Gradual Increase of miR156 Regulates Temporal
Expression Changes of Numerous Genes during Leaf
Development in Rice1[C][W][OA]
Kabin Xie2, Jianqiang Shen, Xin Hou, Jialing Yao, Xianghua Li, Jinghua Xiao, and Lizhong Xiong*
National Key Laboratory for Crop Genetic Improvement and National Center of Plant Gene Research
(Wuhan), Huazhong Agricultural University, Wuhan 430070, China
The highly conserved plant microRNA, miR156, is an essential regulator for plant development. In Arabidopsis (Arabidopsis
thaliana), miR156 modulates phase changing through its temporal expression in the shoot. In contrast to the gradual decrease
over time in the shoot (or whole plant), we found that the miR156 level in rice (Oryza sativa) gradually increased from young
leaf to old leaf after the juvenile stage. However, the miR156-targeted rice SQUAMOSA-promoter binding-like (SPL)
transcription factors were either dominantly expressed in young leaves or not changed over the time of leaf growth. A
comparison of the transcriptomes of early-emerged old leaves and later-emerged young leaves from wild-type and miR156
overexpression (miR156-OE) rice lines found that expression levels of 3,008 genes were affected in miR156-OE leaves. Analysis
of temporal expression changes of these genes suggested that miR156 regulates gene expression in a leaf age-dependent
manner, and miR156-OE attenuated the temporal changes of 2,660 genes. Interestingly, seven conserved plant microRNAs also
showed temporal changes from young to old leaves, and miR156-OE also attenuated the temporal changes of six microRNAs.
Consistent with global gene expression changes, miR156-OE plants resulted in dramatic changes including precocious leaf
maturation and rapid leaf/tiller initiation. Our results indicate that another gradient of miR156 is present over time, a gradual
increase during leaf growth, in addition to the gradual decrease during shoot growth. Gradually increased miR156 expression
in the leaf might be essential for regulating the temporal expression of genes involved in leaf development.
The microRNAs, a class of 20- to 24-nucleotide small
RNAs, play essential roles in developmental regulation in plants and animals. These microRNAs are
perfectly or imperfectly complementary to their targeted mRNAs and repress the target genes by mRNA
cleavage or translation suppression. A few microRNAs and their targeted genes are conserved among
plant species and likely regulate basic processes for
plants (Axtell and Bartel, 2005; Axtell et al., 2007;
Voinnet, 2009). Temporal or spatial accumulation of
these conserved microRNAs is essential for proper
development in plants. In Arabidopsis (Arabidopsis
1
This work was supported by the National Natural Science
Foundation of China (grant nos. 30725021 and 30921091), the National Program on High Technology Development (grant no.
2012AA10A303), and the Project from the Ministry of Agriculture
of China for Transgenic Research (grant no. 2011ZX08009–004).
2
Present address: Department of Plant Pathology, Pennsylvania
State University, State College, PA 16802.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Lizhong Xiong ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.111.190488
1382
thaliana), for instance, the adaxial-abaxial patterning of
leaves and the radial patterning of stems are regulated
by miR165/miR166 (Emery et al., 2003; Juarez et al.,
2004). Organ boundaries are regulated by miR164
(Mallory et al., 2004; Baker et al., 2005; Nikovics et al.,
2006). The regulation of TEOSINTE BRANCHED/
CYCLOIDEA/PCF genes by miR319 and miR159 define
the leaf margin and size (Palatnik et al., 2003; Ori et al.,
2007). The temporal expression of miR156 and miR172
modulates the vegetative-reproductive phase transitions (Aukerman and Sakai, 2003; Lauter et al., 2005;
Schwab et al., 2005; Xie et al., 2006; Chuck et al., 2007;
Gandikota et al., 2007; Wang et al., 2008, 2009, 2011).
miR156 is one of the most conserved and highly
expressed microRNAs in plants. It has been found in
moss, monocotyledons, and dicotyledons. The miR156
targets SQUAMOSA-promoter binding-like (SPL) transcription factor genes in plants. In Arabidopsis, maize
(Zea mays), and rice (Oryza sativa), overexpressing
miR156 resulted in dramatic morphologic changes,
suggesting that miR156 has global regulatory function
in plant development (Schwab et al., 2005; Xie et al.,
2006; Chuck et al., 2007). In Arabidopsis, miR156 and its
target SPL genes define an essential regulatory module
in phase change (Wang et al., 2009; Wu et al., 2009), leaf
trichome development (Yu et al., 2010), male fertilization (Xing et al., 2010), embryonic patterning (Nodine
and Bartel, 2010), and anthocyanin biosynthesis (Gou
et al., 2011). The miR156-targeted SPLs regulate the
expression of several essential regulators, including
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miR156 Gradient in Leaf Development
miR172, LEAFY, FRUITFULL, APETALA1, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1),
TRICHOMELESS1, and TRIPTYCHON (Chuck et al.,
2007; Wang et al., 2009; Wu et al., 2009; Yamaguchi et al.,
2009; Yu et al., 2010). miR156 is dominantly expressed
in juvenile shoot (or shoot apex) and decreased when
the plant matures, and a low level of miR156 promotes
adult phase development (Wu and Poethig, 2006;
Chuck et al., 2007; Wu et al., 2009). This temporal
expression pattern of miR156 is essential for its regulatory roles in phase change.
The leaf is a basic organ and major component of the
shoot. Leaf initiation and maturation progress continuously and are highly coordinated by dynamic
changes of gene expression. Large-scale transcriptome
analysis revealed that multiple expression patterns
rise or fall with age or display age-specific peaks
(Efroni et al., 2008; Breeze et al., 2011). Changes of gene
expression over leaf development may be involved in
regulating the timing of specific development events.
MicroRNAs and trans-acting siRNA have essential
roles in coordinating the developmental processes
temporally and spatially (Pulido and Laufs, 2010).
For example, a gradual decrease of miR164 promotes
leaf senescence (Kim et al., 2009). In addition, compensation through non-cell-autonomous and autonomous effects between old and young leaves (leaf size
and plastochrone length) also affects leaf development
(Ferjani et al., 2007; Tsukaya, 2008; Kawade et al.,
2010). This compensation could occur between leaf
size and plastochrone length, cell proliferation, and
cell expansion. Nevertheless, how temporal and spatial changes of gene expression coordinate leaf development is still undetermined.
Rice is one of most important crops worldwide as
well as being a model organism for monocotyledons.
Modulations of rice development by genetic approaches have important economic significance. Recently, OsSPL14/IPA1, one of the miR156 targets, was
identified as a major regulator of panicle size (Jiao
et al., 2010; Miura et al., 2010). miR156 also has
potential utility to produce new biofuel crops (Chuck
et al., 2011). Our previous study suggested that rice
miR156 targets 11 rice SPL genes, and the interaction of
miR156 and OsSPLs may be spatial and temporal (Xie
et al., 2006). In this study, we found that the miR156
level is increased gradually over time in developing
adult leaves. This is different from the reported temporal expression pattern in Arabidopsis, in which
miR156 expression is gradually reduced in the shoots
during plant growth. Global expression profiling analysis revealed that more than 3,000 genes were affected
with altered expression levels in miR156 overexpression (miR156-OE) rice, and most of these genes were
up-regulated in the old leaves. miR156 also affected
the temporal expression of several plant conserved
microRNAs. Our results imply that a gradual increase
of miR156 expression may be essential for leaf development by attenuating temporal expression changes
of the relevant genes.
RESULTS
Gradual Increase of miR156 Expression in Developing
Leaves of Rice
Our previous data indicated that miR156 is accumulated at different levels at different stages during the
development of the rice panicle (Xie et al., 2006).
miR156 showed a much lower expression level in
3-week-old seedlings than in 3-d-old shoots (data not
shown), which is similar to observations in Arabidopsis. We also noticed that the miR156 level showed
dramatic differences between old and young leaves,
which prompted us to investigate the variation of
miR156 expression level in the developing or growing
leaves from a single plant. In this study, leaves on each
tiller were numbered according to their order of emergence, with L1 indicating the first developed leaf (Fig.
1). By checking the level of miR156 in three sequentially
developed leaves (L3–L5) from the main tiller of 25-dafter-germination (DAG) rice plants, the miR156 level is
approximately 2 times higher in old leaves (L3) than in
young leaves (L5; Fig. 1A). The miR156 level increased
gradually from L5 to L3. At the tillering stage, when rice
boost to branching (40 DAG), miR156 also had a similar
expression pattern in the leaves (increased gradually
from young to old leaf) both in primary tiller and
secondary tiller (L5–L7 in primary tiller and L1–L3 in
secondary tiller; Fig. 1B). It is interesting that the
difference of relative expression level of miR156 between sequential leaves is always between 20% and
30% (Fig. 2, A and B). At the grain-filling stage at which
rice leaves stop growing (100 DAG), miR156 showed a
similar expression level in all the green leaves (L11–L15;
Fig. 1C). The expression level of miR156 showed a
similar dynamic change also in the leaf sheath (Fig. 1,
A2C), although the expression level in the sheath was
lower than that in the corresponding leaf blade. We
further checked the oldest leaves collected at different
shoot developmental times, and miR156 had comparable levels in these leaves (Fig. 1D). According to the
gradually decreased expression pattern of miR156 in
shoot, early emerged or juvenile leaves should have
higher levels of miR156 than later-emerged adult leaves
have. This model cannot explain the comparable high
levels of miR156 in adult leaves that emerged at different shoot stages (Fig. 1, C and D), suggesting that
miR156 might have a different temporal expression
pattern in developing adult leaves.
To further identify the relationship between miR156
level and leaf developmental time, L4, L6, L8, L9, and
flag leaf (L16), with growing times of 3 to 40 d after
emergence, were used to check the miR156 level.
These leaves were initiated at a comparative adult
vegetative stage, except that L4 was from an intermediate juvenile/adult stage. The result clearly showed
that miR156 is increased gradually during leaf development (Fig. 2A). In the flag leaf, expression of miR156
was highly correlated (r2 = 0.990) with the time of leaf
growth (Fig. 2B). Taking the maximal miR156 level as
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Figure 1. Expression of miR156 is gradually increased during leaf development. A to C, Expression levels of miR156 in
sequentially developed leaves at three different stages: seedling (25 DAG), tillering (40 DAG), and graining (100 DAG). As shown
in the photographs of rice plants on the right, the leaf samples were numbered according to the order of emergence from the tiller.
The relative expression levels are indicated at the bottom of the blots except for weak bands. The labeled leaves were initiated at
the adult vegetative phase, except that L3/L4 were intermediate juvenile/adult leaves of the main tiller. D, Comparison of the
maximal miR156 level in leaves at different shoot stages. [See online article for color version of this figure.]
100%, miR156 was increased about 10% every 5 d in
flag leaf. These data indicated that the miR156 level
may positively reflect the developmental time or age
of leaves. The difference of miR156 level in different
leaves at the same shoot age (Fig. 1) is probably due to
leaf age rather than shoot age. However, the miR156
level was lower in leaves than in 3-d-old shoots, which
consist of juvenile leaves (Fig. 2C), suggesting that
opposite temporal expression patterns of miR156 in
developing leaves and shoots (or whole plants) coexist
in the rice plant.
Recent studies suggested that some mature small
RNAs have different expression features than their
primary transcripts (Chitwood et al., 2009). Therefore,
we investigated the expression of the precursors of
miR156 (pri-miR156) during leaf development by reverse transcription-quantitative (RT-q)PCR. The results
indicated that at least pri-miR156d and pri-miR156h
have a similar expression pattern to that of miR156
(Supplemental Fig. S1), suggesting that the temporal
expression of miR156 in leaves was probably regulated
by transcriptional control. We also checked the spatial
expression of miR156 by in situ RNA hybridization
using a locked nucleic acid (LNA) probe. miR156 was
expressed in leaf primordium (P1–P3) and leaf sheath
but was not detected in the shoot apical meristem
(SAM; Supplemental Fig. S2). In leaf elongating leaves
(L3 in 15-DAG seedlings), miR156 was detected in
almost all cells (Supplemental Fig. S2). miR156 has no
obvious specificity of distribution in leaves.
Overexpression of miR156 Attenuates the Temporal
Expression of miR156-Targeted OsSPL Genes
In a previous report, we generated miR156-OE
transgenic rice lines in which pri-miR156d and primiR156h were driven by a maize ubiquitin promoter
(Xie et al., 2006). In these studies, homozygous primiR156d and pri-miR156h overexpression plants
(abbreviated as Md/Mh hereafter) at the T3 or T4
generation were used. By checking age-different leaves
from three lines (Md15, Md43, and Mh9), miR156 levels
in all the leaves of Md/Mh were higher than levels in
the wild type (Fig. 3). In L3 (45 DAG), the miR156 level
was 5 times higher in Md/Mh than that in the wild
type. In L6 or L7 (45 DAG), miR156 was increased
8-fold in Md/Mh compared with the wild type. Despite high expression, temporal changes of miR156
were less obvious in Md/Mh than in the wild type. In
Md/Mh, miR156 was increased only about 30% in the
newly emerged leaf compared with early-emerged old
leaf. These results suggested that miR156 was overexpressed and the pattern of gradual increase in the wild
type was disrupted in Md/Mh.
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miR156 Gradient in Leaf Development
Figure 2. miR156 level is positively correlated
with leaf developmental time. A, Expression of
miR156 during leaf development. Rice leaves L4,
L6, L8, L9, L10, and flag leaf from the main tiller
were collected every 5 d after their emergence. B,
Relative expression of miR156 appears to be
correlated to leaf developmental time in the flag
leaf. C, Comparison of the maximal level of
miR156 in leaves (L4 and L5) and 3-d-old shoot.
We continued to check whether the temporal expression of miR156 affects its target gene expression.
In this assay, sequential leaves from plants at 45 DAG
were used. In contrast to the miR156 expression pattern, real-time RT-qPCR indicated that five of the
miR156-targeted OsSPL genes (OsSPL3, OsSPL12,
OsSPL13, OsSPL14, and OsSPL17) showed obviously
higher expressed levels in young leaves than in old
ones (Fig. 4). Compared with the wild type, the five
target genes showed reduced levels in young leaves of
Mh43 (an Md/Mh plant), and the temporal expression
patterns of the genes disappeared. The expression of
other miR156-targeted OsSPL genes showed no obvious temporal changes both in the wild type and in
Mh43 plants. Because miR156 can function by inhibiting target genes at both the transcript cleavage and
translational levels in Arabidopsis (Gandikota et al.,
2007), it is possible that these OsSPLs may be regulated
by miR156 mainly at the translation level. The expression levels of these OsSPL genes were also checked in
other Md/Mh lines, and similar results were obtained.
These results indicate that the temporal expression
patterns of the miR156-targeted OsSPLs were disrupted
by miR156-OE.
The Temporal Expression of Numerous Nontarget Genes
Was Also Attenuated by miR156-OE
To identify genes whose expression is affected or
indirectly regulated by miR156, Affymetrix 57K DNA
chips were used to check the transcriptomes of old (L3)
and young (L6) leaves (as designated in Fig. 3) of Md/
Mh and the wild type at the tillering stage. Compared
with the wild type, 3,008 genes were significantly
changed in the Md plant (Fig. 5A; Supplemental Tables
S1 and S2). Among these genes, 2,233 and 775 genes
were up- and down-regulated by miR156-OE, respec-
tively; 2,694 and 407 genes were affected by miR156OE in the early emerged (L3) and later emerged (L6)
leaves, respectively (Fig. 5A). Only 93 genes were
affected by miR156-OE in both the L6 and L3 leaves
(Fig. 5A). These results suggest that miR156-OE can
cause predominant up-regulation of genes mainly in
the old leaf.
We further checked the temporal expression of the
genes affected by miR156-OE in the wild-type plants.
Of the genes up-regulated by miR156-OE, 90% showed
increased expression levels in the old leaf compared
with levels in the young leaf (such change is called
time positive) and 4% of them showed decreased
expression (called time negative) in the old leaf (Fig.
5B). Of the genes down-regulated by miR156-OE, 67%
and 18% showed time-positive and time-negative expression changes, respectively (Fig. 5B). To indicate
more explicitly the temporal changes of the miR156regulated genes during leaf development, the expression levels of these genes in old leaves were normalized
to young leaves of the same plant and log transformed
(log-transformed fold change [logFC]). Interestingly,
logFC of these genes ranged from 26 to 22 in wild
type plants but from 22 to 21 in Md plants (Fig. 5C).
Genes with logFC of less than 25 were not found in
Md, whereas about 300 genes were found with logFC
of less than 25 in the wild type. In Figure 5D, a logFCbased heat map of these genes clearly shows that
miR156-regulated genes have fewer temporal changes
in Md than in the wild type. The average logFC values
of miR156-regulated genes are 22.3 and 20.8 in the
wild type and Md, respectively. According to gene
expression patterns, these genes could be grouped into
three clusters (Fig. 5E). In cluster I, 2,660 genes were
affected by miR156-OE in old or young leaves, and the
logFC values of miR156-OE plants (Md) were lower
than those of wild-type plants, suggesting that the
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Figure 3. Expression of miR156 in Md/Mh and wild-type leaves. Wildtype leaves are labeled WT1 to WT3. The RNA samples labeled with
asterisks were also used for microarray hybridization. Relative levels of
miR156 are shown on the bottom of blots except for weak bands.
temporal changes of cluster I genes were significantly
attenuated (Fig. 5E). Cluster II included 247 genes
without temporal expression changes in wild-type
leaves, but these genes showed increased/decreased
expression changes in the Md leaf. Cluster III contained
101 genes showing highly dynamic changes in wildtype or Md leaves, but these are distinct from those in
cluster I or II. Together, these results indicate that
miR156-OE can attenuate the temporal expression
changes of numerous genes between old and young
leaves.
Ten genes, including six genes showing up-regulation
in Md and four genes with distinct temporal expression
patterns in the wild type/Md, were selected to confirm
their expression changes by RT-qPCR. The results
generally agreed with the microarray data (Fig. 6).
Some of the arbitrary relative expression values
showed little difference between microarray data and
RT-qPCR results because of the different data normalization methods used.
Genes Affected by miR156-OE Are Involved in Diverse
Cellular Processes
Gene Ontology enrichment analysis was performed for
all genes regulated by miR156-OE. The results showed
that genes related to the cell wall, development, and
reproduction were overrepresented (Supplemental Table
S3). In addition, genes related to the carbohydrate metabolism process, motor activity, hydrolase activity, transferase activity, catalytic activity, and lipid binding were
also overrepresented. Most of the genes up-regulated by
miR156-OE were predicted with functions related to
metabolism, such as hydrolase activity, transferase activity, and catalytic activity. Genes annotated as transcription
regulators, receptors, and kinases were overrepresented
in the genes down-regulated by miR156-OE. Among the
genes up-regulated by miR156-OE, four genes (LOC_
Os01g07630, LOC_Os03g16010, LOC_Os02g09359, and
LOC_Os08g17410) are homologous to BAK1 (for BRI-1associated protein kinase), which mediates brassinosteroid signaling in Arabidopsis; 13 genes are related to
auxin response or signaling, including five OsIAA
genes (OsIAA14, OsIAA21, OsIAA27, OsIAA30, and
OsIAA31) and five OsSAUR genes (LOC_Os02g24700,
LOC_Os04g56680, LOC_Os09g37500, LOC_Os12g41600,
and LOC_Os09g37480). Gel blotting of small RNA
suggested that miR156 expression was decreased slightly by brassinosteroid treatment but not by other
phytohormones (Supplemental Fig. S3). This result
implies that miR156 may be involved in the regulation
of auxin and brassinosteroid signaling pathways associated with leaf development. Interestingly, stressresponsive genes were overrepresented in the miR156
down-regulated genes (Supplemental Table S3). These
data suggested that miR156-OE affected the expression of genes involved in diverse cellular processes
related to metabolism, development, and stress responses.
Among the genes up-regulated by miR156-OE, 31
genes were specifically expressed in Md leaves (Supplemental Table S4). Five of them were confirmed by
RT-qPCR (Fig. 6). Among these miR156-specific genes,
18 are annotated as unknown function genes. These
unknown genes were predicted to encode small polypeptides (less than 200 amino acids) or no-coding
RNAs. By searching a small RNA database, three
transcripts (AK064111, AK109584, and AK111071)
were found with matches of 23- to 24-nucleotidelong small RNAs, which most likely were small interfering RNAs. In addition to these unknown genes,
other genes activated by miR156-OE are predicted to
function diversely, including stress-related proteins,
F-box proteins, zinc finger proteins, and cytochrome
P450.
miR156-OE Affects the Temporal Expression of Some
Plant Conserved MicroRNAs
To determine whether miR156-OE affects other
microRNAs, 15 plant conserved microRNAs were
investigated for their expression levels in leaves of
wild-type and Md plants by using stem-loop RT-qPCR
(Shen et al., 2010). Of these microRNAs, eight (miR156,
miR159, miR160, miR164, miR169, miR172, miR319,
and miR399) showed greater than 3-fold change between young and old leaves (Fig. 7A). These microRNAs can be categorized into three classes according
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miR156 Gradient in Leaf Development
Figure 4. Temporal expression of miR156-targeted
OsSPLs during leaf development in Md and wildtype (WT) plants. Error bars indicate SD (n = 3).
to their expression patterns in different leaves of the
wild type (Fig. 7B). The microRNAs in class I (miR162,
miR166, miR167, miR168, miR171, miR393, and miR399)
showed strong expression in young leaves with changes
of less than 3-fold between young (L6) and old (L3)
leaves. miR168 has the most stable expression compared
with the others. The microRNAs in both classes II and III
showed greater than 5-fold changes between young and
old leaves; the microRNAs in class II (miR156, miR159,
miR169, and miR319) were strongly expressed in older
leaves, whereas those in class III (miR160, miR164,
miR172, and miR390) were strongly expressed in younger leaves. The expression patterns of these microRNA
clearly show that most microRNAs are expressed temporally during leaf development.
Compared with the wild type, the expression of six
microRNAs (miR159, miR164, miR169, miR172, miR319,
and miR393) was significantly suppressed (more than
2-fold at least in one leaf; Fig. 7A) in Mh. Interestingly,
some of these microRNAs affected by miR156-OE were
found only at specific leaf developmental stages. For
example, miR164 was dramatically suppressed in
young leaves (L5; expanding leaf), miR319 was suppressed specifically in mature leaves, and miR393 was
suppressed most obviously in young leaves (Fig. 7A).
miR172 was suppressed by miR156 in all leaves except
the oldest one, in which miR172 was barely detected.
The result of stem-loop RT-qPCR, with miR164 as an
example, was confirmed by small gel blotting (Fig. 7C).
These results suggest that miR156-OE can also affect the
temporal expression of other microRNAs.
miR156-OE Promotes Leaf Initiation and
Precocious Maturation
Among the genes affected by miR156-OE, 91%
showed expression changes between young and old
leaves, and many of them were related to development
based on their annotation as indicated above. A previous study showed that miR156-OE plants (Md/Mh) had
significantly more leaves and tillers but with smaller
size than wild-type or negative control (NC; no overexpression lines segregated from Md/Mh) plants (Xie
et al., 2006). Therefore, we checked the morphologic
changes in the miR156-OE plants (Md/Mh) in more
detail to determine the role of miR156 in development.
In the early vegetative phase (before 35 DAG), no
difference was apparent between the Md/Mh plants
and wild-type/NC plants. After 35 DAG, the Md/Mh
plants were distinct from the wild-type/NC plants in
plant height (Fig. 8A), leaf number (Fig. 8B), and tiller
number (Fig. 8C). From 35 to 120 DAG, Md/Mh plants
showed a 1.5-fold higher rate of leaf initiation than wildtype/NC plants. The maximum leaf initiation rate of
Md/Mh was 14 to 17 leaves per day, with 0.3 leaves
emerging for a tiller each day on average, which is about
1.5-fold the wild-type value (0.18 leaves per day from
one tiller; Fig. 8D). Both Md/Mh and wild-type plants
burst in leaf/tiller initiation between 40 and 60 d (Fig.
8D). At the grain-filling stage (120 DAG), the leaf
number and tiller number in Md/Mh plants were 250
to 500 and 50 to 100 times, respectively, those in wildtype/NC plants (Fig. 8, B and C). In contrast to the high
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Figure 5. Summary of miR156-regulated genes in different leaves. A, Venn diagram representation of miR156 up- and downregulated genes in L6 (later-emerged young leaf) to L3 (early-emerged old leaf). B, Most miR156-regulated genes have temporal
expression in the wild type (WT). Time positive indicates that gene expression is increased from L6 to L3 in the wild type; time
negative indicates that gene expression is decreased from L6 to L3 in the wild type. C, Distribution of logFC of miR156-regulated
genes in the wild type and Md. LogFC indicates the fold change of each gene from young to old leaf in these plants. D, Heat map
shows logFC of different plants. Color indicates the value of logFC as shown on the bar at bottom. E, Clusters of miR156-regulated
gene expression patterns.
speed of leaf initiation, the size of Md/Mh SAM was
smaller than that of wild-type/NC plants after the fourleaf stage (P , 0.05; Supplemental Table S5). These
results imply that overexpression of the miR156 level
can accelerate the initiation rates of leaf and tiller.
Because the temporal expression changes of miR156OE-affected genes were observed mainly in old leaves,
we checked the maturation status of these leaves.
Except for the early vegetative stage, the width and
length of fully expanded Md/Mh leaves at the other
stages decreased significantly (P , 0.01) compared with
wild-type/NC leaves (Fig. 8, E–J). We further checked
the maturation status of the Md/Mh leaves using
toluidine blue O staining, which has been used to
distinguish the cell walls of juvenile and adult leaves in
maize (Poethig, 2003). Except for the leaves from the
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miR156 Gradient in Leaf Development
In addition to increased leaf numbers with smaller
size, the Md/Mh plants generated more roots with
smaller size than did wild-type plants (Supplemental
Fig. S6). This result implies that miR156 may also
regulate root development, probably by the same
mechanism as present in shoots.
DISCUSSION
In this study, we found that miR156 increased gradually from young leaves to old leaves in rice after the
juvenile stage, and disruption of the miR156 gradient
by overexpression of two miR156 precursors attenuated the temporal expression of thousands of genes.
Figure 6. Confirmation of the results of miR156-regulated genes from
microarray analysis by RT-qPCR. Error bars indicate 95% confidence
interval. WT, Wild type.
very early vegetative stages, all the other leaves showed
no difference in staining (Supplemental Fig. S4, A and
B), although the cell size was decreased slightly in Md/
Mh mature leaves (Supplemental Fig. S4, C and D). The
numbers of trichomes, papilla, and stomata were also
obviously decreased in Md/Mh leaves (Supplemental
Fig. S5). These results suggest that miR156-OE can
result in precocious leaf maturation.
Figure 7. Expression of miR156-regulated microRNAs temporally during
leaf development. A, Relative levels of 15 microRNAs in different leaves
were monitored by stem-loop RT-qPCR. RNAs are from the same samples as
in Figure 2. Error bars indicate SD (n = 3). WT, Wild type. B, Cluster analysis
of microRNAs according to their temporal expression in the wild type. The
relative expression level is indicated by the color scale. C, The stem-loop RTqPCR results were confirmed by small RNA gel blotting of miR164.
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Xie et al.
Figure 8. miR156-OE changed leaf development. A to D, Dynamic changes of plant height, leaf number, tiller number, and leaf
initiation rate of wild-type (WT)/NC and Md/Mh plants (n = 12). Error bars indicate SE. E to J, Comparison of Md/Mh and wildtype/NC plants in leaf shape (length and width) at three representative stages of rice (n = 6). Error bars indicate SD. K, Md/Mh and
wild-type/NC plants at the flowering stage (90 DAG). [See online article for color version of this figure.]
miR156-OE has global impacts on leaf development,
including increased initiation rates of tillers and leaves
and precocious maturation. Our results suggest that
the temporal expression of miR156 has an essential
role in regulating leaf and tiller development.
Two Opposite Gradient Expressions of miR156
The gradual decrease of miR156 in shoots over
plant-growing time regulates phase changing in Arabidopsis (Chuck et al., 2007; Wu et al., 2009). Such a
decreased expression of miR156 also exists in rice (Xie
et al., 2006; Fig. 2C). We found an opposite gradient of
miR156 over time, that is, a gradual increase over the
time of leaf growth and maturation in rice (Figs. 1 and
2). At a specific time point, older (or early-emerged)
leaves have higher levels of miR156 than younger (or
later-emerged) leaves (Fig. 1, A–C), and in a specific
leaf generated after the juvenile phase, miR156 is
increased gradually over its growing time until it
reaches a maximal expression level (Fig. 2, A and B).
The maximal levels of miR156 in different adult leaves
are comparable (Fig. 1, C and D), which also suggests
that miR156 expression in leaves is independent of
shoot or plant age after the juvenile stage. It will be
interesting to reveal how miR156 regulates plant
growth and development through two opposite temporal expression changes over time: decreasing in
shoots (or at the whole plant level) and increasing in
leaves. Such opposite temporal expression patterns in
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miR156 Gradient in Leaf Development
shoots and leaves also exist for miR172, which is
gradually decreased in leaves from young to old (Fig.
7) and increased gradually in shoots (Zhu et al., 2009).
Because unexpanded or young leaves are the major
part of young shoots, the gradual reduction of miR156
in shoots is likely due to the decreased average level of
miR156 for all the unexpanded or young leaves. The
younger leaves with lower miR156 levels may compromise the overall miR156 level in the whole plant.
This is indeed the case when we compare the last three
leaves from the primary tiller and the secondary tiller
(Fig. 1B). Because a growing shoot or plant normally
gains more young leaves and miR156 has low levels in
young leaves, it is not strange that miR156 has a
gradually decreased pattern if it is quantified in shoots
or whole plants. Therefore, we propose that the temporal expression pattern in a shoot or whole plant is
mainly determined by the temporal expression of
miR156 in leaves.
In rice, miR529 shared 70% sequence identity with
miR156 (Jeong et al., 2011). However, we did not detect
any miR529 in adult rice leaves by small RNA gel
blotting, and no cross-hybridization between miR156
probe and miR529 probe was detected either (Supplemental Fig. S7).
miR156-OE Attenuates Temporal Changes of
Gene Expression
In the microarray analysis, old and young leaves at
the same shoot stage were collected to study the
changes of gene expression. Variations between leaf
position and emerging time may also affect differential
gene expression. Here, we used only two representative leaves to measure the temporal gene expression
changes, so the number of genes affected by miR156
during leaf development might be underestimated.
Leaf development is coordinated by a number of
temporal/spatial expressions of genes. In rice, approximately half of the expressed genes show temporal
changes between old and young leaves, and 30% of their
expression is affected by miR156-OE. Besides miR156,
some other plant-conserved microRNAs also show
temporal expression changes in growing leaves. Regardless of the expression patterns in the wild type, the
magnitudes of the temporal changes of these genes have
been attenuated in the miR156-OE plants (Figs. 5–7).
Therefore, the gradual increase of miR156 during leaf
growth and maturation may be essential for maintaining the temporal changes of numerous genes. Nevertheless, high levels of miR156 in Md/Mh leaves, in
which the miR156 levels exceeded the maximal levels of
miR156 in the old leaves of the wild type, did not
completely abolish the temporal changes of most
miR156-regulated genes, suggesting that other factors
should be involved in maintaining the temporal
changes of gene expression.
Because miR156 is increased gradually from young to
old leaves, miR156-OE was supposed to suppress gene
expression predominantly in young leaves. Unexpect-
edly, 1,958 genes that have higher expression levels in
the young leaves of wild-type plants and were expected
to be down-regulated by miR156-OE were actually upregulated specifically in the old leaves of miR156-OE
plants. Related to such unexpected gene expression
changes, Md/Mh leaves showed precocious maturation. The unexpected gene expression changes by
miR156-OE might result from non-cell-autonomous
compensation between leaf maturation and leaf initiation. Non-cell-autonomous compensation has been
reported as an important mechanism for leaf development (Kawade et al., 2010). Misexpression of miR156 in
different domains of the shoot apex also suggested that
miR156 might affect leaf development through a noncell-autonomous effect (Wang et al., 2008). Because
miR156 is increased gradually, it is unclear at which
leaf stage miR156 triggers the non-cell-autonomous
signal and what the threshold is for the miR156 level
to trigger non-cell-autonomous signals.
Temporal Expression of miR156 Affects Genes Involved
in Leaf Development
The overall morphology of Md/Mh was different
from that of other plastochrone mutants of rice, such as
pla1, pla2, pla3, and mori1 (Asai et al., 2002; Miyoshi
et al., 2004; Kawakatsu et al., 2006, 2009). Except for
smaller leaf size, the number of leaves per tiller (three
to four leaves per tiller) in Md/Mh is not changed
compared with wild-type/NC plants, but pla1, pla2,
pla3, and mori1 continuously generate leaves from each
tiller. PLA1 and PLA2 are dominantly expressed in
young leaf primordial, whereas PLA3 has higher expression in old leaves than in young leaves according
to our microarray data. No difference in expression
level was observed for PLA1, PLA2, and PLA3 between
miR156-OE and the wild type in leaves of different
ages, suggesting that miR156 might act in concert with
PLA genes to regulate leaf development.
The expression of miR156 was suppressed by brassinosteroid treatment but not by other phytohormone
treatments (Supplemental Fig. S3). Several genes that
might be involved in brassinosteroid biosynthesis (e.g.
CYP724 and CYP90; Tanabe et al., 2005) or signaling
(BAK1 homologous) were affected by miR156-OE (Supplemental Tables S1 and S2). In rice, brassinosteroid
deficiency resulted in dwarf stature, erect leaves, and
reduced panicle size (Hong et al., 2003; Sakamoto et al.,
2006), and Md/Mh plants showed partial similarity to
these phenotypes, suggesting that the miR156-initiated
regulation networks may have some overlaps with the
brassinosteroid signaling-initiated regulation networks.
Although auxin treatment did not result in obvious
changes of miR156 expression, some auxin-responsive
genes, such as OsIAAs and OsSAUPs, showed significant changes in expression in miR156-OE leaves (Supplemental Table S1). In addition, miR156-OE also
affected the expression of miR164 and miR393 (Fig. 7),
whose targets are thought to be involved in auxin
signaling (Guo et al., 2005; Navarro et al., 2006). In
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Xie et al.
addition, the expression of several target genes of
miR164 and miR393 in rice showed time-dependent
fluctuation in miR156-OE leaves (Supplemental Fig. S8).
These data imply that miR156-initiated regulation networks might have some overlaps with auxin signalinginitiated networks. Because auxin is essential for leaf
initiation and development, and because auxin levels
are also temporally changed in different leaves (Terry
et al., 1986; Avsian-Kretchmer et al., 2002), it will be
intriguing to check the temporal and spatial interactions
between miR156 and auxin signaling, if they exist.
miR156 also temporally regulated the expression of
six plant-conserved microRNAs during leaf development. Temporal regulation of miR172 by miR156 was
reported in Arabidopsis and maize. miR172 is decreased gradually during leaf development (Fig. 7),
whereas a previous study suggested that miR172 is
increased gradually in rice shoots during plant maturation (Zhu et al., 2009). Nevertheless, miR156 always
has opposite expression patterns compared with
miR172. The microRNAs regulated by miR156 might
modulate leaf development according to studies in
Arabidopsis. For example, both miR164 and miR319
have been suggested to be involved in the control of
two distinct leaf developmental events, leaf margin
formation and leaf senescence, in Arabidopsis (Palatnik
et al., 2003; Mallory et al., 2004; Schommer et al., 2008;
Kim et al., 2009). miR156-OE also activated the expression of three small RNA precursors. The function of
these no-coding RNAs in rice is still unclear.
Temporal Expression of miR156 Affects Genes Involved
in Stress Responses
Leaf age affects susceptibility to abiotic and biotic
stresses, and the environmental stresses can also affect
leaf growth and initiation. According to our unpublished
data and other public microarray data, a number of
stress-responsive genes have different expression levels
between old and young leaves. As an essential regulator
of leaf development, miR156 is also up-regulated by
drought, salt, and cold (Shen et al., 2010). The expression
levels of many stress-related genes were affected by
miR156-OE. For example, drought- or disease-responsive
genes, such as dehydration-responsive element-binding
proteins (LOC_Os02g45450, LOC_Os06g03670, LOC_
Os03g09170, and LOC_Os09g20350), NAC transcription
factors (ONAC055 [LOC_Os03g01870] and ONAC122
[LOC_Os11g03300]), and Pathogenesis-related protein1
(LOC_Os07g03730), were down-regulated in old leaves
by miR156 (Supplemental Tables S1 and S2). Thus, environmental stresses might modulate plant growth, at least
in part, by the miR156-initiated regulation networks.
Taken together, we report a gradually increased gradient of miR156 in the developing leaf, which is conserved in plants and may have an important role in leaf
development. Although it is unclear how this gradient is
controlled, our data offer novel insights into the function
of miR156 and other microRNAs with temporal expression patterns in plant growth and development.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
Seeds of rice (Oryza sativa) transgenic plants were surface sterilized and
germinated in Murashige and Skoog medium containing 50 mg L21 hygromycin.
The seedlings were transferred to a greenhouse and grown in polyvinyl chloride
tubes. The leaf number, height, and tiller number were measured every 7 d (n =
12). Rice leaf samples used in this study were collected at approximately 2 PM. To
collect leaves at different developmental series, plants at the same developmental
stage (based on the emergence of the same order leaf on the same day) were
selected and the orders of leaves were labeled. Then, the same order leaves were
sampled at a specific time point (every 5 d). Each plant was sampled only once.
The developmental process and stage system of rice leaf were as described (Itoh
et al., 2005). For rice cv Zhonghua11 (subspecies japonica), which was used in this
study, L1 and L2 are juvenile leaves, L3 and L4 are intermediate juvenile/adult
leaves, and later leaves are adult leaves. RNA samples were prepared using
TRIzol reagent (Invitrogen) following the manufacturer’s instructions.
Histologic Analysis
The tissues were fixed in formaldehyde-acetic acid immediately after being
sliced from plants. Samples were then dehydrated in an ethanol series,
embedded in paraffin, and sectioned. The internode samples were treated
with 0.1% hydrofluoric acid for 3 d before dehydration. Epidermal peels of
rice leaves were prepared using the maize protocol (Chuck et al., 2007). The
1-cm-long segments were collected from the middle of the leaf. Mesophyll was
released by digestion with 0.1% pectinase and 0.5% cellulose, stained in
toluidine blue O, and photographed with a light microscope. Entire shoot
apices were prepared as described (Kawakatsu et al., 2006), and the size of the
SAM was measured using ImageJ (version 1.4; http://rsbweb.nih.gov/ij/).
For scanning electronic microscopy, dehydrated samples were soaked, infiltrated with isopentyl acetate, dried to the critical point, sputter coated with
platinum, and then observed with a scanning electronic microscope (Hitachi S450).
Real-Time RT-qPCR
A one-tube stem-loop RT-qPCR was applied for the quantification of
mature microRNAs. SYBR was adopted to monitor the amplification (Chen
et al., 2005; Varkonyi-Gasic et al., 2007; Shen et al., 2010). Briefly, cDNA was
synthesized in a 5-mL reaction containing 20 ng of total RNA, 13 first-strand
buffer, 10 mM dithiothreitol, 1 mM appropriate stem-loop RT primer (Supplemental Table S5A; except for U6 RNA, for which a specific primer was used),
1 unit of RNase inhibitor (Takara), and 20 units of SuperScript III (Invitrogen).
A pulsed RT reaction (40 cycles of 16°C for 2 min, 42°C for 1 min, and 50°C for
1 s, followed by final reverse transcriptase inactivation at 85°C for 5 min) was
performed with an ABI9700 Thermal Cycler (Applied Biosystems). Real-time
PCR was performed with a Prism7500 system using the SYBR Ex Taq kit
(Takara). In brief, 12.5 mL of SYBR mix (Takara), 1 mM universal reverse primer
(5#-CCAGTGCAGGGTCCGAGGT-3#; except for U6 RNA, for which a specific
primer [Supplemental Table S6] was used), and 1 mM appropriate forward
primers specific to target microRNA or U6 RNA (Supplemental Table S5A)
were added to 96-well plates, and PCR continued in an ABI Prism 7500
(Applied Biosystems). All reactions were performed with three replications.
U6 RNA was used as a reference for data normalization. The stem-loop
primers were designed as described previously (Chen et al., 2005). The relative
expression levels were normalized to U6 RNA and calculated as described
previously (Shen et al., 2010).
Real-time RT-qPCR for mRNA detection was performed as described (Xie
et al., 2006). The expression levels of the genes of interest were normalized to
the reference gene (AK071592; 6-phosphogluconate dehydrogenase), which
has stable expression in different leaves, and the relative expression levels
were calculated by the 22DDCT method (Livak and Schmittgen, 2001; Chen
et al., 2005). Primers for RT-qPCR are listed in Supplemental Table S6.
Microarray Hybridization and Data Analysis
Individual leaves of primary tiller were collected from Md/Mh and wildtype plants. RNA samples were subjected to microarray hybridization after
the miR156 level was checked by small RNA gel blot. The Affymetrix 57K rice
DNA chip, which contains 57,386 probe sets, was used to measure gene
expression in different leaves. Microarray hybridization was performed by
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Copyright © 2012 American Society of Plant Biologists. All rights reserved.
miR156 Gradient in Leaf Development
Capitalbio according to the standard Affymetrix protocol. For each group of
leaf samples, three biologic repeats were used. These data have been deposited into the Gene Expression Omnibus database of the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo/) with accession number GSE14692.
The expression level of each probe set was summarized and normalized
using GeneChip Robust Multiarray Averaging (Gentleman et al., 2004). The
probe sets were then filtered by three steps: (1) assigning presence by MAS5 in
at least four samples; (2) removing the hybridizations with low correlation
(r , 0.95) with other biologic repeats (as a result, samples from two plants
were removed [Supplemental Table S7]); and (3) identifying probe sets with
expression levels that were significantly changed by the LIMMA program in
the R package (Smyth, 2004). In the third step, genes were identified as
significantly changed when changes were greater than 2-fold with P , 0.05.
Annotation of the Affymetrix rice DNA chip was downloaded from the Gene
Expression Omnibus (GPL2025) and modified according the Rice Gene
Annotation Project (http://rice.plantbiology.msu.edu/). Gene Ontology enrichment was analyzed using BiNGO (Maere et al., 2005).
Supplemental Figure S5. Scanning electron microscopic photos of Md43
and wild-type leaf epidermis.
Supplemental Figure S6. Overexpression of miR156 changes the root
development.
Supplemental Figure S7. The miR156 probe and miR529b probes did not
cross hybridization in small RNA gel blotting.
Supplemental Figure S8. Expression of miR164- and miR393-targeted
genes at different developmental time of leaf 6 in wild type and Md43.
Supplemental Table S1. miR156-regulated genes in L3 (old leaf).
Supplemental Table S2. miR156-regulated genes in L6 (young leaf).
Supplemental Table S3. Overrepresnented gene ontology in miR156regulated genes.
Supplemental Table S4. Genes specific expressed in miR156-OE leaves.
Supplemental Table S5. SAM size of Md/Mh and wild type.
Supplemental Table S6. Primers used for real-time PCR.
Small RNA Gel Blot
Briefly, 30 mg of total RNAs was separated on a 20% polyacrylamide gel
containing 8 M urea and then transferred to a Hybond-N+ membrane (GE
Biosciences) using a Mini-Protean System III (Bio-Rad). The entire gel was
blotted to ensure that the high-molecular-weight RNAs transferred to the
membrane. The probes were prepared by end-labeled synthetic DNA oligonucleotides with [g-32P]ATP using T4 polynucleotide kinase (Takara). Hybridization was performed overnight at 50°C in hybridization buffer
containing 53 SSC, 20 mM Na2HPO4 (pH 7.2), 7% SDS, 23 Denhardt’s
solution, and 50 mg mL21 sheared salmon sperm DNA. The blots were washed
three times with low-stringency washing solution (33 SSC, 25 mM NaH2PO4
[pH 7.5], and 1% SDS) and one time with high-stringency washing solution
(13 SSC and 1% SDS). Radioactive signals were detected and quantified using
the FujiFilm phosphorimager system (FJL-5100; Fuji). The probes were stripped
by washing in 1% SDS at 80°C for 30 min and certified by phosphorimager.
Stripped blots with no detectable signals were used to detect other probes. The
relative expression level was normalized to U6 or rRNA signals using ImageJ
(version 1.4). The following DNA oligonucleotides were used: miR156-probe,
5#-GTGCTCACTCTCTTCTGTCA-3#; miR164-probe, 5#-GCACGTGCCCTGCT
TCTCCA-3#; miR529b-probe, 5#-AGCTGTACTCTCTCTCTTCT-3#; and U6probe, 5#-TGTATCGTTCCAATTTTATCGGATGT-3#.
In Situ MicroRNA Detection
miRCURY digoxigenin-labeled LNA probes (Exiqon) were used for in situ
detection of miR156. Rice tissues were collected and embedded as described
above. Transverse and vertical sections (10 mm) were subjected to overnight
hybridization at 50°C in buffer containing 50% formamide, 300 mM NaCl, 10 mM
Tris (pH 7.5), 1 mM EDTA, 5% dextran sulfate, 1% blocking reagent (Roche), 150
mg mL21 yeast tRNA, and 1 pM digoxigenin-labeled LNA probes. Then, slides
were washed in low-stringency washing solution (23 sodium chloride/sodium
phosphate/EDTA) at room temperature and high-stringency washing solution
(0.23 sodium chloride/sodium phosphate/EDTA) at 50°C. The signal was
detected using anti-digoxigenin-alkaline phosphatase antibody (Roche). The
color reaction was stopped after 6 h. Images were captured under the bright field
using a light microscope (Leica).
Microarray data from this article can be found in the National Center for
Biotechnology Information Gene Expression Omnibus database under accession number GSE14692.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Expression of pri-miR156d and pri-miR156h in
different leaves.
Supplemental Figure S2. In situ detection of miR156.
Supplemental Figure S3. Expression of miR156 after phytohormone
treatments.
Supplemental Figure S4. Toluidine O blue-stained leaf epidermal cells of
Md43 and wild type.
Supplemental Table S7. Pearson coefficient of microarray data by pairwise comparison.
ACKNOWLEDGMENTS
We thank Lihong Qing for technical support with the scanning electron
microscopy.
Received November 1, 2011; accepted January 17, 2012; published January 23, 2012.
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