miR396-targeted AtGRF transcription factors are required for coordination of cell division and differentiation during leaf development in Arabidopsis.

Abstract

In plants, cell proliferation and polarized cell differentiation along the adaxial-abaxial axis in the primordium is critical for leaf morphogenesis, while the temporal-spatial relationships between these two processes remain largely unexplored. Here, it is reported that microRNA396 (miR396)-targeted Arabidopsis growth-regulating factors (AtGRFs) are required for leaf adaxial-abaxial polarity in Arabidopsis. Reduction of the expression of AtGRF genes by transgenic miR396 overexpression in leaf polarity mutants asymmetric leaves1 (as1) and as2 resulted in plants with enhanced leaf adaxial-abaxial defects, as a consequence of reduced cell proliferation. Moreover, transgenic miR396 overexpression markedly decreased the cell division activity and the expression of cell cycle-related genes, but resulted in an increased percentage of leaf cells with a higher ploidy level, indicating that miR396 negatively regulates cell proliferation by controlling entry into the mitotic cell cycle. miR396 is mainly expressed in the leaf cells arrested for cell division, coinciding with its roles in cell cycle regulation. These results together suggest that cell division activity mediated by miR396-targeted AtGRFs is important for polarized cell differentiation along the adaxial-abaxial axis during leaf morphogenesis in Arabidopsis.

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Journal of Experimental Botany, Vol. 62, No. 2, pp. 761–773, 2011
doi:10.1093/jxb/erq307Advance Access publication 29 October, 2010
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
miR396-targeted AtGRF transcription factors are required for
coordination of cell division and differentiation during leaf
development in Arabidopsis
Li Wang1,2, Xiaolu Gu1, Deyang Xu1, Wei Wang1, Hua Wang1, Minhuan Zeng1, Zhaoyang Chang2, Hai Huang1
and Xiaofeng Cui1,*
1National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
2College of Life Science, Northwest A & F University, Yangling, Shaanxi 712100, China
* To whom correspondence should be addressed. E-mail: xiaofeng@sippe.ac.cn
Received 11 June 2010; Revised 12 August 2010; Accepted 13 September 2010
Abstract
In plants, cell proliferation and polarized cell differentiation along the adaxial–abaxial axis in the primordium is
critical for leaf morphogenesis, while the temporal–spatial relationships between these two processes remain
largely unexplored. Here, it is reported that microRNA396 (miR396)-targeted Arabidopsis growth-regulating factors
(AtGRFs) are required for leaf adaxial–abaxial polarity in Arabidopsis. Reduction of the expression of AtGRF genes by
transgenic miR396 overexpression in leaf polarity mutants asymmetric leaves1 (as1) and as2 resulted in plants with
enhanced leaf adaxial–abaxial defects, as a consequence of reduced cell proliferation. Moreover, transgenic miR396
overexpression markedly decreased the cell division activity and the expression of cell cycle-related genes, but
resulted in an increased percentage of leaf cells with a higher ploidy level, indicating that miR396 negatively
regulates cell proliferation by controlling entry into the mitotic cell cycle. miR396 is mainly expressed in the leaf cells
arrested for cell division, coinciding with its roles in cell cycle regulation. These results together suggest that cell
division activity mediated by miR396-targeted AtGRFs is important for polarized cell differentiation along the
adaxial–abaxial axis during leaf morphogenesis in Arabidopsis.
Key words: Arabidopsis, AtGRFs, cell differentiation, cell division, leaf polarity, miR396.
Introduction
The development of multicellular organisms relies on the
generation of an appropriate number of cells and timely
acquisition of specialized cell functions. In flowering
plants, leaves are determinate organs, whose development
is mediated by the temporal–spatial regulation of cell
proliferation and differentiation (Gutierrez, 2005; Ramirez-
Parra et al., 2005; Fleming, 2006). Leaf morphogenesis is
conceptually divided into three processes: primordia initia-
tion, establishment of polarity, and leaf expansion (Sinha,
1999). After initiation from the peripheral zone of shoot
apical meristem (SAM), cells in leaf primordia rapidly
divide and are subsequently specified to establish asymmet-
ric growth along the proximo-distal, medio-lateral, and
adaxial–abaxial axes (Hudson, 2000; Bowman et al., 2002).
The establishment of the adaxial–abaxial axis is most
critical for leaf morphogenesis (Sussex, 1954; Waites and
Hudson, 1995; Bowman et al., 2002). In recent years,
several families of transcription factor genes have been
identified as the key determinants of adaxial or abaxial cell
fate. Members of the KANADI (KAN) and YABBY (YAB)
families and two AUXIN RESPONSE FACTOR genes
(ARF3/ETT and ARF4) play important roles in determination
of abaxial cell fate (Eshed et al., 2004; Pekker et al., 2005).
In contrast, three genes in the class III homeodomain-leucine
ª 2010 The Author(s).
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zipper (HD-ZIP III) family, namely PHB, PHV, and REV,
are expressed in the leaf adaxial domain and redundantly
promote adaxial leaf fate (McConnell et al., 2001; Emery
et al., 2003). Transcripts of HD-ZIP III genes are the
targets of microRNA165 and 166 (miR165/166) (Rhoades
et al., 2002; Tang et al., 2003), whereas ARF3/4 are targeted
by a trans-acting small interfering RNA (ta-siRNA), termed
tasiR-ARF (Allen et al., 2005; Fahlgren et al., 2006). More-
over, the Myb-domain protein ASYMMETRIC LEAVES 1
(AS1) and the LOB domain protein AS2 function together
to promote leaf adaxial identity by repressing the abaxial-
expressed KAN genes and miR165/166 (Byrne et al., 2000;
Semiarti et al., 2001; Iwakawa et al., 2002; Kumaran et al.,
2002; Xu et al., 2002, 2003; Lin et al., 2003; Li et al., 2005;
Wu et al., 2008), while KAN genes antagonize the roles of
HD-ZIP III genes (Emery et al., 2003) and directly repress
AS2 expression by binding to its promoter (Wu et al., 2008).
In addition, the 26S proteasome and ribosome proteins
were recently implicated to play important roles in promot-
ing the establishment of leaf polarity at the protein regula-
tion level (Huang et al., 2006; Pinon et al., 2008; Yao et al.,
2008).
In contrast to the adaxial–abaxial axis, only a few genes
were known to be involved in patterning of the proximo-
distal (leaf length direction) and medio-lateral (leaf width
direction) axes of leaves (Tsukaya, 2006). Growth along the
leaf length is governed by ROTUNDIFOLIA3 (ROT3),
which regulates polarized cell expansion (Kim et al., 1998),
and ROT4, which inhibits cell proliferation to regulate
specifically cell number in the leaf length direction (Narita
et al., 2004). Leaf expansion in the width direction is
affected by ANGUSTIFOLIA (AN) and AN3. AN regulates
cell expansion in the width direction probably through
controlling the cortical microtubule arrangement in leaf
cells (Folkers et al., 2002; Kim et al., 2002). AN3, which
encodes a putative coactivator for the Arabidopsis growth-
regulating factor (AtGRF) family of transcription factors
(J. H. Kim et al., 2003), also called AtGIF1 (GRF-
interacting factor1), predominantly controls cell number in
the leaf width direction by modulating cell proliferation
activity (Horiguchi and Tsukaya, 2003; Kim and Kende,
2004; Horiguchi et al., 2005). The Arabidopsis AtGRF
family consists of nine members, while AtGIF1 has another
two homologues, AtGIF2 and AtGIF3. Both AtGRF and
AtGIF genes appear to perform redundant functions to
promote and/or maintain cell proliferation activity in leaves
(J. H. Kim et al., 2003; Kim and Kende, 2004; Horiguchi
et al., 2005; Lee et al., 2009), whereas at least seven AtGRF
genes (AtGRF1–4 and AtGRF7–9) are targeted by miR396,
which is encoded by two loci, MIR396a and MIR396b, and
attenuate cell proliferation activity by repression of expres-
sion of the targeted AtGRF genes (Jones-Rhoades and
Bartel, 2004; Liu et al., 2009; Rodriguez et al., 2010).
Although a number of factors have been found to specify
leaf polarity, the temporal–spatial relationships of cell
proliferation and differentiation during leaf morphogenesis
remain largely unexplored. Recently, a novel gene, AS1/2
ENHANCER7 (AE7), which is required for both leaf
adaxial–abaxial polarity formation and normal cell pro-
liferation, was characterized. It was also found that the
previously characterized 26S proteasome mutant ae3-1
(Huang et al., 2006) and ribosome mutant ae5-1 (Yao
et al., 2008) exhibited not only leaf adaxial–abaxial polarity
but also cell proliferation defects, and it was therefore
proposed that normal cell proliferation may be essential for
establishment of leaf polarity (Yuan et al., 2010). However,
because the 26S proteasome and ribosome are widely
recognized as essential machinery for many biological
processes, and AE7 encodes a member of a functionally
unknown protein family conserved in eukarytoes, whether
this proposal is a general mechanism and how the functions
of these proteins in cell proliferation specifically regulate
leaf polarity need further investigation.
In this work, it is reported that cell proliferation mediated
by miR396-targeted AtGRFs is required for leaf adaxial–
abaxial polarity formation during leaf morphogenesis. It is
further shown that miR396 negatively regulates cell pro-
liferation in leaves by controlling the entry into the mitotic
cell cycle, coincident with its expression in leaf cells arrested
for cell division. Because the cells unable to enter into the
mitotic cell cycle often undergo enlargement and expansion
to start differentiation (Gutierrez, 2005, 2009; De Veylder
et al., 2007; Nieuwland et al., 2009), these data together
with previous results strongly suggest that active cell
division in the primordium is important for leaf adaxial–
abaxial polarity formation, and highlight that miR396-
targeted AtGRFs may mediate coordination processes of
cell division coupled with cell differentiation during leaf
morphogenesis.
Materials and methods
Plant materials and growth conditions
The as1-1, as2-1, and T-DNA insertion mutant atgif1/an3
(SALK_150407) were obtained from the ABRC. All the plant
materials used in this study are in the Columbia-0 (Col-0)
background. Plant growth was according to previous conditions
(Chen et al., 2000).
Plasmid construction and plant transformation
For transgenic miR396 overexpression, the 316 bp MIR396A and
MIR396B precursor fragments were PCR-amplified with genomic
DNA from wild-type Col plants. The DNA fragments were cloned
into a modified pCAMBIA2300 vector, behind the duplicated
cauliflower mosaic virus 35S promoter, to generate the constructs
35S:miR396a and 35S:miR396b. For generation of miR396-
resistant versions of AtGRF9, the miR396 target site in AtGRF9
was altered by introducing synonymous mutations using an
overlap-PCR method. The modified AtGRF9 gene was then cloned
into a modified pCAMBIA1300 vector, behind the duplicated 35S
promoter. For promoter–GUS (b-glucuronidase) constructs, the
3125 bp and 3132 bp 5# upstream sequences of MIR396A and
MIR396B were PCR-amplified and cloned into the entry vector
pENTR/D-TOPO. The resulting DNA fragments were then
recombined into the GATEWAY destination vector pMDC162A
(Curtis and Grossniklaus, 2003), to generate p396a:GUS and
p396b:GUS constructs. All constructs were verified by nucleotide
sequencing for inserts and introduced into the Agrobacterium
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strain GV3101. The transgenic plants were generated by the floral
dip method (Clough and Bent, 1998). The sequences of primers
used for PCR are listed in Supplementary Table S1 available at
JXB online.
Real-time RT-PCR (qRT-PCR)
Total RNAs were extracted from leaves of 13-day-old seedlings
using TRIzol reagents (Invitrogen), and reverse transcription was
performed with 2 lg of total RNA using a kit (Fermentas, Vilnius,
Lithuania). Real-time PCR was performed according to previous
methods (Li et al., 2005). Primer sequences are listed in Supple-
mentary Table S1 at JXB online.
In situ hybridization and GUS staining
In situ hybridizations were performed as previously described (Kim
et al., 1998) using 13-day-old seedlings. Detection of miR396 by
in situ hybridization was performed using the complementary
locked nucleic acid (LNA)-modified DNA probe, 5#-AgTTcAAg
AAaGCtGGaA-3#, where the lowercase letters represent LNAs.
Other probe preparations and GUS staining were according to
previous methods (Li et al., 2005). The sequences of primers used
for probe preparation are listed in Supplementary Table S1 at JXB
online.
Microscopy
Scanning electron microscopy (SEM) and preparation of thin-
section specimens were according to previous methods (Chen
et al., 2000). For differential interference contrast microscopy
(DICM) observation, leaves were first fixed in FAA (formalin:
acetic acid:70% ethanol, 1:1:8) overnight and then cleared in
a chloral solution (chloral hydrate:glycerol:water, 8:2:1) (Tsuge
et al., 1996). The determined unit area for cell counting was
located in leaves taken at 50% above the base and half way
between the midrib and the leaf margin. The unit area was first
photographed and then the number of palisade cells within the
area was counted.
Flow cytometric analysis
A 0.1 g aliquot of 8-day-old leaves was harvested from 13-day-old
seedlings and freshly chopped with a razor blade in a Petri dish
containing 2 ml of nuclear isolation and stain buffer (NIM-DAPI
10, Beckman Coulter, CA, USA). The chopped leaves were then
filtered through a 40 lm mesh and the isolated nuclei were
analysed with a Quanta? SC flow cytometer (Beckman Coulter).
Results from two biological replicates, each with duplicated
samples, were analysed.
Results
miR396-targeted AtGRF transcription factors are
required for establishment of leaf polarity
To explore how cell proliferation defects affect establishment
ofleafpolarity,the transgenic
MIR396a and MIR396b under the control of the cauliflower
mosaic virus 35S promoter (35S:miR396a and 35S:miR396b,
respectively) were first characterized. Compared with the wild
type (Supplementary Fig. S1A at JXB online), transgenic
35S:miR396a (Supplementary Fig. S1B) and 35S:miR396b
(Supplementary Fig. S1C) plants displayed narrow and
slightly small rosette leaves, resembling atgif1/an3 mutants
(Horiguchi et al., 2005) (Supplementary Fig. S1D). Further-
more, similarly to atgif1/an3 (Supplementary Fig. S1H), the
plants overexpressing
size of the palisade mesophyll cells in 35S:miR396a (Supple-
mentaryFig.S1F)and
35S:miR396b
Fig. S1G) plants was clearly bigger than those of the wild-
type (Supplementary Fig. S1E), while the cell numbers in
thoseplants are significantly
Fig. S1I). qRT-PCR analysis revealed that the transcription
levels of the nine AtGRF genes including seven predicated
AtGRF gene targets in 35S:miR396a and 35S:miR396b plants
were all decreased (Supplementary Fig. S1J), indicating that
miR396 negatively regulates the cell proliferation in leaves by
repressing the expression of AtGRF genes, consistent with
reported results (Liu et al., 2009; Rodriguez et al., 2010).
The roles of miR396-targeted AtGRFs in establishment
of leaf polarity were then investigated by introducing the
35S:miR396a and 35S:miR396b constructs into the leaf
polarity mutants as1-1 (Fig. 1A) and as2-1 (Fig. 1E), which
exhibit only weak leaf adaxial–abaxial polarity defects
(Byrne et al., 2000; Semiarti et al., 2001; Iwakawa et al.,
2002; Kumaran et al., 2002; Xu et al., 2002, 2003; Lin et al.,
2003). In contrast, the 35S:miR396/as1-1 (Fig. 1B, C) and
35S:miR396/as2-1 (Fig. 1F, G) transgenic plants produced
stronger leaf polarity defects; the first two rosette leaves
typically appeared lotus like and, in some extreme cases,
needle like. In the F3 progeny of three independent
homozygous lines, >80% of 35S:miR396a/as2 plants pro-
duced lotus-like or needle-like leaves in the first pair of true
leaves, while <5% of as2 plants generated lotus-like leaves
(Fig. 1I), indicating that miR396-AtGRFs and AS1/2 path-
ways synergistically regulate leaf adaxial–abaxial formation.
The transcription levels of AtGRF genes were analysed in
35S:miR396a/as2 transgenic plants by qRT-PCR. Similar to
35S:miR396/Col transgenic plants, the expression levels of
AtGRF genes were decreased in 35S:miR396a/as2 (Fig. 1J),
suggesting that the leaf polarity defects in 35S:miR396a/as2
plants were due to the decreased expression of AtGRF
genes. To support these results, the double mutants an3 as1
and an3 as2 were also generated. The phenotype of the an3
as1 (Fig. 1D) and an3 as2 (Fig. 1H) double mutants largely
resembled that of 35S:miR396/as1 and 35S:miR396/as2
plants, respectively. To confirm this further, the construct
35S:rAtGRF9, which carries the miR396-resistant version of
AtGRF9
(rAtGRF9), was made
35S:miR396a/as2 plants. The miR396 target sequence in
AtGRF genes is located at the end of the region encoding
the conserved WRC domain (Fig. 2A). Disruption of the
miR396-binding sites in AtGRF9 could result in the
accumulation of their stabilized transcripts in transgenic
plants. By qRT-PCR, it was found that the AtGRF9 mRNA
in the double transgenic plants was increased to levels
higher than in the wild type, in contrast to the much lower
accumulation in 35S:miR396a/as2 plants (Fig. 2D). Accord-
ingly, it was observed that the leaf polarity defects (as
indicated by the appearance of lotus-like leaves, Fig. 2C) of
35S:miR396/as2-1 could be rescued by the introduced
rAtGRF9 gene (Fig. 2E, F) but not by an empty vector
(Fig. 2B). Taken together, these results suggested that the
AtGRF–AtGIF complexes are required for establishment of
leaf polarity, and the reduction of AtGRF gene expression
(Supplementary
reduced(Supplementary
and introducedinto
miR396-targeted AtGRFs regulate leaf morphogenesis | 763

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by transgenic miR396 overexpression resulted in the leaf
polarity defects in 35S:miR396a/as2 plants.
Enhanced defects of abaxial–adaxial polarity
in the leaves of 35S:miR396a/as2 plants
To better understand the roles of miR396 and AtGRF genes
in leaf patterning, the leaf phenotype of 35S:miR396a/as2
transgenic plants was analysed in more detail by SEM. The
adaxial epidermis of both wild-type and as2 leaves was
characterized by an undulating surface comprising uni-
formly sized pavement cells (Fig. 3A, D), while smaller
pavement cells interspersed with some long and narrow cells
were noted in the abaxial epidermis (data not shown). The
epidermal cell patterns of 35S:miR396 plants were normal
(data not shown). However, the adaxial surface of the lotus-
like leaves of 35S:miR396a/as2 transgenic plants comprised
a mosaic of both adaxial and abaxial epidermal cells,
characterized by the presence of abaxialized long and
narrow cells in the adaxial surface (Fig. 3B, E). The
extremely narrow and needle-like leaves of 35S:miR396a/
as2 plants exhibited long and narrow abaxial epidermal cells
(Fig. 3C, F), suggesting that this leaf is abaxialized.
The vascular pattern of 35S:miR396a/as2 plants was
further analysed by transverse sectioning through the
blade–petiole conjugation region. In the wild-type or as2
leaves, vascular bundles in this region showed a pattern
whereby xylem develops on the adaxial pole and phloem is
located on the abaxial pole (Fig. 3G). In the 35S:miR396
transgenic plants, the vascular pattern in the expanded
rosette leaves did not show obvious changes (data not
shown). In contrast, the lotus-like leaves of 35S:miR396a/
as2 plants exhibited a phloem-surrounding-xylem structure
(Fig. 3H), while the needle-like leaves of 35S:miR396a/as2
plants exhibited no apparent vascular bundle but numerous
intercellular spaces (Fig. 3I).
To obtain molecular evidence that miR396-targeted
AtGRFs are required for establishment of leaf polarity,
the expression pattern of a YABBY family gene, FIL/YAB1,
was examined by in situ hybridization. FIL is usually
expressed on the leaf abaxial domain of both the wild
type (Fig. 3J) and the as2 mutant (Fig. 3K). In contrast,
FIL was expressed throughout the entire primordium of
the needle-like leaves of 35S:miR396a/as2 plants and
seemed to accumulate to higher levels in the other leaves
(Fig. 3L). These results together indicated that miR396-
targeted AtGRFs are required for leaf patterning and
Fig. 1. miR396-targeted AtGRF transcription factors are required for establishment of leaf polarity. (A–H) Morphological observations of
the as1 (A), 35S:miR396a/as1 (B), 35S:miR396b/as1 (C), an3 as1 (D), as2 (E), 35S:miR396a/as2 (F), 35S:miR396b/as2 (G), and an3 as2
(H) plants. Arrowheads in B–D and F–H indicate the lotus- or needle-like leaves. (I) Lotus- or needle-like leaves were present more
frequently in 35S:miR396a/as2 plants than in the as2 mutant. The first pair of rosette leaves was analysed for the presence of lotus- or
needle-like leaves. n, numbers of plants analysed; (J) qRT-PCR to analyse the transcription levels of AtGRF gens in 14-day-old seedlings
of as2 and 35S:miR396a/as2 plants. Bars¼1 cm in A–H.
764 | Wang et al.

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genetically regulate the expression of leaf polarity-controlling
genes.
miR396 negatively regulates cell proliferation by
controlling entry into the mitotic cell cycle
In Arabidopsis, the proliferating cells in lateral organ
primordium usually divide in the mitotic cell cycle, which
follows the common scheme of G1, S, G2, and M phases
(Inze and De Veylder, 2006; Gutierrez, 2009), to multiplify
and promote organ growth and development. To gain
insights into how the roles of miR396 and AtGRFs in cell
proliferation affect establishment of leaf polarity, the
expression pattern of one of the cell cycle marker genes,
histone H4, was analysed by in situ hybridization. Histone
H4 is predominantly expressed in the proliferating cells in
the DNA replication-related S phase of the cell cycle and
thus might mark the cells undergoing mitotic cell division
(Reichheld et al., 1995, 1998). Compared with the wild type
(Fig. 4A), the number of leaf cells expressing histone H4 was
dramatically reduced in the 35S:miR396a plants (Fig. 4B, C).
In addition to histone H4, the expression of several other
cell cycle-related marker genes was also analysed by qRT-
PCR. Cyclin D3;1 (CycD3;1) is mainly expressed at pro-
liferating cells in the G1phase and drives the G1/S transition
together with cyclin-dependent kinase A (CDKA) (Dewitte
et al., 2003; Menges et al., 2006). CYCA2;1 is involved in
S/G2transition, and CYCB1;1 in G2/M transition (Doerner
et al., 1996; Shaul et al., 1996). The expression of all the
tested marker genes was reduced in 35S:miR396a plants
with a 2- to 4-fold change (Fig. 4D), indicating that the
defective cell proliferation in leaves of 35S:miR396 plants
could be due to the reduced cell division activity. The
expression of cell cycle-related genes was also examined in
as2 and 35S:miR396a/as2 plants. Similarly, the number of
cells expressing histone H4 was reduced (Supplementary
Fig. S2B, C at JXB online), and the mRNA levels of cell
cycle marker genes were reduced even more (5- to 15-fold
change) in 35S:miR396a/as2 plants (Fig. S2D) as compared
with those in the as2 mutant (Fig. S2A, D), indicating that
the cell division activity mediated by miR396-targeted
AtGRFs is important for establishment of leaf adaxial–
abaxial polarity.
To dissect further the phase of the cell cycle affected by
transgenic miR396 overexpression, a flow cytometric analy-
sis was performed with nuclei from leaves of ;8 d old to
examine the nuclear ploidy level. The atgif1/an3 leaves were
analysed as a control. The percentages of 8C and 16C cells
in 35S:miR396 and atgif1/an3 plants were significantly
higher than those in the wild-type plants, whereas the
percentages of 2C and 4C cells were reduced accordingly
(Fig. 4E, F). Because it has been proposed that at this
time point leaf cells have the capacity to divide and 2C and
4C cells could represent the G1and G2cells, respectively
(Takahashi et al., 2008), the ratio of 4C/2C cells in 8-
day-old leaves was compared further. As shown in Fig. 4G,
Fig. 2. Introduction of the miR396-resistant AtGRF9 gene into a 35S:miR396a/as2 plant partially rescued the leaf polarity defects. (A)
Diagram of the miR396 target sites of the wild type and a modified version of AtGRF9. The conserved QLQ motif in AtGRFs is indicated
in yellow, and the WRC motif is shown in blue, with the miR396 target region marked in purple. (B, C) Morphological observation of the
27-day-old seedlings of as2 (B) and 35S:miR396a/as2 (C). The arrowhead in C indicates the lotus-like leaf. (D) qRT-PCR analysis of the
AtGRF9 transcript levels in as2, 35S:miR396a/as2, and 35S:miR396a/as2 expressing the miR396-resistant AtGRF9 gene. (E, F)
Morphology of the 27-day-old seedlings of 35S:miR396a/as2 expressing the miR396-resistant AtGRF9 gene driven by the 35S
promoter. #1, line1; #2, line2. Bars¼1 cm in B, C, E, and F.
miR396-targeted AtGRFs regulate leaf morphogenesis | 765