Arabidopsis mutant sk156 reveals complex regulation of SPL15 in a miR156-controlled gene network
BMC Plant Biology
Arabidopsis mutant sk156 reveals complex regulation of SPL15 in a miR156-controlled gene network
Shu Wei 0 3
Margaret Y Gruber 0
Bianyun Yu 0 2
Ming-Jun Gao 0
George G Khachatourians 5
Dwayne D Hegedus 0
Isobel AP Parkin 0
Abdelali Hannoufa 1 4
0 Agriculture and Agri-Food Canada , 107 Science Place, Saskatoon, SK S7N 0X2 , Canada
1 Agriculture and Agri-Food Canada , 1391 Sandford Street, London, ON N5V 5T3 , Canada
2 Current address: Plant Biotechnology Institute, National Research Council of Canada , 110 Gymnasium Place, Saskatoon, SK S7N 0W9 , Canada
3 College of Tea & Food Science and Technology, Anhui Agricultural University , 130 Changjiang Blvd West, Hefei 230036 , China
4 Agriculture and Agri- Food Canada , 1391 Sandford Street, London, ON N5V 5T3 , Canada
5 Department of Food and Bioproduct Sciences, University of Saskatchewan , 51 Campus Drive, Saskatoon, SK S7N 5A8 , Canada
Background: The Arabidopsis microRNA156 (miR156) regulates 11 members of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) family by base pairing to complementary target mRNAs. Each SPL gene further regulates a set of other genes; thus, miR156 controls numerous genes through a complex gene regulation network. Increased axillary branching occurs in transgenic Arabidopsis overexpressing miR156b, similar to that observed in loss-of-function max3 and max4 mutants with lesions in carotenoid cleavage dioxygenases. Arabidopsis miR156b was found to enhance carotenoid levels and reproductive shoot branching when expressed in Brassica napus, suggesting a link between miR156b expression and carotenoid metabolism. However, details of the miR156 regulatory network of SPL genes related to carotenoid metabolism are not known. Results: In this study, an Arabidopsis T-DNA enhancer mutant, sk156, was identified due to its altered branching and trichome morphology and increased seed carotenoid levels compared to wild type (WT) ecovar Columbia. Enhanced miR156b expression due to the 35S enhancers present on the T-DNA insert was responsible for these phenotypes. Constitutive and leaf primodium-specific expression of a miR156-insensitive (mutated) SPL15 (SPL15m) largely restored WT seed carotenoid levels and plant morphology when expressed in sk156. The Arabidopsis native miR156-sensitive SPL15 (SPL15n) and SPL15m driven by a native SPL15 promoter did not restore the WT phenotype in sk156. Our findings suggest that SPL15 function is somewhat redundant with other SPL family members, which collectively affect plant phenotypes. Moreover, substantially decreased miR156b transcript levels in sk156 expressing SPL15m, together with the presence of multiple repeats of SPL-binding GTAC core sequence close to the miR156b transcription start site, suggested feedback regulation of miR156b expression by SPL15. This was supported by the demonstration of specific in vitro interaction between DNA-binding SBP domain of SPL15 and the proximal promoter sequence of miR156b. Conclusions: Enhanced miR156b expression in sk156 leads to the mutant phenotype including carotenoid levels in the seed through suppression of SPL15 and other SPL target genes. Moreover, SPL15 has a regulatory role not only for downstream components, but also for its own upstream regulator miR156b.
The Arabidopsis miR156 family has eight members and
is highly conserved in the plant kingdom. It has been
identified in 45 plant species . MiR156 is known
to repress SPL (SQUAMOSA PROMOTER BINDING
PROTEIN-LIKE) genes [2-4], which are plant-specific
transcription factors containing the SBP (SQUAMOSA
promoter binding protein) box . In Arabidopsis, 11 SPL
genes are targeted by miR156 [2,4,6,7], and in silico full
genome analysis showed that no other genes in
Arabidopsis have the segment complimentary to miR156 . Some
details of the relationship between the miR156 regulatory
network downstream of SPL genes and flowering
enhancement and flavonoid metabolism have been revealed
[8-10]. But this is clearly an area of research that still
needs strong attention.
Diverse and redundant roles of some individual SPL
genes in plant morphology and development have been
reported. The SPL genes targeted by miR156 can be
grouped into four major clades: SPL3/SPL4/SPL5, SPL2/
SPL10/SPL11, SPL9/SPL15, and SPL6/SPL13 . SPL3,
SPL4 and SPL5 exhibit partially redundant effects on
plant juvenile-to-adult transition [4,11,12]. SPL9 and
SPL3 directly activate MADS box genes that promote
flowering . In addition, SPL9 and SPL15
interchangeably control shoot maturation and leaf initiation .
SPL10 and SPL9 expression in leaf primordia modulated
by miR156 affects initiation of new leaves at the shoot
apical meristem . SPL2, SPL10 and SPL11 were each
able to control leaf lamina shape in association with
shoot maturation in the reproductive phase .
Moreover, SPL genes (represented by SPL9) regulate trichome
development via direct interaction with miR172  and
the MYB transcription factor genes TRICHOMELESS1
(TCL1) and TRIPTYCHON (TRY) .
Increased axillary branching occurs in transgenic
Arabidopsis lines expressing miR156b under the control of a
cauliflower mosaic virus 35S promoter (CaMV35S) ,
similar to that observed in loss-of-function max3 and
max4 mutants with lesions in carotenoid cleavage
dioxygenases, CCD7 and CCD8, respectively [17-19]. These
max mutations cause defective biosynthesis of
strigolactones; a group of carotenoid-derived hormones that
inhibit shoot branching [20,21]. The morphological
similarities between these transgenic and mutant lines
suggest a link between miR156b expression and
carotenoid metabolism. This was partially confirmed when
Arabidopsis miR156b was found to enhance carotenoid
levels and reproductive shoot branching when expressed
in Brassica napus .
In this study, we report on a new Arabidopsis activation
tagged mutant, sk156, with strongly enhanced expression
of miR156b. MiR156b-induced SPL15 suppression was
partially responsible for the increased seed carotenoid
abundance and altered plant morphology observed in
sk156. In addition, we highlight a new SPL15 feedback
loop which controls expression of miR156b, likely via a
physical interaction between the SPL15 SBP domain and
the promoter of miR156b.
Phenotypes of the sk156 mutant
The sk156 mutant was selected from an Arabidopsis
activation-tagged mutant population that was developed
using a T-DNA construct containing four CaMV35S
enhancers . Compared to the parental wild type
(WT) Col-4, sk156 exhibited the following
morphological changes: increased numbers of rosette leaves
which were slightly pale, smaller and rounder, increased
reproductive branching, ectopic expression of trichomes
on flower sepals and shoot tips, delayed bolting, severely
stunted cauline stems, and decreased flower and silique
sizes (Figure 1).
For comparison, we examined the phenotypes of
max3-9  and max4-1  mutants, which had been
confirmed previously to contain defective CCD7 and
CCD8 genes, respectively (Additional file 1). In our
hands, these max mutants showed increased secondary
and tertiary branches (Figure 1G), which was consistent
with an earlier report that max mutants showed
increased inflorescence number compared to WT plants
. However the cauline stems of these two max
mutants were not significantly stunted as in sk156. The
branching pattern of sk156 was also somewhat distinct
compared with max mutants; sk156 had more
quaternary and quinary branches and the max mutants mainly
had increased secondary and tertiary branches.
The abundance of five major carotenoid compounds
was examined in the mature seeds and leaves of sk156
and WT plants. Levels of lutein, -carotene,
violaxanthin, and zeaxanthin were 2.3-, 11.7, 5.5-, and 1.8- fold
higher in sk156 seeds than in WT seeds (Figure 2A and
B). Slight decreases (but not statistically significant) in
the levels of major carotenoids in leaves were found in
sk156 compared to WT (Figure 2C and D).
Cryptoxanthin was undetectable in either tissue of the mutant,
but present at 0.62 g g-1 FW (fresh weight) in WT
seeds and 4-fold higher in WT leaves.
Molecular analysis of sk156
Segregation analysis of heterozygous sk156 mutant
offspring revealed that the ratio of resistant-to-sensitive
seedlings (271:109, respectively) was well within the
expected 3:1 ratio (2, p = 0.05), indicating that there was
a single T-DNA insertion site. This was confirmed by
Southern blot analysis (Figure 3A). Sequencing of the
TDNA-flanking regions indicated that the T-DNA was
located in AT4G30980 (Figure 3B), which encodes a
basic helix-loop-helix (bHLH) family protein bHLH069
[25,26]. CaMV 35S enhancers in the T-DNA were within
close proximity to five other gene loci: AT4G30975
(unknown RNA gene), AT4G30972 (miR156b), AT4G30970
(unknown protein) and AT4g30960 (SOS2-like protein
The sk156 phenotypes were dominant when crossed
with WT, suggesting that the altered phenotypes in
sk156 were tightly linked to enhanced expression of gene
(s) adjacent to the T-DNA, rather than gene disruption.
Thus, transcript levels of genes close to the T-DNA
insertion site were compared in the mutant and WT
plants using quantitative RT-PCR. Transcript levels of
AT4G30972 (miR156b) and the unknown RNA gene
Figure 1 Morphological phenotype of the Arabidopsis sk156 mutant. (A) 30-d-old rosette plants. (B) 8-week-old mature plants. (C) 30-d-old
plant leaves. WT on top, sk156 on bottom. (D) Flowers, showing trichome-bearing sk156 sepals (right). (E) Siliques at 14 days post-anthesis. (F)
Trichome enhancement on cauline stems and leaves of sk156. (G) Enhanced rosette leaf and branch numbers (insert defines branch order). Data
were the mean of thirty 8-week-old plants. MB, main stem; RL, rosette leaves. A Duncan's multiple range test showed significant differences of
the means ( standard deviation) between mutant lines and WT at p < 0.01 (**) and p < 0.05 (*).
AT4G30975 were increased 91.4-fold ( 25.8) and
95.8fold (18.6), respectively, in sk156 (Figure 3C), while the
T-DNA-disrupted AT4g30980 (bHLH069) was repressed
11.1-fold (Figure 3D). No significant changes in
transcript levels were detected for the other adjacent genes
(data not shown). This indicated that the four CaVM
35S enhancers inserted into bHLH069 caused the
activation of miR156b and the unknown RNA gene.
To determine which of the three T-DNA affected
genes was responsible for the altered phenotypes in
sk156, including enhanced seed carotenoid levels, four
types of plants were obtained and examined (Table 1).
These included a bHLH069 knockout mutant S468
(SALK_032468) (Figure 4A), transgenic sk156
overexpressing a 35S:bHLH069 cDNA (Figure 4B) transgenic
WT carrying 35S:AT4G30975 cassette (A975, the
unknown RNA gene) (Figure 4C and D), and transgenic
Arabidopsis WT carrying a 35S:miR156b cassette
(T156b) (Figure 4E and F) . Transcript analysis
showed that expression of bHLH069 was defective in
Figure 2 Carotenoid levels in sk156 mutant. (A) and (B)
Carotenoid levels in mature seeds. (C) and (D) Carotenoid levels in
leaves of 30-d-old plants. A Duncan's multiple range test showed
significant differences of the means (standard deviation) between
mutant lines and WT at p < 0.01 (**) and p < 0.05 (*).
both S468 and sk156 but enhanced in sk156
overexpressing bHLH096 (Figure 4A). Morphology was
indistinguishable between sk156 and sk156 over-expressing
bHLH096 (Figure 4B) and between the SALK mutant and
WT (data not shown). Additionally, over-expression of
the unknown RNA cDNA in WT (line A975) (Figure 4C)
did not produce the sk156 mutant phenotype (Figure 4D).
However, the 35S promoter-driven miR156b
overexpression resulted in morphological characteristics in line
T156b similar to those of sk156 (Figure 4F).
Carotenoid analysis further confirmed the role of
enhanced miR156b expression in generating the sk156
phenotypes (Figure 4G and H). No significant differences
in seed carotenoid abundance were found between the
defective bHLH069 mutant S468 and WT (p 0.05).
Complementation of sk156 with bHLH069 did not
restore sk156 seed carotenoid levels back to WT levels,
nor did over-expression of the unknown RNA in WT
result in enhanced seed carotenoid levels. Only in line
T156b overexpressing miR156b were seed carotenoid
levels raised to levels found in sk156 seeds (Figure 4G
and H). These data clearly indicate a role for miR156b in
the phenotypes of sk156.
Due to similar enhanced transcript levels of miR156b
and AT4G30975 in the sk156 mutant, yet unchanged
morphology and seed carotenoid accumulation in the
AT4G30975 over-expression lines (line A795, Table 1),
we examined the relationship between miR156b
transcripts and AT4G30975 cDNA. These two transcripts
are separated by a 17 bp intergenic fragment (TAIR,
version 9). Sequencing of cDNA obtained from the
leaves of 4-week-old WT plants revealed a miR156b
transcript which included both the 17 bp fragment
and the full segment of the AT4G30975 transcript
(sequencing data not shown). These data show that
enhanced expression of miR156 is the reason for the
phenotypic changes in the sk156 mutant line,
regardless of AT4G30975 co-transcription.
Suppression of SPL15 is involved in the sk156 phenotype
Arabidopsis Columbia has 11 SPL genes (two loci for
SPL13) that are regulated by miR156 [7,9]. To determine
which SPL geneswere most likely involved in altered
carotenoid profiles of sk156 seeds, mutant lines with
confirmed T-DNA insertion knockouts for all miR156-target
SPL genes (except SPL5 where mutants were unavailable)
were obtained (Additional file 2). The mutant lines were
compared for morphology and carotenoid differences
with their corresponding ecotype Columbia and
Wassilevskija controls. While levels of carotenoids were
modestly increased in three spl15 mutant lines (Figure 5A
insert), they were less affected in the remaining spl
mutants (Figure 5A). Even in the spl15 mutant lines, the
enhanced total carotenoid levels (21.5 1.7g/g.FW)
(Figure 5 insert) were not as high as the sum of each
individual carotenoid compound observed in sk156
(37.8 5.5g/g.FW) (Figure 6C and D).
The three different SPL15 knockout mutants,
CS852117, CS856815 and SALK_138712 (Figure 5A
insert) all exhibited reduced SPL15 transcript levels
similar to levels found in the sk156 mutant and the
miR156b over-expression line T156b (Figure 5B;
Table 1). However, other spl mutants had normal
cauline stem lengths (data not shown), while spl15
mutants had slightly stunted stems (Additional file 3)
as reported previously . Assays using rapid
Figure 3 T-DNA insertion site and affected genes in sk156 mutant. (A) Southern blot analysis of sk156 showing a single T-DNA insertion.
Lane 1, digested with BamHI. Lane 2, digested with HindIII. (B) Schematic of T-DNA inserted in the third intron of bHLH069 shown by r (empty
triangle). Boxes, exons; Lines, introns and intergenic regions. Thick grey arrows show promoter regions and their transcription direction. (C)
Transcript levels for miR156b and unknown RNA gene (AT4G30975) in sk156 relative to WT (set at 1) using qPCR. (D) Transcript levels for bHLH069
in sk156 relative to WT (set at 1) using qPCR. A Duncan's multiple range test in (C) and (D) showed significant differences of the means
( standard deviation) between sk156 and WT Col-4 at p < 0.01 (**) and p < 0.05 (*).
amplification of 5' complementary DNA ends (5'-RACE)
indicated that three cleavage sites exist within the region
of the native SPL15 transcript that is complementary to
the miR156 mature sequence, with one site being used
more frequently than the other two (Figure 6A).
Collectively, these results suggest that SPL15 is a direct target of
miR156 and that it plays a role in the morphology and
seed carotenoid phenotypes found in sk156 mutant line.
Expression of miR156-insensitive SPL15m affects the
phenotype of sk156
Since spl15 mutants had several phenotypes similar to
those of sk156, two different SPL15-complemented
sk156 lines were developed to determine whether the
WT phenotype could be restored in a miR156b-enhanced
sk156 background (Table 1). The first transgenic sk156
carried a 35S:SPL15n (native SPL15) cassette to avoid
any endogenous transcriptional regulation which might be
present if using a native SPL15 promoter (Figure 6A).
The second carried 35S: (mutated SPL15), which
contains 11 mutated sites within the SPL15 segment
complementary to mature miR156 so that SPL15m
becomes insensitive to miR156 regulation (Figure 6A).
PCR analysis using gene-specific primers confirmed
both pSKI015 T-DNA and either SPL15n or SPL15m
within these transgenic lines (Additional file 4).
Sequencing of these PCR products confirmed that all
SPL15m-expressing lines had a SPL15-mutated
segment (Figure 6A) as expected. SPL15 transcript levels
(sum of SPL15m and endogenous SPL15) within the
35S:SPL15m+ lines were increased by 86.2-fold to
617.4-fold compared to WT (Figure 6B). No significant
differences were found in the amount of lutein,
vialoxanthin, zeaxanthin and -carotene in seeds between these
35S:SPL15m-transformed plants and wild type control
plants (Figure 6C and D). The transgenic plants showed a
largely restored WT morphology in leaf and branch
numbers, bolting time, cauline stem length, flower and silique
size, and trichome number in reproductive shoots
and tissues (Figure 6E). Only leaf morphology was
different between transgenic and non-transgenic lines
(Figure 6F). This contrasted with miR156-controlled
4.3 fold less expression for SPL15
Modified pBI121, 35S:At4g30980 cDNA,
Modified pBI121, 35S:miR156b,
Modified pBI121, 35S:At4g30975 cDNA,
pDs-Lox, CS852117,Col background
pDs-Lox, CS856815, Col background
(miR156 sensitive), sk156 background
Modified pBI121, 35S:SPL15m
(miR156 insensitive), sk156 background
Modified pBI121, AS1:SPL15n
(miR156 sensitive), sk156 background
(miR156 insensitive), sk156 background
Modified pBI121, SPL15:SPL15m
(miR156 insensitive), sk156 background
Gene expression vs. WT control
91.4- and 95.8-fold higher expression
for miR156b, At4g30975
708.4 fold higher expression for bHLH069
2134.6 fold higher expression for miR156b
332.5 fold higher expression for At4g30975
4.3 fold less expression for SPL15
5.2 fold less expression for SPL15
4.7 fold less expression for SPL15
4.7 fold less expresson for SPL15.
22.8-613.6 fold higher expression of SPL15
3.2 fold less expression for SPL15
4.6 fold higher expression for SPL15
1.6 fold higher expression for SPL15
Slightly stunted cauline
stem and increased
Similar to sk156, slightly
changed leaf color
Table 1 Summary of transgenic and mutant Arabidopsis lines characterized in this study
pROK2, SALK_032468, Col-0 background
11.1 fold less expression for AT4G30980
35S:SPL15n+ lines (Table 1), which displayed sk156
Since constitutive expression of SPL15m
predominantly restored sk156 phenotypes, native SPL15n and
mutated SPL15m under the control of the young leaf
primordia promoter from the ASYMMETRIC LEAVES 1
(AS1) gene  were used to generate two additional
complemented sets of sk156 lines. This was to find out
whether SPL15 functions at the leaf primordia within
the shoot apex as occurs with certain SPL genes
controlling plastochron length . PCR analyses using
promoter-specific and gene-specific primers (Additional
file 5, see materials and methods) confirmed the
presence of the transgene in these transgenic plants
(Additional file 6). SPL15m transcript levels were increased
in AS1 promoter-driven SPL15m transgenic sk156 lines,
but not in AS1 promoter-SPL15n lines, compared to
WT and sk156 mutants (Figure 7A). As with SPL15m
expression directed by the 35S promoter, expression
of SPL15m under the control of the AS1 promoter in
the sk156 background restored WT morphology at
different growth stages, whereas AS1:SPL15n did not (Figure 7B,
C, and D). Moreover, the levels of seed carotenoids in
these ASI:SPL15m plants were not significantly different
from those in WT (p > 0.05), while seed carotenoid
abundance was significantly higher in plants carrying the
AS1:SPL15n cassette (p > 0.05) (Figure 7E and F). These
changes in the phenotype of transgenic sk156 plants
expressing the miR156-insensitive AS1:SPL15m
suggested that SPL15m could function independently of
miR156b and that expression of SPL15 is effective at the
leaf primordia within the shoot apex.
Since the native SPL15n appeared to be ineffective at
restoring the WT phenotype in the enhanced miR156b
environment of sk156, another set of complemented
sk156 lines was generated to express the
miR156-insensitive SPL15m under the control of the native SPL15
promoter (Table 1). Morphological traits and carotenoid
levels remained unchanged for these transgenic SPL15:
SPL15m plants compared to uncomplemented sk156,
except that the leaf shape was similarly elongated as were
35S:SPL15m plants compared to WT plants (Figure 6F).
These data suggested that overwhelming miR156b levels
within the sk156 mutant negatively regulated the
expression of the SPL15n under its native promoter and
prevented restoration of WT seed carotenoid abundance
Figure 4 Involvement of miR156b, bHLH069, and the unknown RNA AT4G30975 in the phenotypes of the sk156 mutant. (A) Transcript
levels of bHLH069 in WT (set at 1), sk156, mutant line S468 (SALK_032468), and in sk156 transformants complemented with a 35S:bHLH069
construct relative to WT (set at 1). Insert, schematic of T-DNA insertion site (triangle) in bHLH069 (AT4G30980) in S468 shows above. Exons (black
boxes), untranslated regions (light grey boxes), promoter direction (dark arrow). (B) Morphology of 45-d-old transgenic sk156 complemented with
35S:bHLH069. Sk156, and WT plants are also shown. (C) Transcript levels of unknown RNA gene AT4G30975 in WT (set at 1), sk156, and 35S:
AT4G30975cDNA-complemented sk156 (A975) plants. Schematic of AT4G30975 expression construct shows above. (D) Morphology of 35-d-old
transgenic plant A975 compared to sk156. (E) Transcript levels of miR156 in WT, sk156, and 35S:miR156b-complemented sk156 (T156b) plants.
Schematic of 35S:miR156b expression construct shows above. (F) Morphology of 40-d-old transgenic plants from sk156 and
35S:miR156b-overexpressing T156b lines compared to WT. (G) and (H) Seed carotenoid levels in sk156, T156b, A975, S468, 35S:bHLH069/sk156, and WT plants. A
Duncan's multiple range test showed significant differences of the means ( standard deviation) compared to WT at p < 0.05. Means containing
the same letter for the same compounds are not significantly different.
Figure 5 Involvement of SPL15 in phenotypic alteration of
sk156. (A) Carotenoid levels in SPL loss-of-function mutants
specifying nine SPL genes, including SPL15. SPL15 SALK lines in
insert show details on specific carotenoid changes. (B) SPL15
transcript levels in sk156, SPL15-related Salk lines, and T156b relative
to WT (Col, set at 1). Insert shows schematic diagrams of T-DNA
insertion sites r in SPL15 SALK mutants CS2117, CS6815 and S8712.
Duncan's multiple range test showed significant differences (*) of
the means ( standard deviation) between sk156 and WT (Col or
WS) at p < 0.05.
and plant morphology. Moreover, the inability of SPL15:
SPL15m plants to restore most of the WT phenotype as
did 35S:SPL15m+ lines suggested that the enhanced seed
carotenoid level and morphological traits observed in
the sk156 mutant were the collective result of miR156
suppression of at least some other SPL genes rather than
SPL15 alone and that SPL15 functions redundantly with
some other SPL gene products.
Transcription of miR156b is affected by over-expression
Wu and Poethig  reported earlier that expression of
miR156-insensitive SPL3 under the control of the
CaMV35S promoter causes reduced levels of miR156
transcripts in Arabidopsis. To investigate the ability of
SPL15m to affect expression of its regulatory miR156
genes, we examined miR156b transcript levels in the
sk156 mutant and in complemented sk156 carrying the
35S:SPL15m cassette (Table 1). Primers for premature
miR156b rather than mature miR156 sequence were
used so that measurement of transcripts originating
from other miR156 genes would be excluded. In
30-dayold plants, miR156b premature transcript levels were
reduced from 82.2-fold above WT levels in sk156 leaves
down to a mean of 7 5.5 fold above WT in the leaves
of these complemented lines, with a minimum of
1.4fold in line 7 and a maximum of 19.6-fold in line 9
(Figure 6G). Thus, miR156b transcription was depressed
substantially such that ~50% of the complemented lines
had WT levels. A reduction of miR156b transcript level
concurrent with an increase in SPL15m transcripts
suggests that SPL15 may have two regulatory functions; one
controlling feedback regulation of its cognate regulator
miR156 and one controlling downstream genes, such as
miR172, APETALA2-LIKE (AP2-like) transcription
factors TOE1, and TOE2, in the miR156 controlled gene
Physical interaction between the SPL15 DNA binding
domain and GCAT motifs in the miR156b promoter
To investigate whether SPL15 interacts directly with the
miR156b promoter to affect transcription, we identified
putative SPL15 binding sites in the miR156b promoter based
on the consensus sequence for SBP binding domain
(Figure 8A). To define the consensus sequence, we
compared cis elements previously identified as SBP box
interacting sequences in Chlamydomonas reinhardtii [28,29],
Antirrhinum majus  and A. thaliana [5,11,31,32] using
WebLogo . The consensus SBP domain binding
sequence was determined to be 5-NNGTACR-3, where
frequently N = C and R = A (Figure 8A). This consensus
sequence was used to search for putative SPL binding sites
in the promoters of miR156 genes. Putative SPL binding
sites with a GTAC core sequence were repeatedly present
in the miR156b promoter [-1 to -1500], according to the
gene transcript start site revealed by Schwab . Of these,
a region containing three repeats of the core sequence
appeared between -200 bp to -220 bp (Figure 8B)
suggesting that miR156b expression could be directly controlled by
SPL15. This was tested using a His-tagged SPL15
recombinant SBP DNA-binding domain (Figure 8C and D) in an
electrophoretic mobility shift assay with a labeled DNA
fragment (46-mer) containing a motif from -200 bp to -220
bp from the miR156b promoter. The recombinant SBP
domain of SPL15n specifically bound to this region of the
miR156b promoter to yield a less mobile promoter
fragment (Figure 8E), and competition with 100-fold unlabeled
miR156b promoter DNA was required to displace the SBP
domain. This result suggested that a direct physical
Figure 6 Expression of mutated SPL15m in sk156 results restores a WT phenotype and down regulates miR156b transcription. (A)
Complementarity of miR156 with SPL15 sequences. Arrows show three cleavage sites (one used 10 out of 13 times) in SPL15 mRNA due to
interaction with miR156b. SPL15m shows 11 nucleotides mutated in DNA sequence but unchanged in amino acid sequence. (B) SPL15 transcript
levels in independent transgenic sk156 lines carrying 35S:SPL15m compared to WT (set at 1) and sk156. Error bars are standard deviations of the
means. (C) and (D) Restoration of WT-like carotenoid levels in dry seeds of transgenic sk156 expressing SPL15m. Cryptoxanthin was undetectable
in the mutant. (E) Restoration of WT-like plant growth in 35-d-old plants of sk156 expressing SPL15m. (F) Distinctive elongated leaf morphology of
SPL15m-transformed plants compared to WT plants. (G) miR156b transcript levels in different transgenic sk156 lines expressing SPL15m compared
to WT (set at 1) and sk156. A Duncan's multiple range test was conducted to separate significantly different means. Panels B, G, standard
deviation; Panels C, D, standard error; p < 0.01 (**) and p < 0.05 (*).
interaction can occur between the SPL15 DNA binding
domain and the miR156b promoter through the GTAC motif.
Enhanced expression of miR156b in sk156
In this study, we highlight a new Arabidopsis mutant,
sk156, with enhanced expression of miR156b due to the
insertion of a T-DNA activation tag. The elevated miR156b
transcript levels in sk156 are responsible for the full
spectrum of morphological phenotypes observed for this
mutant. Increased seed carotenoid levels and enhanced
branching are consistent with phenotypes observed in B.
napus expressing AtmiR156b under the control of the
CaMV35S promoter . These results confirm the role for
miR156b in seed carotenoid accumulation, and are in
agreement with morphological changes reported in earlier
Differences in transcriptional regulation for the native
miR156 promoter, the CaMV35S promoter, and the
enhanced miR156b native promoter, in addition to different
T-DNA insertion sites, can lead to substantial differences in
transcript levels between individual transgenic lines and,
consequently, to variation in phenotype. For example,
miR156b transcription driven by the CaVM 35S promoter
was more than 2000-fold higher than native miR156b
transcription in WT plants, whereas miR156b transcription in
the sk156 mutant was ~80-fold higher than in WT plants.
Such differences in miR156b expression levels may result in
distinct phenotypes between the sk156 mutant and 35S:
miR156b lines. For example, numerous small rosettes and
Figure 7 Leaf primodium-dependent miR156-insensitive SPL15m restores WT phenotypes when expressed in sk156. The sk156
background was transformed with native miR156-sensitive SPL15n or miR156-insensitive SPL15m expression cassettes each controlled by either an
AS1 or 35S promoter. (A) SPL15 transcript levels by qPCR ( standard deviation) in transgenic sk156 plants complemented with AS1:SPL15m, AS1:
SPL15n, 35S:SPL15n or SPL15:SPL15m compared to WT (set at 1) and sk156 (p < 0.05). (B) and (C) Morphology at bolting (32 d) and maturity (50 d)
for plant expressing SPL15n or SPL15m under the control of AS1 promoter compared to sk156. (D) Morphology of 42 d flowering lines for sk156
plant carrying 35S:SPL15n or SPL15:SPL15m compared to sk156. (E) and (F) Carotenoid levels ( standard error) in seeds of sk156 complemented
with AS1:SPL15m, AS1:SPL15n, 35S:SPL15n or SPL15:SPL15m compared to those of WT and sk156. Panels A, E, F: A Duncan's multiple range test was
conducted to separate significantly different means for more than 10 independent transgenic lines (each measured with triplicated seed batches)
relative to WT plants at p < 0.05 (*).
tiny leaves were produced in sk156 (Figure 1C), and
comparatively much smaller and many more rosette leaves were
noted in the 35S:miR156b lines in this and previous studies
Strigolactone-independent seed carotenoid increase in
Our data indicated that morphological traits and seed
carotenoid profiles are affected in sk156. A link between
altered carotenoid profiles and branching was previously
observed in max mutants [17,19] and in an Arabidopsis
histone methyltransferase (SDG8)-defective mutant .
This was largely due to the effect on the biosynthesis of
carotenoid-derived strigolactone branching inhibitors
[20,21]. In the current study, we also demonstrated that
the elevated levels of seed carotenoids in Arabidopsis
mutant sk156 were due to the enhancer-driven
expression of miR156b. However, morphological phenotypes of
the T1 progeny of sk156 crossed with WT (in which
CCD7 and CCD8 are transcribed normally) and of
Figure 8 Interaction of the miR156 SBP-binding motif (GTAC core sequence) with the SBP protein domain of SPL15. (A) Consensus DNA
sequence present in the promoter region of six previously reported genes interacting with SBP DNA binding domains. CCR1-bx represents the
GTAC motif binding to SBP protein CCR1 from Chlamydomonas reinhardtii [28,29]; SBP1/2-bx1 represents the GTAC motif binding to SBP1 and
SBP2 in Antirrhinum majus ; SPL3-bx1 and -bx2 [5,11], SPL7-bx ; and SPL14-bx  represents the GTAC motif binding to corresponding
SPL proteins from Arabidopsis thaliana. Yellow boxes, 100% conserved; green boxes, 67-83% conserved. The degree of conservation is indicated in
the schematic by the height of the letters (measured as bits). (B) GTAC core repeats present in the miR156b promoter sequence (from bp -1 to
-1700). (C) SDS-PAGE analysis of His-tagged recombinant SBP peptide expressed in E. coli. Lane 1, protein marker; lane 2, protein extract of
noninduced E. coli cells carrying the SBP domain in pET28a; lane 3, protein extract of induced E. coli carrying the SBP domain. (D) Western blot of
resolved proteins in C using anti-His antibody. (E) Electrophoretic mobility shift assay illustrating His-tagged SBP peptide bound to a labeled
miR156b promoter fragment containing tandem repeats of the GTAC core. Lane 1, labeled DNA plus SBP peptide. lane 2, labeled DNA only; lane
3, labeled DNA plus SBP protein and 100-fold excess unlabeled DNA. Black arrow shows the shifted band in lane 1.
transgenic WT overexpressing CCD7 or CCD8 (data not
shown) remained almost the same as in sk156.
Exogenous application of the artificial strigolactone GR24
(2'epi-5-deoxystrigol) to sk156 seedlings for six weeks by
supplementing the chemical to the in vitro plant
growing in MS media did not affect sk156 phenotypes
(data not shown). These data suggest that ectopic
expression of the miR156b-induced branching phenotype
in sk156 might not be directly related to the
strigolactone pathway. In silico analysis revealed that no known
carotenoid biosynthesis or catabolism genes in
Arabidopsis possess sequences complimentary to the mature
sequence of miR156b . As well, transcript levels of
-ring carotenoid hydroxylase, E-ring carotene
hydroxylase, lycopene -cyclase, lycopene E-cyclase, phytoene
desaturase, phytoene synthase or -carotene desaturase
were not significantly different in leaves and siliques of
sk156 and WT (data not shown). Thus, miR156b likely
affects the seed carotenoid pathway indirectly through the
regulation of SPL networks as demonstrated in the
miR156-regulated accumulation of anthocyanin via
several SPL genes . However, we cannot exclude the
possibility of changes to seed carotenoid enzyme
activities in the absence of transcription changes, such as
occured with phytoene synthase in etiolated Arabidopsis
. Also, the impact on seed carotenoid accumulation
may not necessarily be due to biosynthesis, but rather to
improved carotenoid sequestration and storage as
demonstrated in the Or mutant of Brassica oleracea . These
possibilities point to the need for additional investigation
to unravel the biological basis for increased carotenoid
accumulation in sk156 seeds.
In sk156, a substantial increase in seed carotenoid
levels was detected in all except cryptoxanthin. This
minor carotenoid, however, was reduced in sk156 seeds.
Cryptoxanthin may be more efficiently converted to
zeaxanthin in sk156 than in WT Arabidopsis. This
possibility could arise potentially due to differences in
transcription of the related genes between WT and
sk156 and should be tested in the future. For example,
in Zea mays, a -carotene hydroxylase variant,
ZmBCH1, converts -carotene to -cryptoxanthin and
zeaxanthin, whereas ZmBCH2 can only convert
carotene to -cryptoxanthin . Also, in Arabidopsis
two variants of -carotene hydroxylase were found to
function differently .
A slight decrease in carotenoid abundance was
observed in leaves of the sk156 line even though
carotenoid levels were increased in sk156 seeds. Transgenic
B. napus in which the gene encoding lycopene E-cyclase
was constitutively down-regulated also showed increased
carotenoids in seeds, but not in leaves . Different
mechanisms control carotenoid metabolism in green
and non-green tissues, including seeds [42-44]. In
developing chloroplasts, the synthesis of carotenoids, such as
lutein, -carotene, zeaxanthin and violaxanthin, is
regulated in concert with the light-regulated assembly of
light-receiving antennae and photosystem centers into
which the carotenoids are integrated [45,46]. In contrast,
carotenoid compounds in non-green tissue plastids vary
widely in quantity and composition and their synthesis
is not necessarily regulated similarly to chloroplasts. In
seeds, carotenoids are stored in elaioplasts (lipid storing
plastids) or amyloplasts (starch storing plastids) [42,47].
Evaluating the impact of miR156b on the transcription
of genes specifying differentiation into these
carotenoidrich plastid types could lead us to understand why
miR156b regulates carotenoid accumulation differently
in seeds and leaves of sk156.
SPL15 regulation of the morphology and carotenoid
alterations in sk156
Our 5 RACE assays showed that SPL15 mRNA was
cleaved at three cleavage sites in the segment located
near the middle of the coding sequence and is
complementary to mature miR156, suggesting a role for
miR156-directed cleavage in SPL15 transcript
processing. However, distinct morphologies were often difficult
to find between many of the Arabidopsis mutants
defective in individual SPL genes both in our study and in
others . In the present study, spl15 mutants exhibited
slightly stunted shoot growth and a modest increase in
carotenoid levels, which were some (but not the full
spectrum) of the phenotypes of sk156, while the other
spl mutants examined in this study did not exhibit any
discernible phenotypes. Consistently, phenotypes
specified by the sk156 mutant were largely restored to WT
phenotypes in sk156 complemented with SPL15m
expressed under the control of the CaVM35S promoter
or the AS1 promoter. These pieces of evidence clearly
suggest that miR156 suppresses SPL15 in the sk156
mutant and causes much of its phenotypic iteration.
Other SPL genes must contribute somewhat to the
sk156 mutant phenotype development, since spl15
mutants did not show the entire spectrum of sk156
phenotypes and sk156 was not fully restored to a WT
phenotype by SPL15m expressed from a native SPL15
promoter. These data not only indicate that SPL15 is
functionally redundant with other miR156-targeted SPL
genes, but that this redundancy probably is limited by
the endogenous regulation of temporal and spatial gene
expression. Our data derived from AS1 promoter-driven
SPL15m experiments show that the leaf primordium
within the shoot apex is a crucial site for SPL15m
expression to restore sk156 phenotypes. This is consistent
with previous findings that the shoot apex is a
predominant expression site for some SPL genes [2,5,13] and
that AS1 promoter-driven miR156 promotes leaf
initiation by suppressing SPL genes at the shoot apical
meristem . AS1 is expressed only in emerging lateral
organ primordia, and in seeds its expression is
detectable only in limited subepidermal cells corresponding to
cotyledon initials at the heart stage . In our hands,
AS1:GUS + plants did not show visible GUS staining in
seeds but did show staining in shoot tips, suggesting that
SPL15m transcript level might not be significantly
elevated in AS1:SPL15m+ seeds compared to sk156. The
morphological changes in transgenic sk156 expressing
35S/AS1:SPL15m point to functional redundancy of
SPL15 with at least SPL9 and SPL10 on plastochron
length , SPL9 on shoot development , and SPL3,
SPL4, and SPL5 on flowering stage transition [4,12].
Expression of miR156b-insensitive SPL15m modifies
leaf shape in the sk156 lines carrying 35S:SPL15m or
SPL15p:SPL15m constructs. A role for SPL15 in leaf
morphological traits was also revealed by Usami et al
. The enhancement of miR156b expression in sk156
possibly prevents the plants from maintaining sufficient
SPL transcript levels required for normal control of leaf
architecture, including those from SPL15. These
findings are consistent with reduced expression of 10 SPL
genes in the inflorescences of Arabidopsis lines
overexpressing miR156b . Expression of SPL15 gene in
leaf primodia was also important for rescuing WT-like
seed carotenoid and morphology traits in sk156. This
was supported partially by the finding that leaf
primodia are a site where SPL factors control the rate of leaf
SPL15 feedback regulation of miR156b
Feedback loops, in which a miRNA-targeted
transcription factor either increases or decreases the expression
of its cognate miRNA, have been reported in animals
[50,51]. However, the mechanism underlying this
feedback regulation is unclear, particularly in plants. In our
hands, complementation of sk156 with SPL15m leads to
increased levels of SPL15 transcripts and decreased
levels of miR156b transcripts. The reduction in miR156b
expression due to increased SPL15 was consistent with
the inverse correlation of SPL3 and mature miR156
transcripts in WT Arabidopsis at the early vegetative stages
(19- and 26-d-old) . These data suggest that negative
feedback regulation by SPL15 may exist for miR156b. A
recent study on the regulation of miR156a and miR172b
in early vegetative stages proposed feedback regulation
between the miRNAs and their targets but did not
further investigate the underlying mechanism .
All SPL family members contain a conserved SBP DNA
binding domain [5,30] with variations outside the domain.
Moreover, DNA sequences in promoters of SPL-regulated
genes contain a GTAC core [5,28,29,31,32,52], which
is a key element for transcription regulation . Our
gel shift assay demonstrated a direct physical
interaction between the SPL15 SBP domain and the
promoter region of miR156b containing three repeats of
the GTAC core sequence. This suggests a mechanism
for the underlying feedback regulation of miR156b by
its target SPL15. Support for this finding comes from
reports by Birkenbihl et al  and Yamasaki et al
 which indicated that such an interaction also
exists between the SBP domain for SPL1, -3, -7 and
-8 and the GTAC element.
In the present study we showcase a new mutant, sk156,
in which a T-DNA insert containing four CaMV35S
enhancers hyper-induced miR156b. We documented
phenotypes for several sk156 lines complemented with
different expression cassettes for miR156b-sensitive and
miR156b-insensitive SPL15, and showed for the first
time a direct interaction between SPL15 and miR156b.
These data and the inverse pattern of miR156b and
SPL15 expression in mutant sk156 lead us to conclude
that negative feedback regulation of miR156b by SPL15
exists. A second finding is that miR156b regulates seed
carotenoid abundance in Arabidopsis differently than in
leaves although the underlying molecular basis remains
a subject for further investigation.
Plants and growth conditions
The sk156 mutant was isolated from a T-DNA
activation-tagged mutant population of Arabidopsis Col-4
. The population was generated at the AAFC
Saskatoon Research Centre using the pSKI015 binary vector
containing a T-DNA with a Bar gene and 4 tandem
CaMV enhancers . SALK and FLAG T-DNA
insertion lines with knockouts in SPL genes were obtained
from the Arabidopsis Biological Resource Center and
the FST (flanking insertion sites) project
(www.arabidopsis.org; Additional file 2). max mutants were kindly
provided by Dr. M-S Peng (University of Guelph) and
transgenic Arabidopsis lines over-expressing miR156b
under the control of the CaMV35S promoter were
provided by Dr. R. S. Poethig (University of Pennsylvania).
To recover homozygous mutant lines, segregating
offspring grown were grown on half-strength
MurashigeSkoog (MS) basal medium (Sigma, M5519-50L) with
0.8% agar and a selective agent (7.5 mg/L
phosphinothricin or 50 mg/L kanamycin) and were screened by PCR
using primers specific for each insertion site (Additional
file 5). Homozygous plants were transplanted into
growth chamber pots for further characterization.
For phenotypic analysis, plants were grown in 32-well
flats containing Co-Co Mix soil mixture consisting of
compacted coconut fibre/peat moss/vermiculite (1/3/3,
v/v/v) and 15-9-12 "Osmocote PLUS" controlled release
fertilizer (Scotts Company LLC). Plant age was recorded
from the time seeds were imbibed. All plants were kept
in a controlled environment growth chamber under a
16/8 h light/dark photoperiod with a light intensity of
230 E/min/m2 and temperature of 20oC/17oC. Leaf and
branch number means were calculated from 30
individual plants of mutant or WT lines. A Duncan's multiple
range test was conducted to show significant differences
between the means at p < 0.01 and p < 0.05.
Carotenoid extraction and quantification
For carotenoid analysis, leaves of 28-day-old plants were
excised and samples immediately ground in liquid
nitrogen. Mature seeds harvested from different plants were
kept at -80oC for further analysis. Tissues were
maintained at -80oC before extraction to minimize
carotenoid degradation. Approximately 100 mg of leaf tissue
powder and 150 mg of homogenized seeds were used
for carotenoid extraction. Carotenoid extraction and
HPLC quantitative analysis were carried out as
previously described for seeds . HPLC chromatograph
peaks were identified by comparing their retention
times and absorption spectra to authentic standards,
and quantified using standard curves corresponding to
carotenoid compounds. Pure chemical standards for
-carotene and lutein were purchased from Sigma.
cryptoxanthin, zeaxanthin and violaxanthin were
purchased from CaroteNature (Lupsingen, Switzerland).
Each sample was pooled from the leaves or seeds of
6-10 different plants. Six replicates (two biological
replicates with three technical replicates) were used
for carotenoid analysis. A Duncan's multiple range test
was used to separate statistically different means at
p < 0.01 and p < 0.05.
Molecular characterization of the sk156 mutant
Plant genomic DNA was extracted to determine T-DNA
copy number in the sk156 mutant. Southern analysis
was conducted using 15 g of genomic DNA digested
with BamHI and HindIII. The 1.4 kb fragment was
excised from a pSKI015 plasmid using HindIII and
EcoRI, labeled with 32P, and used as a hybridization
probe. To identify the T-DNA insertion site of sk156,
genomic DNA was digested with each of four blunt-end
restriction enzymes provided in the GenomeWalk kit
(Clontech), ligated with adaptors, and flanking regions
amplified by nested PCR with the T-DNA specific
primers pSKI015-GW-LB1 and pSKI015-GW-LB2
according to the manufacturers instructions (Additional file 5).
Cloned GenomeWalk PCR products were sequenced
using the pSKI015-GW-LB2 primer (Additional file 5)
and the T-DNA insertion site was determined by
BLAST analysis to the TAIR sequence database (Version 9)
segregation of plants with T-DNA inserts was
completed in triplicate by recording T-DNA insertion
and the number of surviving seedlings that
geminated out of ~100 seeds growing in half-strength MS
media containing 7.5 mg/L phosphinotricin or 50 mg/L
kanamycin, followed by 2 analysis.
Constructs and plant transformation
Total RNA for cDNA synthesis was extracted from
3week-old Arabidopsis thaliana ecotype Columbia plants
using the QIAGEN RNeasy kit (Qiagen) according to
the manufacturers instructions. DNase I was used for
on-column DNA digestion to minimize genomic DNA
contamination. First-strand cDNA was synthesized by
reverse transcription of 300 ng of total RNA in a final
reaction volume of 20 L using random primers and
200 units of SuperScript II Reverse Transcriptase
(Invitrogen). Full length cDNA of miR156b, SPL15, AT4G30975
and bHLH069 was amplified using PlatinumW Taq High
Fidelity DNA Polymerase (Invitrogen) and gene specific
primer pairs (miR156b_XbaI_F/ miR156b_SacI_R; pXbaI_
SPL15F/ pSacI _SPL15R; p30975BamHI_F /p30975SacI_R;
and pbHLH_F / pbHLH_R, respectively), with addition of
restriction enzyme sites at the 5 and 3 ends of the genes
(Additional file 5). SPL15m was generated by introducing
11 mutations into the predicted miR156 binding site using
the PCR primers pSPL15m851F and pSPL15m865R
containing mutated sequences (Additional file 5). The AS1
promoter was isolated from A. thaliana Columbia
genomic DNA using pAS1_HindIII_F and pAS1_XbaI_R
(Additional file 5) according to Wang et al , while a 3 kb
DNA fragment upstream of the SPL15 transcription start
site including both the proximal promoter and distal
promoter regions  was amplified by PCR using the
primers pSPL15Pr3_HindIII_F and pSPL15Pr3_XbaI_R
(Additional file 5) and used as the native SPL15 promoter.
Amplified PCR fragments were cloned into
pCR2.1TOPO vector (Invitrogen) and verified by DNA
sequencing. The fragments were retrieved with corresponding
restriction enzymes and inserted into the binary vector
pBI121 digested with the same pair of restriction enzymes
(replacing the original gusA gene or CaMV35S promoter
in the vector). The resulting binary expression vectors
contained an nptII gene and transgenes under the control
of either the CaMV35S promoter, the AS1 promoter, or
the native SPL15 promoter and were introduced into
Agrobacterium tumefaciens GV3101pMP90 and used to
transform Arabidopsis using the standard floral dip
method. Putative transgenic lines that survived antibiotic
selection were transplanted into the greenhouse for
5RACE was carried out using the FirstChoice RLM
RACE Kit (Ambion) following the manufacturer's
instructions specially for the detection of miRNA
degradation products . Briefly, total RNA was isolated
from 3-week-old seedlings as described above. Five
micrograms of total RNA was ligated to the RNA
adapter and a random-primed reverse transcription
reaction was performed to synthesize cDNA. A second
round of PCR was carried out using a nested adapter
primer and primers specific for SPL15 (Additional file
5). The RACE products were cloned into the pGEM T
Easy vector (Promega) for sequencing.
Quantitative Real Time PCR analysis
Total RNA extraction, on-column genomic DNA
digestion, and first-strand cDNA synthesis were conducted as
described above. Real time quantitative RT-PCR (qPCR)
analyses were performed with gene specific primers
listed in supplemental Table S1. To quantify miR156b,
primers based on the stem sequence of the pre-miR156b
hairpin structure were designed to measure premature
miR156b according to Schmittgen et al.  (Additional
file 5). qPCR mixtures contained 10 l of diluted cDNA,
12.5 l of 2X SYBR Green qPCR Master Mix (Cat No.
11735-040, Invitrogen) and 200 nM of each
genespecific primer in a final volume of 25 l. qPCR
reactions were conducted using the StepOne Plus system
and software (Applied Biosystems) using a relative
standard curve method and default reaction parameters.
The relative index of the standard curve was over 98%.
Control PCR reactions with no templates were also
performed for each primer pair. The specificity of
amplicons was verified by melting curve analysis (60 to 95oC)
after 40 cycles and by agarose gel electrophoresis. All
samples were assayed in triplicate from two independent
RNA preparations. Mean expression values of all
replicates were calculated and normalized to the expression
of PEROXIN 4 (PEX4, set at 1), a suitable endogenous
reference gene because of its stable and low level
expression in Arabidopsis . All PCR reactions displayed
efficiencies between 87 and 115%. Normalized means
were analyzed for significant differences by a Duncans
test (p < 0.05).
Expression and extraction of SBP-domains
For recombinant protein expression in Escherichia coli,
a coding sequence fragment encoding the SPL15 SBP
domain (80 amino acid residues) plus 5 amino acid
residues both upstream and downstream  was amplified
by PCR using the primers p15SBP-BamHI-F and
p15SBP-SalI-R with addition of BamHI at the 5 end and
SalI and a TGA stop codon at the 3 end (Additional file
5). The PCR product was digested with BamHI and SalI
and cloned into the pET28a vector (Novagen) between
the BamHI and SalI sites. The resulting chimeric
recombinant protein fused with a His tag (16.3kD) was
expressed in E. coli strain Rosetta 2(DE3) pLacI
(Clontech) by induction with 0.8 mM
isopropyl--d-thiogalactopyranoside. Cells were harvested by centrifugation and
resuspended in BugBusterW Master Mix (Novagen) to
lyse the cells. Recombinant His-tagged SBP proteins
were detected in the inclusion bodies using anti-His
antibody (G-18) (Santa Cruz Biotechnology) in Western
blot assays. Proteins in inclusion bodies were solubilized
with 1.5% sarkosyl (N-laurylsarcosine) according to
Frangioni and Neel , and recombinant SBP protein
recovered and re-folded using a protein refolding kit
(TB234, Novagen) according to the manufacturers
instructions. Protein concentration was measured in
lysates by a NanoDrop 1000 spectrophotometer (Thermo
Fisher Scientific) and proteins separated in 13% SDS
polyacrylamide gels. The protein was stored at 4C until
use in gel shift assays.
Electrophoretic mobility shift assays
The ability of SPL15 SBP to bind to the miR156b
promoter DNA was examined using electrophoretic
mobility shift assays. A 46 residue DNA fragment, which
included the three repeated GTAC core sequences close
to the transcriptional start site of miR156b (shown in
Figure 8B), was synthesized using primers R156b-bx1-U
and R156b-bx1-L (Additional file 5), hybridized and
labeled with digoxigenin using a DIG Gel Shift kit
(second generation) (Roche). The labeled DNA fragment
(~20 fmol) was incubated for 30 min at 25C with or
without the SPL15 SBP protein (~500 ng) in 20 L of
reaction buffer containing 10 mM Tris (pH 7.5),
50 mM KCl, 5 mM MgCl, 5 mM DTT, 2.5% glycerol,
and 0.05% NP-40. Then, a 100-fold excess of the
unlabeled promoter DNA fragment was added to the
reactions. After incubation, the mixtures were separated by
polyacrylamide electrophoresis (7.5% gel) at 4oC for
1.5 h (0.8 V/cm2) in 0.5 X TBE (44.5 mM Tris base,
44.5 mM boric acid, and 1 mM EDTA at pH 8.0).
DNA was blotted onto a nylon membrane and
mobility changes for the labeled miR156b promoter
fragment detected with digoxigenin-specific antibodies.
Additional file 1: Schematic diagram of disrupted carotenoid
cleavage dioxygenase genes CCD7 and CCD8 in the max3-9 and
max4-1 mutants used in this study. Boxes represent exons and lines
represent introns. Triangles show the T-DNA insertion sites. The max
mutants were previously reported by (Booker et al. 2004).
Additional file 2: SALK and FLAG T-DNA insertion lines for
miR156targeted SPL genes.
Additional file 3: Reduced lengths of cauline stem basal internodes
in three spl15 mutants compared with WT Arabidopsis. Length of the
cauline stem basal inter-node was measured from the rosette core up to
the first visible basal node for WT, three spl mutants and sk156 plants
grown for 6 weeks.
Additional file 4: Confirmation by PCR of transgene presence in
sk156 lines transformed with a 35S:SPL15m gene. The miR156
insensitive SPL15m contained 11 mutated nucleotides as described in
Materials and Methods. Primer sequences are listed in Additional file 5.
(A) Activation-tag from pSKI015 T-DNA detected in transgenic sk156
plants carrying 35S:SPL15m cassette (lanes 0-11) and in the sk156
background alone (lanes 12 and 13). Primers SK2222-F (430bp upstream)
and SK2222-R (1830bp downstream) flanking the T-DNA insertion site
and primer pSKI015-GW-LB2 (439bp to the T-DNA left border) were used
to detect the insert. In WT lane, no T-DNA insert was detected and only a
fragment close to 1.9kb was present due to genomic DNA amplified with
the primers flanking the T-DNA insertion site. In homozygous sk156
plants which did not carry 35S:SPL15m, a single T-DNA fragment (869bp)
was generated. 1kb, 1-kb Plus DNA ladder (Invitrogen); WT, Col-4;
pSKI015, plasmid containing the activation tag present in sk156. (B)
Transgene SPL15m detected in the transgenic sk156 lines carrying 35S:
SPL15m (lanes 0-14), but not in sk156 alone using primers 35SF3 and
Additional file 5: Primers used in this study.
Additional file 6: PCR confirmation of transgene presence in four
different miR156-sensitive or miR156-insensitive transgenic
Arabidopsis populations used in this manuscript. (A) Lanes 2-8, PCR
product (818bp) for 7 transgenic plants carrying a 35S:AT4G30975 cassette
in a WT background using primers 35S-F3 and p795-3R. P, binary plasmid
pBI121 containing 35S:AT4G30975 as a positive control. WT, Col-4. (B), (C)
and (D) Transgene PCR product (636bp) carrying AS1:SPL15m, AS1:SPL15n
and SPL:SPL15m cassettes in a sk156 background, respectively, using
primers SPL15-871F and NosTer-R6. P, plasmid containing 35S:SPL15 as a
positive control. Black arrows points to the DNA marker. Primer
sequences are listed in Additional file 5. 1kb, 1-kb Plus DNA ladder
SW designed the experiments and conducted the majority of the
experimentation, analyzed data, and drafted the manuscript; IAP Parkin
constructed and provided the Arabidopsis activation-tagged population; BY
was involved in construction of SPL15 binary vectors; MJG performed
computational sequence analysis of the miR156b promoter binding motif;
MYG, and AH provided critical feedback on experimental concepts. MYG,
GGK, DDH and AH revised the manuscript. All authors read and approved
the final manuscript.
Submitting author: Shu Wei, PhD in Plant Sciences and currently Professor in
Plant Molecular Biology and Biotechnology at Anhui Agricultural University,
China, working on, but not limited to plant microRNA regulated gene
networks and metabolic pathway engineering.
We thank Dr. Mingsheng Peng, University of Guelph, for kindly providing us
with the max mutants and Dr. R. S. Poethig, University of Pennsylvania, for
transgenic Arabidopsis plants over-expressing the miR156b gene. GR24
(2'epi-5-deoxystrigol) was a kind gift from Professor S. Yamaguchi at the RIKEN
Plant Science Center, Japan and Prof. M. Sasaki at Kumamoto University,
Japan. This work was supported by the Genome Canada-funded project
Designing Oilseeds for Tomorrows Markets.
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