The role of WRKY transcription factors in plant abiotic stresses.

Abstract

The WRKY gene family has been suggested to play important roles in the regulation of transcriptional reprogramming associated with plant stress responses. Modification of the expression patterns of WRKY genes and/or changes in their activity contribute to the elaboration of various signaling pathways and regulatory networks. Furthermore, a single WRKY gene often responds to several stress factors, and then their proteins may participate in the regulation of several seemingly disparate processes as negative or positive regulators. WRKY proteins also function via protein-protein interaction and autoregulation or cross-regulation is extensively recorded among WRKY genes, which help us understand the complex mechanisms of signaling and transcriptional reprogramming controlled by WRKY proteins. Here, we review recent progress made in starting to reveal the role of WRKY transcription factors in plant abiotic stresses. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress.

Full text

Page 1

Physiologia Plantarum 136: 223–236. 2009
Copyright © Physiologia Plantarum 2009, ISSN 0031-9317
Ectopic expression of miR396 suppresses GRF target gene
expression and alters leaf growth in Arabidopsis
Dongmei Liua,b, Yu Songa,b, Zhixiang Chenc,∗and Diqiu Yua,∗
aKey Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, 650223, Yunnan,
China
bGraduate School of the Chinese Academy of Sciences, Beijing 100049, China
cDepartment of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana, IN 47907-2054, USA
Correspondence
*Corresponding author,
e-mail: zhixiang@purdue.edu and
ydq@xtbg.ac.cn
Received 20 August 2008; revised 24
February 2009
doi: 10.1111/j.1399-3054.2009.01229.x
MicroRNAs (miRNAs) are comprised of approximately 21 nucleotide (nt)
RNAs that play important regulatory roles in growth and development by
targetingmRNAsforcleavageortranslationalrepression.InArabidopsis,more
than one hundred miRNAs have been identified but the biological functions
of only a limited number of them have been determined by molecular genetic
analysis. miR396 is a miRNA conserved among the dicot and monocot plants.
In Arabidopsis, miR396 has two loci (MIR396a and MIR396b) and targets
six Growth-Regulating Factor (GRF) genes encoding putative transcription
factors with roles in plant leaf growth. Using a northern blot hybridizations
approach, we have found that MIR396 is predominantly expressed in leaf
and seedling. To further analyze the role of miR396 in the regulation of
target genes and leaf growth, we have generated transgenic Arabidopsis
plants that constitutively overexpress MIR396a or MIR396b. These transgenic
plants have narrow-leaf phenotypes due to reduction in cell number. Ectopic
overexpression of MIR396 represses expression of not only six GRF genes but
also GIF1 encoding a GRF-interacting transcription coactivator with a role
in cell proliferation in leaf. In addition, transgenic MIR396-overexpressing
plants have lower densities of stomata and are more tolerant to drought
than wild-type plants. These results strongly support the belief that miR396
plays an important role in plant leaf growth and development, most likely by
repressing GRF gene expression.
Introduction
MicroRNAs (miRNAs) are short, usually comprised of
about 21 nucleotide RNAs with important regulatory
roles in plants and animals (Jones-Rhoades et al. 2006).
Mature miRNAs are excised by Dicer-like enzymes
from their much longer RNA precursors that contain
imperfect hairpins (Bartel and Bartel 2003, Bartel
2004). miRNAs recognize their regulatory target mRNAs
through base pairing (Jones-Rhoades and Bartel 2004,
Abbreviations – GRF, growth-regulating factor; miRNA, MicroRNAs; PCR, polymerace chain reaction; RT PCR, reverse
transcriptase polymerace chain reaction.
Jones-Rhoades et al. 2006). In animals, miRNAs can
pair to the 3?untranslated regions of mRNAs and
repress their translation (Jones-Rhoades et al. 2006).
In plants, miRNAs are usually loaded into the RNA-
induced silencing complex (RISC), where they function
as guide RNAs through base pairing to the coding
regions of target mRNAs, and target their cleavage
(Jones-Rhoades and Bartel 2004, Jones-Rhoades et al.
2006).Many plant miRNA families are conservedamong
different plant species and are believed to play important
Physiol. Plant. , 2009
223

Page 2

roles in plant growth, development and responses to
environmental conditions (Jones-Rhoades et al. 2006,
Zhang et al. 2006).
In Arabidopsis, more than one hundred miRNAs have
been identified. Over the past several years, some of
these miRNAs have been genetically and molecularly
analyzed for their roles in the regulation of their target
genes, many of which encode transcription factors with
roles in plant growth and development (Aukerman and
Sakai 2003, Bartel and Bartel 2003, Carrington and
Ambros 2003, Chen et al. 2004, Duan et al. 2006, Guo
et al. 2005, Laufs et al. 2004, Llave et al. 2002, Palatnik
et al. 2003, Reinhart et al. 2002). For example, miR319
can guide mRNA cleavage of several genes encoding
TCP transcription factors that control leaf development
(Palatnik et al. 2003). miR159 directs the cleavage of
mRNA encoding GAMYB-related transcription factors
that are involved in the gibberellin-promoted activation
of LEAFY and floral transition as well as in the regulation
of anther development (Achard et al. 2004). miR172
acts as a translational repressor of APETALA2, which
regulates flowering time and specification of floral whorl
identity(Chen2004).miR824,arecentlyevolvedmiRNA
conserved in the Brassicaceae, targets sequence-specific
degradation of AGAMOUS-LIKE 16 (AGL16) mRNA,
which encodes a member of the MADS box transcription
factor family with an important role in the regulation of
the density and development of stomatal complexes
on the epidermis of Arabidopsis (Kutter et al. 2007).
These results indicate important roles of plant miRNAs
in regulating organogenesis. Some miRNAs also play
important roles in plant responses to biotic and/or
abiotic stress (Llave 2004, Sunkar and Zhu 2004). It
has been recently shown that expression of miR398
is repressed by oxidative stresses, whereas miR159 is
induced by drought stresses (Reyes and Chua 2007).
In addition, the level of miR393 is induced by a
defense-inducing peptide derived from the flagellin of
the bacterial pathogen Pseudomonas syringae (Navarro
et al. 2006). miR393 negatively regulates mRNAs for the
F-box auxin receptors TIR1, AFB2 and AFB3. Repression
of auxin signaling restricts P. syringae growth. Thus,
miRNA393 promotes plant disease resistance through
suppression of auxin signaling.
miR396 is a family of miRNAs recently identified in
Arabidopsis and rice (Jones-Rhoades and Bartel 2004).
In Arabidopsis, miR396 has two loci (MIR396a and
MIR396b) and their expression has been detected by
both northern blot analysis and a polymerase chain
reaction (PCR)-based assay (Jones-Rhoades and Bartel
2004). Based on a refined computational procedure,
it has been predicted that miR396 targets seven of
the Growth-Regulating Factor (GRF) genes as well
as two additional genes (At2g40760 and At4g27180)
encoding a Rhodenase-like protein and a kinesin-like
protein, respectively (Jones-Rhoades and Bartel 2004).
However, the predicted six GRF genes, except GRF4,
as targets of miR396 have been confirmed by detecting
mRNAfragmentsdiagnosticofmiRNA-directedcleavage
in plants using a modified form of 5’-RACE (rapid
amplification of cDNA ends) (Jones-Rhoades and Bartel
2004).
Plant GRF
genes encode a family of putative
transcription factors with roles in plant leaf growth
(Kim et al. 2003). OsGRF1 is induced in internodes
of deepwater rice in response to GA and submergence
and, therefore, may be involved in the regulation of
stem growth (van der Knaap et al. 2000). In Arabidopsis,
there are nine members of the GRF gene family with
most of them strongly expressed in actively growing
and developing tissues (Kim et al. 2003). Transgenic
plants that overexpress AtGRF1 and AtGRF2 have larger
leaves and cotyledons while the triple null mutants for
AtGRF1-AtGRF3 have smaller leaves and cotyledons
than wild-type plants (Kim et al. 2003). The altered
leaf growth in the overexpresison and triple mutants
was associated with an increase and decrease in cell
size, respectively (Kim et al. 2003, Kim and Kende
2004). Likewise, the atgrf5 mutant plants exhibit narrow-
leaf phenotypes due to decreased cell number and
overexpression of AtGRF5 enhances cell proliferation
in leaf primordial and increased leaf growth (Horiguchi
et al. 2005). Using yeast two-hybrid screens, it has
been shown that both AtGRF1 and AtGRF5 interact with
GRF-interacting factor1 (GIF1), a functional homolog of
the human SYT transcription coactivator (Horiguchi et
al. 2005, Kim and Kende 2004). Like grf mutants, the
loss-of-function gif1 mutant and transgenic RNAi plants
developed narrower leaves due to a reduction in cell
numbers along the leaf-width axis than did wild-type
plants (Kim and Kende 2004). Combinations of gif1 and
grf mutations showed a cooperative effect (Kim and
Kende 2004). Overexpression of AtGIF1, on the other
hand, has larger leaves and cotyledons than wild-type
plants (Horiguchi et al. 2005). Thus, GRFs and GIF1
function as complexes of transcription factors involved
in regulating the growth and shape of leaves (Kim and
Kende 2004).
Although it has been predicted and verified that
miR396 targets six GRF genes, there has been no
molecular genetic analysis that directly address the role
of miR396 in the regulation of GRF gene expression and
plant leaf growth. In the present study, we have used
the northern blot hybridizations approach to analyze the
expression of MIR396. To analyze the role of miR396
directly in the regulation of target genes and leaf growth,
224
Physiol. Plant.136, 2009

Page 3

we have generated transgenic Arabidopsis plants that
constitutively overexpress MIR396a or MIR396b. These
transgenic plants have narrow-leaf phenotypes due to
reduction in cell number. The narrow-leaf phenotype
of the transgenic plants was associated with repressed
expression of not only predicted six GRF target genes
but also GIF1, which encodes a GRF-interacting protein.
In addition, transgenic MIR396-overexpressing plants
have lower densities of stomata and are more tolerant
to drought than wild-type plants. These results strongly
support the role of miR396 in the regulation of plant leaf
growth and development through repressing GRF gene
expression.
Materials and methods
Plant material and growth conditions
Arabidopsis thaliana accession Columbia gl-1 was used
as wild type and is the genetic background for transgenic
plants. The grf triple mutants and an3 mutant were
identified in the Wassilewskija (Ws) accession and
the Columbia (Col), respectively. Seeds were surface-
sterilized and sown on plates containing MS media (3%
sugars, 0.01% vitamin B1 and 0.05% vitamin B6) and
0.9% agar. Plates were stratified in darkness at 4◦C
for 2 days and then transferred to a tissue culture box
at 28◦C for about 6–7 days. Seedlings (6–7 days old)
grown on MS agar plates were transferred to soil under
normal conditions (14-h light, 23◦C/10-h dark, 20◦C).
Three-week-old wild-type plants grown on soil under
normal conditions were treated by drought stress.
Microscopic observations of leaf cells
The fifth leaves were separated from the Vector,
35S:MIR396a(T2),35S:MIR396b(T2),an3,Wswildtype,
grf1-2 grf2 grf3 plants. The leaves and palisade cells in
the subepidermal layer were observed by confocal laser
scanning microscopy (Zeiss LSM). The image of leaf
cells in the subepidermal layer were chloroplast auto
florescence images taken by confocal laser scanning
microscopy. Palisade cells in the center of the leaf blade,
betweenthemidveinandtheleafmargin,wereanalyzed.
The number of palisade cells per unit area (mm2) of this
region was determined.
Plasmid construction
A 544bp genomic sequence containing the MIR396a
foldback was amplified from Arabidopsis genomic DNA
by PCR using two primers (5’-TGCTGTAAAAGAATGAC
CCTT-3?and 5’-AAACTCATAGACAGAAGTTAGGGTT-
3’) and cloned into pUCm-T Vector (Fermentas), giving
pT-MIR396a. The sequence of the amplified DNA
fragment was verified by sequencing. A SacI-XbaI
fragment from pT-MIR396a containing the MIR396a
sequence was then subcloned into the XbaI and SacI
sites of pOCA30 between the CaMV 35S promoter and
the NOS 3’poly (A) signal to generate the 35S:MIR396a
construct. The MIR396b sequence was amplified by PCR
using two primers (5’-TCTTTCAGTCCCACGCTACT- 3?
and 5’-TGGATCTAAAGAGTTATCCTGTGT -3’) and the
35S:MIR396bconstructwasgeneratedbyusingthesame
procedure used for the 35S:MIR396a construct.
Arabidopsis transformation
The constructs 35S:MIR396a and 35S:MIR396b were
transformed into Agrobacterium and selected on Luria-
Bertani medium containing gentanycin at 40 μg/ml or
spectinomycin at 100 μg/ml. Arabidopsis transforma-
tion was performed using the floral-dip procedure as
previously described (Clough and Bent 1998). Transfor-
mants were identified by screening on medium con-
taining 50 μg/ml kanamycin. We obtained 19 indepen-
dent 35S:MIR396a transgenic lines and 23 independent
35S:MIR396b transgenic lines, respectively. T2transfor-
mants of the 35S:MIR396 transgenic plants were used in
all experiments unless otherwise indicated.
Northern blot hybridizations
Total RNA was extracted from plant tissues with Trizol
reagent (Invitrogen). Total RNA was separated on 1.5%
formaldehyde-MOPS agarose gels and blotted onto
Nylon membranes. Hybridization was performed at
68◦C with PerfectHybTMPlus buffer (Sigma-Aldrich).
Probes were labeled with32P-dATP by Klenow fragment
(Takara). Blots were washed once in 2×SSC and 0.5%
SDSfor10 minat 68◦C,twicein0.5×SSCand0.1% SDS
for 20 min at 68◦C, and once 0.1×SSC and 0.1% SDS
for 20 min at 68◦C.
For analysis of small RNAs, 20 μg total RNA
was separated on a denaturing 15% polyacrylamide
/1×TBE/7 M urea gel and transferred to Nylon transfer
membranes. Hybridization was performed at 35◦C with
PerfectHybTMPlus buffer (Sigma-Aldrich). Probes were
labeled with
transferase (Takara). Blots were washed twice in 2×SSC
and 1% SDS for 20 min at 35◦C.
The intensity of the hybridization bands was assessed
with a Bio-profile Bio 1D image analyzer (Vilber
Lourmat, Marne La Vall´ ee, France).
32P-dATP by terminal deoxynucleotidy
Physiol. Plant.136, 2009
225

Page 4

RT-PCR analysis
Total RNA was extracted from 3-weeks-old plants
using the protocol described by Chomczynski and Sac-
chiand (Chomczynski and Sacchi 1987) and treated
with DNAse (Ferments). cDNA was synthesized from
2 × 10−7g RNA by reverse transcription with 2 ×
10−8g primer using OneStep reverse transcriptase
polymerace chain reaction (RT-PCR) kit (Ferments).
PCR was run for approximately 27 to 30 cycles
using AtActin2 genes internal standards. RT-PCR was
performed using the following gene-specific primer:
AtGRF1, 5’-GATTACCTCCCATGGGAAAA-
5’-TCCTCCTAAACCATATCCTGAGT-3’; AtGRF2, 5’-
CATCTTTAGCTATCTTCCTCCCA- 3?and 5’-GTAAGAA
GGTGGAGGAATCTGG-3’; AtGRF3, 5’-AGATGAAGC
AAGAAAGCAACA- 3?and 5’-AGGAAATTTGGATAAA
AACCAAA- 3?; AtGRF7, 5’-TTTACTGTATTGGCAACA
ACAGA- 3?and 5’-TACGATTCGATATCACCAGAGA-3’;
AtGRF8, 5’-ACTGAGGTGGTTTACGAAAGAA- 3?and
5’-AGCTTCTGCTGCAGCTACTAGTA-3’; AtGRF9, 5’-A
CGTGGTTGTTTTCGTTTACA- 3?and 5’-CACCTGGT
GAAAACAAAGACA-3’. Each RT-PCR experiment was
repeated at least three times with similar results.
3?
and
Analysis of stomatal density
For the analysis of stomatal density, epidermis was
separated from the mature rosette leaves of control and
miR396-overexpressing Arabidopsis transgenic plants.
After removing mesophyll cells with a little brush, the
epidermis was stained with 0.5% safranine (in 95%
ethanol) for 10–20 s, and excess dye was rinsed off with
water. The stomatal number from a view field of a given
area was counted under a light microscopy and the
density was calculated.
Water loss measurement
For water loss measurement, 38 fully expanded rosette
leaves of the control and miR396-overexpressing
transgenicplantsweredetachedandweighedat different
times to determine the rate of water loss.
Drought treatment
Plants were grown for 3 weeks under normal conditions
(14-h light, 23◦C /10-h dark, 20◦C) with daily watering.
Watering was then withheld for 14 days for drought
treatment, followed by rewatering. The survival rates
of plants were scored 4 days after rewatering. The
experimentswererepeatedfivetimeswithsimilarresults.
Ten plants were tested in each experiment.
Results
Sequences and expression of miR396
The family of miR396 miRNAs was previously identified
by a computational screen of the Arabidopsis and rice
genome sequences (Jones-Rhoades and Bartel 2004). In
Arabidopsis, the stem-loop hairpins for miR396 miR-
NAs are encoded by two transcribed genes: MIR396a
(At2g10606) and MIR396b (At5g35407). Based on their
mapped 5?ends determined from PCR of small cDNAs
and the mobility on northern blots, miR396a (UUC-
CACAGCUUUCUUGAACUG) and miR396b (UUC-
CACAGCUUUCUUGAACUU) are 21 nt in length and
differ only in their last nucleotide (Jones-Rhoades and
Bartel 2004).
miRNAs negatively regulate gene expression by
degrading coding mRNAs or suppressing gene trans-
lation by perfect or near-perfect complement to target
mRNAs (Carrington and Ambros 2003). miR396 shares
nearly perfect complementarity with seven members of
the Arabidopsis growth-regulating factor (AtGRF) gene
family except GRF5 and GRF6 (Fig. 1). And Jones-
Rhoades and Bartel (2004) validated the predicted seven
GRF genes except GRF4 as targets of miR396 by 5’-
RACE experiments. Predicted and confirmed targets of
miR396 include six members of the Arabidopsis AtGRF
gene family that encodes putative transcription factors
and play regulatory roles in growth and development of
leaves and cotyledons (Jones-Rhoades and Bartel 2004,
Jones-Rhoades et al. 2006). GRF proteins interact with
AN3 (also known as GIF1), a putative transcription coac-
tivator also with a role in plant leaf growth (Horiguchi
et al. 2005, Kim and Kende 2004). It has been shown
that AN3 and AtGRF1- AtGRF3 were expressed in as
seedlings of 5-day-old plants (Horiguchi et al. 2005).
To examine the expression profile of miR396 in wild-
type Arabidopsis tissues, samples of total RNA (20 μg)
from roots, seedlings, rosette leaves, siliques and inflo-
rescences were analyzed. Fig. 2 illustrated that miR396
was broadlyan expressionprofileandstronglyexpressed
in 6-day-old seedlings and rosette leaves.
Plant growth phenotypes of transgenic
MIR396-overexpressing plants
To analyze the biological function of miR396 directly,
we tried to overexpress miR396 in transgenic Arabidop-
sis plants. We amplified the MIR396a and MIR396b
precursor sequences (Fig. 1) by PCR, inserted them
behind the enhanced Cauliflower mosaic virus (CaMV)
35S promoter in a plant transformation vector (pOCA30)
and transform the constructs into Arabidopsis. Trans-
genic 35S:MIR396a and 35S:MIR396b plants were then
226
Physiol. Plant.136, 2009

Page 5

Fig. 1. The degree of miR396 complementarity to all AtGRFs. Free energies of duplex structures were calculated using RNAhybrid software
(http://bibiserv.techfak.uni-bielefeld.de/rnahybrid).
Physiol. Plant.136, 2009
227

Page 6

Fig. 2. Expression pattern of miR396 in Arabidopsis. RNA gel blot
analysis was performed with total RNA (20 μg per lane) isolated from
6-day-old seedlings, roots, rosette leaves, siliques and inflorescences.
Ethidium bromide staining of 5S RNA / tRNA is shown at the bottom as
a loading control.
analyzed by RNA blot analysis for levels of MIR396
precursor transcripts and miR396 miRNAs. As shown
in Fig. 3(A), MIR396a and MIR396b precursor tran-
scripts were undetectable in control transgenic plants
(transformed with an empty vector) but were accumu-
lated to high levels in the transgenic 35S:MIR396a and
35S:MIR396b plants, respectively. Likewise, although
low levels of miR396 were present in control plants,
they were higher in the transgenic 35S:MIR396a and
35S:MIR396b plants (Fig. 3(A)). Thus, overexpression
of the synthetic MIR396a and MIR396b sequences
Table 1. Leaf phenotype of transgenic plants. Plants aged 3 weeks old
plants were used. Average rates and standard errors were calculated
using results of five replicated experiments. The experiments were
repeated five times with similar results. The numbers of plants examined
in the five replicated experiments were 42, 36, 38, 40 and 37. Fully
expanded fifth leaves are characterized using leaf index (the ratio of the
length to the width of leaf blade).
Transgenic plants Slightly narrow leaves (%)Narrow leaves (%)
35S::MIR396a
35S::MIR396b
17.6 ± 0.8
0
82.4 ± 2.1
100
enhanced production of MIR396a and MIR396b precur-
sor transcripts, which underwent appropriate maturation
to generate elevated levels of miR396 in transgenic
Arabidopsis.
As shown in Table 1 and Fig. 3(B), the most
striking phenotype of the transgenic 35S:MIR396a
and 35S:MIR396b plants were their narrow leaves.
In addition, transgenic 35S:MIR396a or 35S:MIR396b
plants were substantially shorter than control plants at
mature, seed-setting stages (Fig. 3(C)). It should be noted
that although the width and length of the leaf blades
and the length of petioles of transgenic 35S:MIR396a
and 35S:MIR396b plants were all reduced to various
extents, the most prominent phenotype was the reduced
leaf width (Table 2 and Figs 3(B) & 4). Thus, when
(A) (B)
(C)
Fig. 3. Narrow-leaf and dwarf phenotypes of transgenic plants overexpressing miR396 precursors. (A) Expression level of MIR396 (precursor) and
miR396 in transgenic plants (T1, 3 weeks old). For miRNA gel blots, 20 μg of total RNA was used. Four replicated experiments were performed and the
results were similar. (B)transgenic miR396 overexpression plant leaves were narrower than vector transgenic plants (T2; 4 weeks old). (C) transgenic
miR396 overexpression plants were shorter than vector transgenic plants (T2; 6 weeks old).
228
Physiol. Plant.136, 2009

Page 7

Table 2. Effect of miR396 on leaf development. Fully expanded fifth leaves are characterized (n = 10). Data are mean ± SD. Leaf index is the ratio
of the length to the width of leaf blade. The experiments were repeated three times with similar results.
Genotype Leaf length (mm)Leaf width (mm) Leaf indexLeaf petiole (mm)
Vector
35S::MIR396a
35S::MIR396b
24.2 ± 2.1
19.8 ± 2.8
20.6 ± 2.7
10.5 ± 0.9
7.2 ± 1.0
7.1 ± 0.9
2.3 ± 0.2
2.7 ± 0.2
2.9 ± 0.2
19.7 ± 3.7
16.3 ± 2.4
15.5 ± 2.4
(A) (B)
(C)
Fig. 4. Comparison of leaf phenotypes among vector control plants (Col), 35S:MIR396a (T2), 35S:MIR396b (T2), an3 mutant, Ws wild type and
grf1/2/3 triple mutants. [(A), 3-week-old plants were used. Fully expanded first and second leaves are characterized; (B) and (C): 4-week-old plants
were used. 1, Col; 2, 35S:MIR396a; 3, 35S:MIR396b; 4, an3; 5, grf1-2 grf2 grf3; 6, Ws]. The data shown are the mean ± SD. Three replicated
experiments were performed and the results were similar.
compared with those of control plants, the length of
the fifth true leaves of transgenic 35S:MIR396a and
35S:MIR396b plants was reduced on average by only
about 15%, but the width was educed by more than
30%. As a result, the leaf index (the ratio of the leaf
length to the leaf width) was increased from 2.3 ± 0.2 in
control plants to 2.7 ± 0.2 and 2.9 ± 0.2 in transgenic
35S:MIR396a and 35S:MIR396b plants, respectively
(Table 2). Furthermore, in transgenic 35S:MIR396a
plants, the expression levels of miR396 were correlated
withtheextentofnarrow-leafphenotype.Approximately
17%
35S:MIR396atransgenic
increased levels of miR396 and their leaves were only
slightly narrower than those of control plants (Table 1,
Fig. 5). The narrow-leaf phenotypes of transgenic
35S:MIR396aand 35S:MIR396b plants were very similar
plants had slightly
to those of an3 mutant or GIF1-RNAi transgenic plants
and mildly similar to those of the grf1/2/3 triple null
mutant (Fig. 4) (Kim et al. 2003, Kim and Kende 2004).
Repression of putative target genes by
overexpressed miRNA396
Predicted and confirmed targets of miR396 include six
members of the AtGRF gene family (AtGRF1-AtGRF3,
and AtGRF7-AtGRF9) (Jones-Rhoades and Bartel 2006).
To examine the effect of miR396 overexpression on
expression of the putative target genes, we analyzed
their transcript levels in the transgenic MIR396-
overexpressing plants using both RNA blotting and/or
RT-PCR. RNA gel blot analysis showed that in
35S:MIR396 transgenic plants, miR396 levels were
increased by several-fold when compared with that
Physiol. Plant.136, 2009
229

Page 8

(A)
(B)
Fig. 5. The phenotype of mildly narrow leaves were due to mild increase of miR396 expression in 35S:MIR396a transgenic plants. (A) Leaves of
transgenic plant line 11 was mildly narrower than those of control plants (4-week-old plants, T2). (B) Northern analysis of miR396 in transgenic line
11. For miRNA gel blots, 20 μg of total RNA was used. Three replicated experiments were performed and the results were similar.
(A)
(B)
(C)
Fig. 6. Overexpression of miR396 repressed GRF1- GRF3, GRF7-GRF9
expression. (A) Northern analysis of GRF1- GRF3. (B) Northern analysis of
miR396.(C) RT-PCRanalysis ofGRF1-GRF3, GRF7-GRF9. Total RNAfrom
3-week-old transgenic plants (T2) of vector control, 35S::MIR396a and
35S:MIR396b wasextracted. For RNA and miRNAgel blotanalysis, 20 μg
of total RNA was used. Four replicated experiments were performed with
similar results. For RT-PCR, cDNA was synthesized from 2 × 10−7g RNA
by reverse transcription with 2 × 10−8g primer using OneStep RT-PCR
kit (Ferments). PCR was run for about 27 to 30 cycles using AtActin2
genes internal standards. At least three replicated experiments were
performed with similar results.
in the control plants harboring only an empty vector.
In the same transgenic plants, the levels of AtGRF1,
AtGRF2 and AtGRF3 transcripts were 2–14-folds lower
than those in control plants (Fig. 6). Similar results
were obtained from analysis using RT-PCR (Fig. 6). In
addition, AtGRF7, AtGRF8 and AtGRF9 mRNA levels
were reduced in transgenic 35S:MIR396 plant leaves
relative to those in control plants (Fig. 6). miR396 shares
nearly perfect complementarity with AtGRF4, we also
tested the expression of AtGRF4 by RT-PCR. The results
showed that AtGRF4 mRNA levels were also reduced
in transgenic 35S:MIR396 plant leaves compared with
control plants (Fig. 6).
GRFs and AN3 physically interact and functionally
cooperate in regulating the development of leaf shape
(Horiguchi et al. 2005, Kim and Kende 2004). Therefore,
we also analyzed the transcript levels of AN3 in
transgenic 35S:MIR396a and 35S:MIR396b transgenic
plants. Both RNA gel blot analysis and RT-PCR analysis
showed that the expression levels of AN3 were
significantly reduced in the transgenic 35S:MIR396a and
35S:MIR396b plants (Fig. 7).
Effects of MIR396 overexpression on leaf cell
number and size
AtGRFs play a regulatory role in growth and develop-
ment of leaves in a functionally redundant manner (Kim
et al.2003,Kim and Kende2004).TheN-terminalregion
of AN3 protein was involved in the interaction with
230
Physiol. Plant.136, 2009

Page 9

(A)
(B)
Fig. 7. Overexpression of miR396 repressed the expression of AN3.
Total RNA of various 3-week-old plants (T2) was extracted and analyzed
for gene expression by RT-PCR. Three replicated experiments were
performed with similar results.
GRF1 (Kim and Kende 2004). In addition, the narrow-
leaf phenotype of an3, as well as that of the grf1/2/3
triple mutant, resulted from a reduction in cell numbers
along the leaf-width axis (Kim and Kende 2004). Since
AtGRF1-AtGRF3 and AN3 mRNA levels were decreased
in transgenic 35S:MIR396a and 35S:MIR396b plants,
the narrow-leaf phenotype of these transgenic plants
might result from reduces cell proliferation as observed
in the grf and an3 mutant plants. To examine this pos-
sibility, we compared the paradermal views of palisade
cells of the fifth leaves from control, 35S:MIR396a and
35S:MIR396b transgenic plants. We observed that the
palisade cells were actually larger in 35S:MIR396a and
35S:MIR396b transgenic plants than those in the vector
control plants (Fig. 8). Thus, the narrow-leaf phenotype
of the transgenic miR396-overexpression plants were
caused by reduced cell proliferation but not reduced
cell expansion. As a result, the cell density in the leaf-
width direction of palisade cells in the subepidermal
layer was significantly reduced in both 35S:MIR396a
and 35S:MIR396b transgenic plants when compared
with that of empty vector control plants (Fig. 8). These
phenotypes of transgenic miR396-overexpressing plants
were strikingly similar to the an3 mutant plants and
mildly similar to those of the grf1/2/3 triple null mutant
(Fig. 8).
Reduced stomata density and increased drought
tolerance in miR396 overexpressing plants
Stomatal density is affected by cell density (number
of cells per unit area) (Wang et al. 2007). The
reduction of cell density in transgenic 35S:MIR396a
and 35S:MIR396b plants suggested that miR396 may
affect stomatal density. Indeed the stomata density
of transgenic 35S:MIR396a and 35S:MIR396b plants
were decreased by 26.7 and 36.2%, respectively, when
(A)
(B)
Fig. 8. Overexpression of miR396 decreased cell number but increased
cell volume. For all experiments, 3-week-old plants were used.
(A) Paradermal views of palisade cells in the subepidermal layer in
fifth leaves. Vector, 35S:MIR396a (T2), 35S:MIR396b (T2), an3, Ws wild
type, grf1-2 grf2 grf3. Bar, 20 μm. (B) Palisade cell number of fifth
leaves. Vector, 35S:MIR396a (T2), 35S:MIR396b (T2), an3, Ws wild-type,
grf1-2 grf2 grf3 (n = 4). The data shown are the mean ± SD. Three
replicated experiments were performed and the results were similar.
compared with that of control plants (Fig. 9). Stomatal
density affects water transpiration, but it could not be
accurately correlated with transpiration rate (Wang et
al. 2007). Therefore, ‘stomatal index’ defined as a
proportion of stomatal cells to total epidermal cells,
has been more often used (Wang et al. 2007). We
found that stomatal indexes of transgenic 35S:MIR396a
and 35S:MIR396b plants were decreased by 19.3 and
30.3%,respectively,whencomparedwiththatofcontrol
plants (Fig. 9). Thus, overexpression of miR396 reduces
stomatal index.
Reduction of stomatal index decreases transpiration
rate (Wang et al. 2007). We found that transpiration
rate of transgenic 35S:MIR396a and 35S:MIR396b
plants were decreased when compared with that of
control plants (Fig. 10(C)). Therefore, overexpression
Physiol. Plant.136, 2009
231

Page 10

(A)
(B)
Fig. 9. Stomata number decreased in transgenic 35S:MIR396 plants.
(A) Stomatal number and stomatal index of the fifth leaves of transgenic
control, 35S:MIR396a (T2) and 35S:MIR396b (T2) plants (n = 10).
The data shown are the mean ± SD. Three replicated experiments
were performed and the results were similar. (B) Paradermal views of
stomata in the fifth leaves of transgenic control, 35S:MIR396a (T2) and
35S:MIR396b (T2) plants (Bar, 20 μm). For all experiments, 3-week-old
plants were used.
of miR396 may improve drought tolerance of the
transgenic plants. To test this possibility, 3-week-old
plants grown on soil were held without watering for
14 days before watered again (Fig. 10). While only
45% of the control plants survived after drought
treatment, 88% of transgenic 35S:MIR396a plants and
100% of transgenic 35S:MIR396b plants survived. Thus
overexpression of miR396 increased drought tolerance
in transgenic 35S:MIR396a and 35S:MIR396b plants.
In light of the enhanced drought tolerance in
MIR396-overexpressed plants, we examined whether
the expression of miR396 was responsive to drought
stress. Wild-type plants (Col-0) (3 weeks old) grown in
soil were subjected to drought stress by withholding
watering and the levels of miR396 were determined
by RNA blot analysis. As shown in Fig. 11, the level
of miR396 was slightly increased in 1 day of watering
withholding and was further elevated 2 days later. The
levels of miR396 remained substantially higher after 4
and 5 days of drought stress than those detected prior
to drought stress treatment. These results indicated that
the level of miR396 was induced by drought stress. We
also analyzed the expression of AtGRF1-AtGRF3 and
AN3 in response to drought stress. Although the levels of
transcripts for these genes somewhat fluctuated during
the period of drought treatment, the transcript levels for
AtGRF1, AtGRF3 and AN3 were significantly lower after
4 days of watering withholding than those detected at
the beginning of drought stress. These results suggested
that increased expression of miR396 might have effects
on expression of its target genes during drought stress.
(A)
(B)
(C)
Fig. 10. Overexpression of miR396 increased drought tolerance in
transgenic plants. (A) Photographs of control, 35S:MIR396a (T2) and
35S:MIR396b (T2) transgenic plants before drought, after drought
and rewatering treatments. (B) Survival rates of transgenic plants of
control, 35S:MIR396a (T2) and 35S:MIR396b (T2) exposed to drought
stress. 3-week-old plants grown on soil were held without water for
14 days before the plants were watered again. Average survival rates
and standard errors were calculated from 50 plants in 5 replicated
experiments. (C) Water loss percentages from leaves of vector control
plants, 35S::MIR396a (T2) and 35S::MIR396b (T2) transgenic plants.
Excised leaves from vector control or MIR396-overexpressing plants
were assayed for water loss. Both vector and transgenic plants (T2)
were grown under normal conditions for 3 weeks and then subjected
to drought stress. Data represent means ± SD of four independent
experiments (38 measurements per point).
To further analyze the molecular basis of enhanced
drought tolerance of the MIR396-overexpressing plants,
232
Physiol. Plant.136, 2009

Page 11

Fig. 11. Expression of miR396, AN3 and AtGRF during drought stress (3-week-old wild-type plants). Total RNA of various 3-week-old plants was
extracted. For RNA and miRNA gel blots, 20 μg of total RNA was used. Band signals of the RNA blots were quantified through image analysis and
shown for each of the analyzed genes. Three replicated experiments were performed and the results were similar.
we also analyzed expression of drought stress-induced
COR15,COR47 andRD22.
transgenic 35S:MIR396a and 35S:MIR396b plants were
subjected to drought stress by withholding watering for
3, 6 and 9 days. As shown in Fig. 12, the control and
transgenic plants exhibited similar induction patterns of
these genes after the drought treatment.
Control plants and
Discussion
In the present study, we have examined the expression
of miR396 and generated transgenic Arabidopsis plants
that constitutively overexpress MIR396a or MIR396b
to analyze their role in the regulation of target genes
and leaf growth. A plant leaf blade has three axes, the
proximo-distal (longitudinal), central-lateral (transverse)
and adaial-abxial axes. Polarized cell proliferation and
cell expansion along the three axes determine the final
size and shape of plant leaves (Tsukaya 2003). Genetic
studies in Arabidopsis have provided evidence that cell
proliferation in leaf primordial is controlled in an axis-
dependent manner (Tsukaya 2006). In addition, polar
cell expansion along the longitudinal axis is regulated
independently of that along the transverse axis (Tsukaya
2006). Transgenic MIR396-overexpressing plants have
narrow-leaf phenotypes largely due to reduction in
cell number (Figs. 3 and 9). These results indicate that
miR396 negatively regulates cell proliferation in leaf
primordial that ultimately determine the cell number
along the longitudinal axis. This phenotype is consistent
with the preferential expression of miR396 in seedlings
and leaves (Fig. 2). Interestingly, we observed that the
narrow-leaf phenotype of the miR396-overexpressing
plants was associated not only with reduced cell number
but also increased cell size (Fig. 8). This is a typical
compensation phenotype in which a decrease in the cell
number is accompanied by an increase in the cell size
(Horiguchi et al. 2006). The increased cell expansion
mayresultfromacompensatorymechanismforinhibited
cell proliferation in the narrow-leaf plants.
We have also shown that the miR396-overexpressing
plants also exhibited repressed expression of six GRF
genes (Figs 6), supporting that six GRF genes are targets
of miR396 based on computational prediction and PCR-
based cleavage assays. Molecular genetic analysis using
both loss-of-function mutants and transgenic overex-
pression lines has indicated that plant GRF transcription
factorsregulateplantleafgrowth(HoriguchiandTsukaya
Physiol. Plant.136, 2009
233

Page 12

Fig. 12. Overexpression of miR396 did not affect the expression of
drought stress-induced COR15, COR47, RD22 during drought. Control
and transgenic plants (3-weeks old) were subjected to drought stress
and total RNA of was extracted at indicated days after initiating drought
treatment. For RNA gel blots, 20 μg of total RNA was used. The
experiment was repeated once with similar results.
2004, Kim et al. 2003, Kim and Kende 2004). Loss-of-
function mutants for some of the Arabidopsis GRF genes
exhibit the narrow-leafphenotypessimilar to those of the
transgenic MIR396-overexpressing plants. These results
strongly suggest that miR396 plays an important role
in plant leaf growth and development most likely by
repressing GRF gene expression. Interestingly, genetic
analysis with triple mutant for AtGRF1-AtGRF3 indicates
that they play a role in leaf growth by regulating cell
expansion (Kim et al. 2003). On the other hand, similar
molecular genetic analysis with AtGRF5 suggests that it
plays a role in leaf growth primarily by regulating cell
proliferation in leaf primordial (Horiguchi et al. 2005).
These studies suggest that different members of the GRF
gene family play distinct roles in polarized cell prolifera-
tion and cell expansion along the longitudinal axis. The
narrow-leaf phenotype of the MIR396-overexpressing
plants was associated not only with reduced cell num-
ber but also increased cell size. The effects on both cell
proliferation and cell expansion by overexpression of
miR396 may result in repression of multiple GRF genes
in these transgenic plants. However, the opposite phe-
notypes in reduced cell number but increased cell size
of the transgenic MIR396-overexpressing plants suggest
a regulatory mechanism that can compensate reduced
cell proliferation through increased cell expansion. This
would in turn suggest that cell proliferation and cell
expansion are not completely independently regulated.
In addition to GRF genes, we observed repressed
expression of AtGIF1 in the miR396-overexpressing
Arabidopsis plants (Fig. 7). The miR396-overexpressing
plants showed the narrow-leaf phenotype closely similar
to an3 mutant plants. This is very similar to phenotypes
of GIF1-RNAi transgenic plants reported by Kim
and Kende (2004). AtGIF1 encodes a GRF-interacting
protein that is homologous to the human transcription
coactivator SYT (Horiguchi et al. 2005). Mutations and
overexpression of AtGIF1 led to altered leaf growth
similar to those caused by mutations and overexpression
of some GRF genes, suggesting that AtGIF1 and some
GRF transcription factors function cooperatively in the
regulation of plant leaf growth (Horiguchi et al. 2005,
Kim and Kende 2004). Since AtGIF1 gene contains no
sequence complementary to miR396, its repression by
overexpression of the miRNA is probably indirect. As six
GRF genes are directly targeted by miR396, repressed
expression of AtGIF1 by overexpression of miR396 may
be mediated by repressed expression of some of the
targeted GRF genes. Thus, in addition to their physical
interactionsthat may be important for their transcription-
activating activities, AtGRF and AtGIF1 factors may
functionally interact in the regulation of leaf growth
through transcriptional activation of their respective
genes.
Unexpectedly,we have
MIR396-overexpressing plants are more tolerant to
drought than wild-type plants (Fig. 10). To examine the
molecular basis for the enhanced drought tolerance, we
have compared wild type and miR396-overexpressing
plants for expression of drought stress-regulated COR15,
COR47 and RD22 genes and found no significant differ-
ence (Fig. 12). These results suggest the overexpression
of miR396 does not induce drought tolerance by activat-
ingplantdroughtstress responsepathways.Significantly,
we have observed that leaves of MIR396-overexpressing
plants have lower densities of stomata than wild-type
plant leaves (Fig. 9). Stomatal density is affected by cell
density (Wang et al. 2007). In transgenic 35S:MIR396a
and 35S:MIR396b plants, overexpression of MIR396
causedthereductionofcell densityby repressingexpres-
sion of not only six GRF genes but also GIF1 encoding a
GRF-interacting transcription coactivator with a role in
cell proliferation in leaf. The ‘stomatal index’ is defined
as a proportion of stomatal cells to total epidermal
cells (Wang et al. 2007). Overexpression of miR396
reduces stomatal index in transgenic 35S:MIR396a and
35S:MIR396b plants (Fig. 9). This suggested that the
reduction of stomatal density affected stomatal index.
Stomatal index is accurately correlated with transpira-
tion rate (Wang et al. 2007).Reduced stomatal index
would reduce water loss during drought, thereby mak-
ing plants more tolerant during water shortage. The
observed effects on stomata number by overexpression
of miR396 suggest that this miRNA may play important
roles not only in cell proliferation and cell expansion but
also in cell differentiation in plant leaf growth. The capa-
bility of drought tolerance of atgif1/2/3 triple mutants
and an3 mutants was not altered under our experi-
mental condition due to only slight reduction in the
stomata density (data not shown). Therefore, miR396-
overexpressing plants have both reduced stomata index
found thattransgenic
234
Physiol. Plant.136, 2009

Page 13

and increased drought tolerance relative to those of con-
trol transgenic plants, probably due to combined effects
of the decreased expression level of AtGRF gene family
and AN3 gene.
Acknowledgements – We thank Dr. G. Horiguchi at the
National Institute for Basic Biology at Japan for Arabidopsis
grf5, grf9 and an3-4 mutants, and Jeong Hoe Kim at
Department of Energy Plant Research Laboratory, Michigan
State University for Arabidopsis grf1-2, grf2,grf3 and grf1-3
triple mutants. We also thank Zhou X., Fu Q. Li L.,
Jiang X. for technical assistance in paradermal view of
stomata. This study was supported by the National High
Technology Research and Development Program of China
(863 Program) (2006AA02Z129), National Natural Science
Foundation of China (90408022), Science Foundation of
Yunnan Province (2004C0051M), and ‘Hundred talents’
Program of the Chinese Academy of Sciences.
References
Achard P, Herr A, Baulcombe DC, Harberd NP (2004)
Modulation of floral development by a gibberellin-
regulated microRNA. Development 131: 3357–3365
Aukerman MJ, Sakai H (2003) Regulation of flowering time
and floral organ identity by a microRNA and its
APETALA2-like target genes. Plant Cell 15: 2730–2741
Bartel DP (2004) MicroRNAs: genomics, biogenesis,
mechanism, and function. Cell 116: 281–297
Bartel B, Bartel DP (2003) MicroRNAs: at the root of plant
development? Plant Physiol 132: 709–717
Carrington JC, Ambros V (2003) Role of microRNAs in
plant and animal development. Science 301: 336–338
Chen XM (2004) A microRNA as a translational repressor
of APETALA2 in Arabidopsis flower development.
Science 303: 2022–2025
Chen J, Li WX, Xie DX, Peng JR, Ding SW (2004) Viral
virulence protein suppresses RNA silencing-mediated
defense but upregulates the role of MicroRNA in host
gene expression. Plant Cell 16: 1302–1313
Chomczynski P and Sacchi N (1987) Single-step method of
RNA isolation by acid guanidinium thiocyanate-
phenol-chloroform extraction. Anal Biochem 162:
156–159
Clough SJ and Bent AF (1998) Floral dip: a simplified
method for Agrobacterium-mediated transformation of
Arabidopsis thaliana. Plant J 16: 735–743
Duan CG, Wang CH and Guo HS (2006) Regulation of
microRNA on plant development and viral infection.
Chin Sci Bull 51: 269–278
Guo HS, Xie Q, Fei JF, Chua NH (2005) MicroRNA directs
mRNA cleavage of the transcription factor NAC1 to
downregulate auxin signals for Arabidopsis lateral root
development. Plant Cell 17: 1376–1386
Horiguchi G, Tsukaya H (2004) Role of ANGUSTIFOLIA3
and its interacting proteins for cell proliferation-
dependent leaf morphogenesis. Plant Cell Physiol 45:
S163–S163
Horiguchi G, Kim GT, Tsukaya H (2005) The transcription
factor AtGRF5 and the transcription coactivator AN3
regulate cell proliferation in leaf primordia of
Arabidopsis thaliana. Plant J 43: 68–78
Horiguchi G, Ferjani A, Fujikura U, Tsukaya H (2006)
Coordination of cell proliferation and cell expansion in
the control of leaf size in Arabidopsis thaliana. J Plant
Res 119: 37–42
Jones-Rhoades MW, Bartel DP (2004) Computational
identification of plant MicroRNAs and their targets,
including a stress-induced miRNA. Mol Cell 14:
787–799
Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs
and their regulatory roles in plants. Annu Rev Plant Biol
57: 19–53
Kim JH, Kende H (2004) A transcriptional coactivator,
AtGIF1, is involved in regulating leaf growth and
morphology in Arabidopsis. Proc Natl Acad Sci USA
101: 13374–13379
Kim JH, Choi DS, Kende H (2003) The AtGRF family of
putative transcription factors is involved in leaf and
cotyledon growth in Arabidopsis. Plant J 36: 94–104
van der Knaap E, Kim JH and Kende H (2000) A novel
gibberellin-induced gene from rice and its potential
regulatory role in stem growth. Plant Physiol 122:
695–704
Kutter C, Schob H, Stadler M, Meins F and Si-Ammour A
(2007) MicroRNA-mediated regulation of stomatal
development in Arabidopsis. Plant Cell 19: 2417–2429
Laufs P, Peaucelle A, Morin H, Traas J (2004) MicroRNA
regulation of the CUC genes is required for boundary
size control in Arabidopsis meristems. Development
131: 4311–4322
Llave C (2004) MicroRNAs: more than a role in plant
development? Mol Plant Physiol 5: 361–366
Llave C, Xie ZX, Kasschau KD and Carrington JC (2002)
Cleavage of Scarecrow-like mRNA targets directed by a
class of Arabidopsis miRNA. Science 297: 2053–2056
Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N,
Estelle M, Voinnet O and Jones JDG (2006) A plant
miRNA contributes to antibacterial resistance by
repressing auxin signaling. Science 312: 436–439
Palatnik JF, Allen E, Wu XL, Schommer C, Schwab R,
Carrington JC, Weigel D (2003) Control of leaf
morphogenesis by microRNAs. Nature 425:
257–263
Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel
DP (2002) MicroRNAs in plants. Genes Dev 16:
1616–1626
Reyes JL, Chua NH (2007) ABA induction of miR159
controls transcript levels of two MYB factors during
Physiol. Plant.136, 2009
235

Page 14

Arabidopsis seed germination. Plant J 49: 592–606
Sunkar R and Zhu JK (2004) Novel and stress-regulated
microRNAs and other small RNAs from Arabidopsis.
Plant Cell 16: 2001–2019
Tsukaya H (2003) Organ shape and size: a lesson from
studies of leaf morphogenesis. Curr Opin Plant Biol 6:
57–62
Tsukaya H (2006) Mechanism of leaf-shape determination.
Annu Rev Plant Biol 57: 477–496
Wang Y, Chen X, Xiang CB (2007) Stomatal density and
bio-water saving. J Intergrat Plant Biol 49: 1435–1444
Zhang BH, Pan XP, Cannon CH, Cobb GP, Anderson TA
(2006) Conservation and divergence of plant microRNA
genes. Plant J 46: 243–259
Edited by D. Van Der Straeten
236
Physiol. Plant.136, 2009