fig. 2D) and its localization was restricted to the vasculature and apex when stolons started to swell (Fig. 2E).
Altogether, these experiments show that: (1) miR172 is present in the vascular bundles of all the organs studied; (2) miR172 levels are highest under tuber-inducing SD conditions; and (3) miR172 distribution changes and its levels increase in stolons at the onset of tuberization.
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miR172 affects flowering time in potato
To understand the function of miR172 in potato, we generated
plants that constitutively express miR172 from the cauliflower mosaic
virus 35S promoter. Seven 35S::miR172 lines and a control
line transformed with an empty vector were selected for analysis. The level of
miR172 in the transgenic lines was examined by RNA blot and RT-qPCR.
No change in the abundance of miR172 was observed in the control line
compared to the wild type. By contrast, an increase in the amount of this
miRNA was clearly visible in all the 35S::miR172 lines (see Fig. S3
in the supplementary material).
Since miR172 is a regulator of flowering time
(Aukerman and Sakai, 2003;
Chen, 2004;
Lauter et al., 2005), we
investigated the flowering time of potato 35S::miR172 plants. All the
35S::miR172 lines flowered earlier than control plants under LDs when
flowering time was estimated by counting the number of leaves at flowering
(Fig. 4A), and most lines were
also statistically different from the controls in the number of days to
flowering (Fig. 4B). Similar
results were obtained under SD+NB and SD conditions (see Fig. S4 in the
supplementary material). These results indicate that miR172 promotes
flowering in potato, as previously shown in other plant species.
Overexpression of miR172 promotes tuberization in a photoperiod-dependent manner
Given that the regulation of flowering and tuberization shows several
similarities (Suárez-López,
2005;
Rodríguez-Falcón et al.,
2006), we investigated whether the increase in miR172
levels could have an impact on tuber induction in potato. Under strongly
inductive SD conditions, no significant differences in tuberization time were
observed between 35S::miR172 lines and the controls
(Fig. 5A,B). In moderately
inductive SD+NB conditions, several 35S::miR172 lines (6, 8 and 22)
showed an early tuberization phenotype
(Fig. 5A,B). In addition,
35S::miR172 lines can tuberize in LD conditions, which are not
inductive for the wild type; Fig.
5C shows the underground part of two 35S::miR172 lines in
comparison to control plants after 3 months in LD conditions. Therefore,
miR172 promotes tuber development and is probably involved in the
photoperiodic control of tuberization.
|
|
), the
tuberization time of anti-PHYB plants was determined under the three
photoperiods. Anti-PHYB plants tuberized at the same time under SDs
and LDs, indicating that they lost the repression of tuberization under LDs
completely (see Fig. S5 in the supplementary material). Tuberization of
anti-PHYB plants was very slightly delayed under SD+NB conditions
compared with SDs or LDs (see Fig. S5 in the supplementary material).
Therefore, the photoperiodic control of tuberization is more severely affected
in anti-PHYB than 35S::miR172 plants.
The effect of miR172 on tuberization is graft transmissible
The presence of miR172 in vascular bundles
(Fig. 2), the change in
distribution in stolons at the onset of tuberization
(Fig. 2) and the opposite
effect of PHYB on miR172 levels in leaves and stolons
(Fig. 3) suggested that this
miRNA might be involved in long-distance signaling to control tuberization.
Therefore, we tested whether the tuberization phenotype of
35S::miR172 plants is graft transmissible. 35S::miR172
scions grafted onto wild-type stocks (35S::miR172/WT grafts)
tuberized as early as 35S::miR172/35S::miR172 controls
(Fig. 5D), showing that the
effect of miR172 on tuberization is transmissible through grafts and
indicating that miR172 overexpression in aerial organs is sufficient
to promote tuberization. By contrast, WT/35S::miR172 grafts tuberized
like WT/WT controls (Fig. 5D),
indicating that the overexpression of miR172 in underground parts is
not sufficient to affect this process.
Identification of RAP1, a potato gene with a miR172 binding site
All of the target genes of miR172 identified so far belong to the
AP2-like family (Aukerman and
Sakai, 2003; Schmid et al.,
2003; Chen, 2004;
Lauter et al., 2005;
Chuck et al., 2007). In order
to further investigate the role of miR172 in potato, we used an
approach to identify potato genes possessing a miR172 binding site,
which therefore represent candidate genes involved in the miR172
pathway. The miR172 sequence was used as a primer in a 3' RACE
reaction, leading to the amplification of a cDNA fragment containing a
miR172 binding site. The full-length cDNA sequence, as well as
validation of the miR172 binding site, were obtained by subsequent
5' RACE. Using this approach, we identified a gene encoding a 454 amino
acid protein showing strong homology with AP2 and AP2-like proteins. This
protein was named RAP1 (RELATED TO APETALA2 1) and it contains two AP2 domains
highly similar to those of previously characterized members of the AP2 family
(Fig. 6A). The RAP1
coding sequence displays a miR172 binding site, and alignments with
known targets of this miRNA indicated a partial conservation of mismatches
between miR172 and its targets
(Fig. 6B). This suggests that
RAP1 is a likely target of miR172 in potato.
Transcript levels of RAP1 are regulated by miR172 and PHYB
To determine whether miR172 regulates RAP1, RT-qPCR
analyses were performed. In wild-type plants grown under SD+NB or LD
conditions, the mRNA abundance of RAP1 was higher in leaves than in
stems and stolons (Fig. 7A).
Therefore, RAP1 transcript levels show an inverse correlation with
miR172 accumulation, suggesting that this miRNA can target
RAP1 mRNA for degradation. The only significant difference in
RAP1 mRNA abundance between SD+NB and LD conditions was observed in
leaves, with higher levels under LD than SD+NB conditions
(Fig. 7A).
To test whether RAP1 is a target of miR172, the levels of RAP1 transcript were analyzed in 35S::miR172 plants. RAP1 mRNA abundance was significantly lower in 35S::miR172 than in wild-type leaves (Fig. 7C), indicating that miR172 negatively regulates RAP1. By contrast, RAP1 mRNA levels were not significantly altered in the stems and stolons of 35S::miR172 plants (Fig. 7C).
Then, levels of RAP1 mRNA were examined in anti-PHYB plants, as these plants showed altered miR172 accumulation (Fig. 3). Levels of RAP1 were drastically reduced in all analyzed organs of anti-PHYB plants in contrast with the controls (Fig. 7D). Nevertheless, anti-PHYB plants still have lower levels of RAP1 in stems and stolons than in leaves (Fig. 7D). These results indicate that PHYB regulates RAP1.
|
;
Banerjee et al., 2006b).
RT-qPCR experiments showed that the levels of BEL5 mRNA were higher
in leaves and stems than in unswollen stolons of wild-type plants under SD+NB
and LD conditions (Fig. 7B),
and an increase in BEL5 in stolons correlated with the onset of tuber
development in SD+NB conditions (Fig.
7B; compare stolons with swollen stolons). This is consistent with
a previous analysis of BEL5 transcript abundance
(Chen et al., 2003). Wild-type
leaves and stems showed a higher accumulation of BEL5 mRNA under LD
than SD+NB conditions, whereas the opposite was found in stolons
(Fig. 7B).
Since miR172, PHYB and BEL5 influence tuberization, we examined
whether miR172 and PHYB have an effect on BEL5. Under SD+NB
conditions, the levels of BEL5 mRNA were higher in all organs of
35S::miR172 than wild-type plants
(Fig. 7E). Therefore,
miR172 overexpression leads to the upregulation of BEL5. In
addition, in anti-PHYB lines the levels of BEL5 were
increased in stems and stolons when compared with the wild type, whereas in
leaves transcript levels were decreased in one of the transgenic lines,
anti-PHYB-10 (Fig.
7F). In other experiments, a reduction in BEL5 mRNA was
also observed in the leaves of transgenic line anti-PHYB-4.
Interestingly, there was a remarkable increase in BEL5 levels in
anti-PHYB stolons at the onset of tuberization
(Fig. 7F, compare stolons with
swollen stolons). These results indicate that PHYB controls BEL5
transcript abundance in several organs. The reduction in BEL5 mRNA
levels in the leaves of anti-PHYB plants and the increase in mRNA
levels in stolons when these start to swell is consistent with the reported
movement of BEL5 mRNA (Banerjee et
al., 2006b).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Chen, 2004;
Lauter et al., 2005;
Chuck et al., 2007;
Zhao et al., 2007). We show
here that, in potato, miR172 affects not only flowering time, but
also the timing of tuberization, another developmental process related to
reproduction.
We have overexpressed a member of the miR172 family that had been
previously identified in potato (Aukerman
and Sakai, 2003). Recently, this miR172 member has been
named miR172b, and four additional potato miR172 precursors
have been computationally identified
(Zhang et al., 2009). The
predicted secondary structure of pre-miR172b shown in Fig. S1 in the
supplementary material is identical to that reported by Zhang et al.
(Zhang et al., 2009). The
sequence of potato miR172b is identical to that of Arabidopsis
miR172a and b and maize miR172a-d. However,
overexpression of this miRNA in potato does not cause any obvious floral organ
identity phenotype (not shown), suggesting that the function of this miRNA has
partially diverged between potato and other species.
|
),
indicating that the regulation of miR172 by PHYB is conserved in
different plant species.
miR172 promotes flowering in Arabidopsis by negatively
regulating AP2-like flowering repressors, such as TOE1, TOE2, SMZ and SNZ
(Aukerman and Sakai, 2003;
Schmid et al., 2003;
Jung et al., 2007). Since this
miRNA accelerates flowering and tuberization in potato (Figs
4 and
5), we propose that
miR172 would negatively regulate flowering and tuberization
inhibitors in this species. We have cloned a potato AP2-like gene,
RAP1, that contains a miR172 target site. There is an
inverse correlation between the abundance of RAP1 transcript and
miR172 in several organs of wild-type plants
(Fig. 1;
Fig. 7A). Moreover,
downregulation of RAP1 in miR172-overexpressing leaves
(Fig. 7C) strongly suggests
that RAP1 is a target of miR172 and that this miRNA induces
the degradation of RAP1 mRNA. The finding that RAP1
transcript abundance is not reduced in 35S::miR172 stems and stolons
(Fig. 7C), which do overexpress
miR172 (see Fig. S3B in the supplementary material), does not exclude
the possibility that miR172 can regulate RAP1 in these
organs by blocking its translation. In fact, miR172 can induce both
mRNA degradation and translational inhibition in other species
(Aukerman and Sakai, 2003;
Kasschau et al., 2003;
Chen, 2004;
Lauter et al., 2005;
Schwab et al., 2005;
Chuck et al., 2007). It is
also possible that feedback regulation of miR172 targets by their
products masks changes in target mRNA abundance, as has been shown in
Arabidopsis (Schwab et al.,
2005).
RAP1 transcript levels are downregulated in 35S::miR172
and anti-PHYB plants (Fig.
7), both of which tuberize earlier than wild-type plants
(Jackson et al., 1996)
(Fig. 5). In addition,
35S::miR172 plants exhibit early flowering
(Fig. 4; see Fig. S4 in the
supplementary material). We hypothesize that RAP1 might be involved in the
repression of tuberization and/or flowering downstream of PHYB and
miR172.
Silencing of PHYB completely abolishes the repression of tuberization under
LDs (Jackson et al., 1996)
(see Fig. S5 in the supplementary material), indicating that PHYB is essential
for the inhibition of tuberization under this photoperiod. miR172
levels are altered in anti-PHYB plants
(Fig. 3), strongly suggesting
that miR172 acts downstream of PHYB to control tuberization. However,
changes in miR172 levels are not sufficient to explain the strong
effect of PHYB on tuberization, as the tuberization phenotypes of
35S::miR172 plants are weaker than those of anti-PHYB plants
and, in addition, 35S::miR172 plants are still partially sensitive to
day length (Fig. 5; see Fig. S5
in the supplementary material). Similarly, Arabidopsis plants
overexpressing miR172 also show a residual photoperiodic flowering
response (Aukerman and Sakai,
2003). Given that BEL5 promotes tuberization
(Chen et al., 2003;
Banerjee et al., 2006b), we
examined the possibility that miR172 and PHYB could regulate this
gene. We found increased BEL5 mRNA levels in all organs of
35S::miR172 plants and in stems and stolons of anti-PHYB
plants (Fig. 7E,F). Therefore,
BEL5 might also contribute to the regulation of tuber induction by
miR172 and PHYB. Taken together, these results suggest that the
regulation of BEL5 by PHYB is at least in part mediated by miR172.
Our results do not rule out that additional genes might act downstream of PHYB
and miR172 to mediate their effects on tuber formation.
The presence of miR172 in the vasculature of wild-type potato
plants (Fig. 2), the detection
of miR172 in the phloem sap of Brassica napus
(Buhtz et al., 2008) and the
graft transmission of the tuberization phenotype of 35S::miR172
plants (Fig. 5D) show that
either this miRNA is mobile or it regulates long-distance signals to induce
tuberization. Our grafting experiments, together with the analysis of
RAP1 and BEL5 mRNA levels in miR172-overexpressing
plants, indicate that miR172 can act at least in the leaves to
promote tuberization and regulate the mRNA levels of these two genes. Although
overexpression of miR172 in the stocks of WT/35S::miR172
grafts does not promote tuberization (Fig.
5D), this does not rule out the possibility that endogenous
miR172 can act in these organs. The clear correlation of tuber
induction with an increase in miR172 levels in stolons is consistent
with this idea. It is possible that the overexpression of miR172 in
stocks is not sufficient to counteract repressive signals coming from the
wild-type scions.
It has been established that the FT protein is a component of the
long-range signals for flowering (Turck et
al., 2008). However, its counterpart for tuberization has not been
identified so far. It has been reported that BEL5 mRNA is graft
transmissible and its movement correlates with tuber induction
(Banerjee et al., 2006b).
Interestingly, anti-PHYB plants show a reduction in BEL5
transcript levels in leaves in comparison with wild-type plants and an
increase in the levels in stolons when these start to swell
(Fig. 7F). Similarly, we have
observed reduced miR172 levels in leaves and increased levels in
swollen stolons of anti-PHYB plants
(Fig. 3). One interpretation of
these results is that PHYB promotes miR172 and BEL5
expression in leaves and represses their expression in stolons. However, this
is inconsistent with the fact that PHYB represses tuberization, whereas
overexpression of miR172 and BEL5 in aerial organs promotes it.
Therefore, we hypothesize that BEL5 mRNA and miR172 move
from leaves to stolons under SDs to induce tuberization. Under LD and SD+NB
conditions, this movement would be repressed by PHYB, thereby preventing or
delaying tuberization. Previously it had been proposed that PHYB induces the
production of a graft-transmissible inhibitor of tuberization
(Jackson et al., 1998). Our
data suggest that PHYB might inhibit the transport of promoters of
tuberization. Although the idea that miRNAs can act as systemic signals is
still under debate (Voinnet,
2009), at least one miRNA has been shown to be a long-distance
signal (Pant et al.,
2008).
|
). miR172 promotes tuberization probably via the
upregulation of BEL5 in leaves and stolons. Moreover, this miRNA
inhibits RAP1. We postulate that the upregulation of BEL5 by
miR172 might be mediated by a miR172 target gene, perhaps
RAP1, which would repress BEL5. Interestingly, the
BEL5 promoter contains two CAACA sequence motifs (in reverse
orientation, TGTTG) that have been described as binding sites for the AP2
domain of an AP2-like transcription factor
(Kagaya et al., 1999), and
RAP1 has two AP2 domains. Increased miR172 levels lead to the
inhibition of its targets (e.g. RAP1) and therefore probably to a release of
BEL5 repression. This release would lead to more BEL5 mRNA
in stolons, therefore inducing tuberization. However, we cannot rule out an
effect of miR172 target genes on tuberization through a
BEL5-independent pathway. In addition, PHYB regulates RAP1 probably through
miR172, but also through a miR172-independent pathway, as
the effect of PHYB on RAP1 mRNA levels is much more dramatic than
that of miR172 (Fig.
7). This complex network probably confers robustness to the
regulation of tuberization by reinforcing signals, as PHYB acts under LDs to
repress the function of two tuberization inducers, BEL5 and miR172,
and to promote the expression of a putative negative regulator of BEL5 (RAP1),
ensuring that tuberization does not occur under LD conditions. This work establishes that miR172 is another regulator shared by flowering and tuberization, shows that this miRNA affects a long-distance signaling pathway and strongly suggests that it acts between PHYB and BEL5 to control tuber induction. In Arabidopsis, several miR172 target genes are involved in the regulation of flowering time. Further research will help to determine whether different miR172 targets are specialized for the control of flowering and tuberization.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/17/2873/DC1
|
Footnotes |
|---|
. by a JAE fellowship from the Spanish Scientific
Research Council (CSIC).
* Present address: INRA-CNRS, UMR1165, Unité de Recherche en
Génomique Végétale, 2 rue Gaston Crémieux, F-91057
Evry, France 
Present address: IRD, UMR DIAPC, IRD/CIRAD Palm Development Group, 34394
Montpellier, France
Present address: College of Natural and Agricultural Sciences, University
of California, Riverside, CA 92521, USA
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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4)
plants grown under LDs for 39 days. U6 snRNA was used as a normalization
control. Three independent experiments were performed and a representative
result is shown. (B) miR172 levels were quantified by RT-qPCR
in the stolons of plants grown under LDs for the indicated times.
Anti-PHYB stolons were swollen after 39 days under LD conditions,
whereas all other stolon samples were not swollen. Mean values calculated from
triplicate samples and s.e.m. are indicated. Relative miR172 levels
are given, with maximum levels set to 1. All samples were collected 4 hours
after dawn.
