Rice histone deacetylase genes display specific expression patterns and developmental functions.

Abstract

Histone deacetylases (HDAC) are important in plant gene expression. Here we show that the expression of rice HDAC genes is both tissue/organ-specific, and most of them are responsive to drought or salt stresses. Over-expression of several rice HDACs did not produce any visible phenotype, whereas down-regulation of a few HDAC genes affected different developmental aspects. Specifically, down-regulation of HDA703 by amiRNA reduced rice peduncle elongation and fertility, while inactivation of a closely related homolog HDA710 by RNAi affected vegetative growth. HDA704 RNAi altered plant height and flag leaf morphology. Down-regulation of HDT702 led to the production of narrowed leaves and stems. These data suggest that rice HDAC genes may have divergent developmental functions compared with closely related homologs in Arabidopsis.

Full text

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Rice histone deacetylase genes display specific expression patterns
and developmental functions
Yongfeng Hua, Fujun Qina, Limin Huanga, Qianwen Suna, Chen Lia, Yu Zhaoa, Dao-Xiu Zhoua,b,*
aNational Key Laboratory for Crop Genetic Improvement, Huazhong Agricultural University, 430070 Wuhan, China
bInstitut de Biotechnologie des Plantes, Université Paris sud 11, 91405 Orsay, France
a r t i c l ei n f o
Article history:
Received 28 July 2009
Available online 5 August 2009
Keywords:
Chromatin
Epigenetic modification
Histone acetylation
Acetyltransferase
Rice development
a b s t r a c t
Histone deacetylases (HDAC) are important in plant gene expression. Here we show that the expression
of rice HDAC genes is both tissue/organ-specific, and most of them are responsive to drought or salt stres-
ses. Over-expression of several rice HDACs did not produce any visible phenotype, whereas down-regu-
lation of a few HDAC genes affected different developmental aspects. Specifically, down-regulation of
HDA703 by amiRNA reduced rice peduncle elongation and fertility, while inactivation of a closely related
homolog HDA710 by RNAi affected vegetative growth. HDA704 RNAi altered plant height and flag leaf
morphology. Down-regulation of HDT702 led to the production of narrowed leaves and stems. These data
suggest that rice HDAC genes may have divergent developmental functions compared with closely
related homologs in Arabidopsis.
? 2009 Elsevier Inc. All rights reserved.
Histone modifications including acetylation, methylation, phos-
phorylation, ubiquitination and others are shown to play an impor-
tant role in chromatin function and gene regulation. Specific
patterns of histone modifications establish the so-called ‘‘histone
code” that determines open or repressed states of the cognate
DNA sequences. Histone acetylation/deacetylation appears as a
key switch for inter-conversion between permissive and repressive
states of chromatin domains for gene expression. The homeostatic
balance of nucleosomal histone acetylation is maintained by antag-
onistic action of histone acetyltransferases (HAT) and histone
deacetylases (HDAC). Plant HDAC can be grouped into four classes.
Among them, three have primary homology to three yeast HDAC
groups: RDP3, HDA1 and SIR2 [1]. The fourth group known as the
HD2 class is found in plants only [2].
During the last years, HDAC function has been most studied in
Arabidopsis. Inactivation of Arabidopsis HD1 (AtHD1/HDA19) by
expressinganantisenseconstructandbyaT-DNAinsertionmutation
(athd1-t1) induces pleiotropic developmental abnormalities [3,4]. In
addition, AtHD1/HDA19 is shown to form an antagonistic couple
with the histone acetyltransferase GCN5 involved in the regulation
of several pathways including photomorphogenic seedling develop-
ment [5]. Recent data show that AtHD1/HDA19 is involved in expres-
sionofgenesrespondingtojasmonicacidandethylenesignaling[6].
Two other Arabidopsis RPD3 type genes AtRPD3A and AtRPD3B are
showntoplayanimportantroleinplantgrowth,asdown-regulation
of the genes delays flowering [7]. Treatment of wild type seedlings
withthespecificHDACinhibitor,Trichostatin-A(TSA),andmutations
in HDA18 induce ectopic production of root hairs [8]. Furthermore,
HDACgenesarefoundtobeinvolvedinepigeneticregulation,asAtH-
DA6 is shown to be involved in RNA interference-mediated DNA
methylation [9]. In addition, AtHDA6 and HDT2 are involved in
nucleolar dominance in allopolyploid hybrids [10].
Rice genome contains at least 19 HDAC genes (Table 1). The
function of most of them has not been studied, except for a few
genes [11–13]. Here we report expression analysis and functional
studies by transgenics of several rice HDAC genes. Our data sug-
gests that individual rice HDAC genes have specific development
functions that may be divergent from the Arabidopsis homologs.
Materials and methods
Plant materials. The rice variety Zhonghua11 (Oryza sativa spp.
japonica) and Minghui63 (Oryza sativa spp. indica) were used for
transgenic plant production in this study.
Expression profile analysis of rice HDAC genes. The data of 33 rice
tissues or organs were extracted from the CREP database (http://
crep.ncpgr.cn) for HDAC expression analysis. For HDAC responses
to various abiotic stress, the data were extracted from GEO data-
base (GSE6901, http://www.ncbi.nlm.nih.gov/geo) [14]. Expression
values corresponding to each gene were analysed using the Cluster
3.0 program, and visualized by the Treeview program [15].
Construction of binary vector. To generate the RNAi constructs,
the cDNA fragments were amplified with gene-specific primers
0006-291X/$ - see front matter ? 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2009.07.162
Abbreviations: HDAC, histone deacetylase; HAT, histone acetyltransferase; GFP,
green fluorescence protein; amiRNA, artificial microRNA
* Corresponding author. Address: Institut de biotechnologie des Plantes, Univer-
sité Paris sud 11, 91405 Orsay, France. Fax: +33 1 96153324.
E-mail address: dao-xiu.zhou@u-psud.fr (D.-X. Zhou).
Biochemical and Biophysical Research Communications 388 (2009) 266–271
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Page 2

and then sequentially cloned into the vector pDS1301 with KpnI/
BamHI and SacI/SpeI [16]. For construction of over-expression vec-
tors, HDAC full-length cDNA were amplified by RT-PCR. The PCR
products (digested by KpnI/BamHI) were inserted into the binary
vector pU1301, which was modified based on pCAMIA1301 (CAM-
BIA) and contains a maize ubiquitin promoter [12]. The HDAC1-3
artificial microRNAs (amiHDAC1-3) were constructed as described
[17]. AmiHDAC1-3sequences
50-TGTTTCAGGAATTCGCTCTGC,
were engineered into the miR319a precursor in vector RS300 by
site-directed mutagenesis and the resulting artificial microRNAs
were cloned into pU1301 (digested by KpnI/BamHI)
Reverse transcription and real time PCR. For gene expression anal-
ysis, total RNA isolation and RT-PCR were performed as described
previously [18]. Real time PCR analysis was performed using
gene-specific primers and SYBR Premix Ex TaqTM(Takara, Dalian)
on a real time PCR 7500 system (Applied Biosystems). The rice
actin gene was used as internal control. The relative expressions
of each gene in transgenic plants and wild type were calculated
using the 2?DDCtmethods as described [19].
RNA gel blot analysis. Fifteen micrograms of total RNA was trans-
ferred onto the Nylon membrane (Hybond-XL, Amersham). Gene
specific-probes were labeled with32P-dCTP and hybridized to the
RNA blots. The fragments for probes were digested from the
over-expression vectors with appropriate restriction enzymes.
Proteingelblotanalysis.Histoneproteinswereextractedfromthe
wildtypeandtransgenicriceleaves(2 g)withthebufferasdescribed
[20]. The histone pellet was separated by SDS–PAGE, detected with
the acetylated H4 antibody purchased from Millipore, and peroxi-
dase (HRP)-conjugated goat anti-rabbit IgG as secondary antibody.
Light microscopy. Flag leaves were harvested from mature
plants. The procedures of dehydration, clearing, infiltration and
embedding were carried out as described [21]. The microtome sec-
tions (10 lm) were mounted on glass slides for imaging.
(50-TTCCATGTTACTTGCTGCAGC,
50-TTGATCTTCATCTGGCTCACG)
Results and discussion
Expression profiles of rice HDAC genes
To study the expression profiles of rice HDAC genes, we
analyzed the Collection of Rice Expression Profiles (CREP) that is
established by a serial Affimetrix microarray analysis of transcripts
from various organs at different developmental stages (http://crep.
ncpgr.cn). Several genes were found to have a specific expression
pattern. For instance, HDA710 was more expressed in germinating
and young seedlings as well as in stamens, whereas HDA703 was
highly expressed in calli and in imbibed seeds. HAD714 and
HDA706 were found to be mainly expressed in shoots and leaves,
whereas HDA716 showed a strong expression in developing endo-
sperm and germinating seeds. The leaf/shoot specificity of HDA714
is consistent with its protein localization in chloroplasts and mito-
chondria [22], suggesting that this HDAC may play a role in plant
specific functions. Three groups of closely related genes (group 1:
HDA705, HDA702, HDA709, HDA710 and HDA711; group 2:
HDA704 and HDA713; group 3: HDT701 and HDT702) showed a
very similar expression pattern with a relatively high level in
developing panicles and calli. However, the expression patterns
of the two members of the Sir2-like HDACs, SRT701 and SRT702
were found to be different. The two proteins are likely to have dis-
tinct functions, as SRT701 (OsSRT1) is nuclear localized and is
shown to be involved in histone H3K9 deacetlyation required for
transposon repression in rice [12], whereas SRT702 is recently
shown to be localized in the mitochondria [22].
In order to know whether rice HDAC genes are regulated by abi-
otic stresses, we analyzed the microarray data obtained from rice
treated with drought, salt and cold stresses [14]. Two genes
(HDA703 and HDA710) are induced, while nine others (i.e.
HDA701, HDA702, HDA704, HDA705, HDA706, HDA712, HDA714,
HDA716, HDT701 and HDT702) are clearly repressed by drought
and salt (Fig. 1). Two additional genes (HDA709 and SRT702) are in-
duced by drought, but repressed by salt. However, cold treatment
seemed to have less impact on rice HDAC gene expression. Abscisic
acid (ABA) is the major plant hormone in water stress signaling and
regulates water balance and osmotic stress tolerance [23]. It has
been shown that ABA down-regulates the expression of AtHD2C
(also known as AtHDT3) [24]. In a recent report it is shown that
HDT701, HTD702, SRT701 and SRT702 are also repressed by ABA
treatment [25]. The present analysis revealed that the expression
of most of the rice HDAC genes is responsive to drought and salt
stresses, with a large proportion of the genes being repressed. Pre-
vious results show that Arabidopsis HDAC (i.e. AtHD1/HAD19, AtH-
DT3) are involved in stress responses [6,24]. Abiotic stresses
Table 1
Rice HDAC genes studied (+) or not studied (?) by transgenics in this work.
GeneLoc. No.GenBankOver-expression RNAiAmiRNA
HDA701
HDA702 (OsHDAC1)
HDA703 (OsHDAC3)
HDA704
HDA705
HDA706 (OsHDAC6)
HDA707
HDA709
HDA710 (OsHDAC2)
HDA711
HDA712
HDA713
HDA714 (OsHDAC10)
HDA716
SRT701 (OsSRT1)
SRT702 (OsSir2b)
HDT701
HDT702
LOC_Os01g40400
LOC_Os06g38470
LOC_Os02g12350
LOC_Os07g06980
LOC_Os08g25570
LOC_Os06g37420
LOC_Os01g12310
LOC_Os11g09370
LOC_Os02g12380
LOC_Os04g33480
LOC_Os05g36920
LOC_Os07g41090
LOC_Os12g08220
LOC_Os05g36930
LOC_Os04g20270
LOC_Os12g07950
LOC_Os05g51830
LOC_Os01g68104
NM_192536
AK068051
AK120027
AK111557
AK111861
AK100812
NM_188519
AK064282
AK103097
AK066667
AK102786
AK111892
AK072557
AK068179
AK060337
AK067069
AK072845
XM_463594
?
?a
?a
+
?
+c
?
+
?a
+
+
?
?e
?
+f
+d
+
+
?
+b
?
+
?
+
?
+
?
+
+
?
?
?
+f
+
+
+
+
+
+
aOver-expression of these genes is studied in Jang et al. [13].
bKnock-out of this gene is studied in Chung et al. [11].
cLocalized in chloroplasts [22].
dlocalized in mitochondria [22].
eLocalized in both chloroplasts and mitochondria [22].
fStudied in Huang et al. [12].
Y. Hu et al./Biochemical and Biophysical Research Communications 388 (2009) 266–271
267

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rapidly induce the expression of a large number of genes. Down-
regulation of HDAC genes may be important in stress-inducible
gene expression in rice. This is in agreement with the hypothesis
that histone acetylation/deacetylation switch is an important
mechanism of short-term regulated gene expression [26].
Functional analysis of rice HDAC genes
To study developmental function of rice HDAC genes, we pro-
duced over-expression and/or RNAi or artificial microRNA (ami-
RNA) transgenic plants of several genes including HDA702,
HDA703, HDA704, HDA706, HDA709, HDA710, HDA711, HDA712,
HDT701, HDT702, SRT701 and SRT702 (Table 1). For each transgene,
at least 20 independent lines were obtained and the expression of
the transgenes or the endogenous genes was tested by Northern or
RT-PCR (Fig. S1). Among these genes, SRT701 is previously shown
to be required for transposon repression and to be involved in
the safeguard against genome instability and cell death [12]. How-
ever in most cases, over-expression of the other HDAC genes did
not produce any morphological phenotype, whereas down-regula-
tion of HDA703,HDA710, HDA704 and HDT702 affected different as-
pects of plant development.
Function of rice homologs of AtHD1/HDA19
HDA702, HDA710 and HDA703 correspond respectively to OsH-
DAC1–3 as previously described [13]. Phylogenetic analysis indi-
cates that the 3 rice genes belong to the same clade as AtHD1/
HDA19, with HDA702 as the closest homolog of AtHD1/HDA19
[25]. AtHD1/HDA19 is the most studied HDAC gene in Arabidopsis.
It is shown that over-expression of HDA702/HDAC1enhances root
growth [13]. As the three genes are highly conserved (more than
75% of amino acid sequence identity), it is suggested that the three
genes might have redundant function [13]. We therefore designed
amiRNAs to try to knock-down simultaneously two or all of the
three members (Fig. 2A). Examination of the target gene expression
Fig. 1. Expression analysis of the rice HDAC genes. Upper: A hierarchical cluster display of relative expression levels of the rice HDAC genes in 33 samples representing
different organs or tissues at different developmental stages of the Minghui 63 cultivar. 1–5: Calli at different induction stages from cultured embryos. 6: Plumule at 48 h after
emergence in the dark. 7: Plumule at 48 h after emergence in the light. 8: Radical at 48 h after emergence in the dark. 9: Radical at 48 h after emergence in the light. 10: 72 h
imbibed seed. 11: Embryo and radicle after germination. 12: Seedling leaves and roots at three-leaf stage. 13: Shoots of seedlings with two tillers. 14: Roots of seedlings with
two tillers. 15: Stem at day 5 before heading. 16: Stems at heading stage. 17: Leaves from plants at young panicle stage 3. 18: Leaves at young panicle stage 7. 19: Flag leaves
at day 5 before heading. 20: Flag leaf at day 14 after heading. 21: Sheaths at young panicle stage 3. 22: Sheaths at young panicle stage 7. 23 : Hulls one day before flowering.
24: Stamens one day before flowering. 25: Young panicle at stage 3. 26: Young panicles at stage 4. 27: Young panicles at stage 5. 28: Panicles at stage 7. 29: Panicles at heading
stage. 30: Spikelets, 3 days after pollination. 31: Endosperm, 7 days after pollination. 32: Endosperm, 14 days after pollination. 33: Endosperm, 21 days after pollination.
Lower: Expression changes of HDAC genes responding to abiotic stresses including drought, salt and cold in seven-day-old light-grown seedlings. Color bar at the base
represents log2 expression values: green, representing low expression; black, medium expression; red, high expression.
268
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revealed that HDA703 mRNA was clearly reduced in plants trans-
formed by all the three amiRNA constructs (Fig. 2B). The amiRNA
effect on HDA702 and HDA710 was less clear. Western blot analysis
revealed that acetylated histone H4 was increased in amiRNA
plants with reduced HDA703 expression (Fig. 2C), suggesting that
HDA703 may be involved in histone H4 acetylation in rice. The
amiRNA plants displayed no abnormal growth during the vegeta-
tive stage. However, the amiRNA plants showed reduced elonga-
tion of the peduncle resulting in partially wrapped panicles.
These plants were partially or completely sterile (Table 2). In addi-
tion, the seeds displayed an awn (Fig. 2D, Table 2). These pheno-
types suggest that HDA703 may have a function during plant
reproductive development and seed morphology. Both wrapped
panicles and awns can be found in natural wild rice and are absent
from the cultivars. The appearance of wrapped panicles and awns
in the transgenic seeds suggests that histone acetylation/deacety-
lation balance may have been altered during rice domestication.
In order to study the function of HDA710, RNAi was designed to
target a specific region of the gene. Amongst the transgenic lines,
HDA702
HDA710 HDA710
HDA703
1
1
1
2
2
2
23
2 3
A
AmiHDAC1
12 14
AmiHDAC2
1 18 19
AmiHDAC3
56
WT
HDA702
HDA702
HDA710
HDA703
Actin
145217
2
1
2
3
B
WT149111418
C
AcH4
Enriched histone
D
AmiHDAC
WT
AmiHDAC
WT
Fig. 2. Down-regulation of HDA702, HDA710 and HDA703 by amiRNA. (A). Target
sites of three amiRNAs on HDA702, HDA710 and HDA703. Numbered triangles (1–3)
represent target sites of amiHDAC1, amiHDAC2 and amiHDAC3, respectively.
Arrows indicate the primers for expression tests of the three genes in transgenic
plants. (B). Expression changes of HDA702, HDA710 and HDA703 in different
transgenic lines tested by RT-PCR. (C). Detection of H4 acetylation alteration in
transgenic plants by western blots. Enriched histone was revealed as loading
controls. (D). Phenotypes of AmiRNA transgenic plants. Left panel, comparison of
peduncle length between wild type and a transgenic plant, indicated by arrows;
right panel, comparison of seed morphology.
Fig. 3. Down-regulation of HDA710 by RNAi induced a semi-dwarf phenotype. (A).
Comparison between wild type (left) and a HDA710 RNAi transgenic plant (right) at
seedling (left panel) and mature stages (right panel). (B). Detection of HDA710
expression in semi-dwarf plants (SD1 and SD2) and normal transgenic plants (N1).
Table 2
Phenotype survey of amiRNA lines.
Transgenic plantsSeed-setting ratePlant heightPeduncle Grain
AmiHDAC1-1
AmiHDAC1-2
AmiHDAC1-3
AmiHDAC1-4
AmiHDAC1-5
AmiHDAC1-6
AmiHDAC1-7
AmiHDAC1-8
AmiHDAC1-9
AmiHDAC1-10
AmiHDAC1-11
AmiHDAC1-12
AmiHDAC1-13
AmiHDAC1-14
AmiHDAC1-15
AmiHDAC1-16
AmiHDAC1-17
AmiHDAC1-18
AmiHDAC1-19
AmiHDAC1-10
AmiHDAC1-20
AmiHDAC1-21
AmiHDAC1-22
AmiHDAC1-23
AmiHDAC2-1
AmiHDAC2-2
AmiHDAC3-1
AmiHDAC3-2
AmiHDAC3-3
AmiHDAC3-4
AmiHDAC3-5
AmiHDAC3-6
AmiHDAC3-7
AmiHDAC3-8
EL
EL
N
EL
N
N
N
N
L
L
EL
S
M
N
N
N
N
L
L
M
N
N
M
L
L
M
N
M
M
M
EL
EL
L
EL
Semi-dwarf
Semi-dwarf
Short
Short
Semi-dwarfWrappedBig grain
Short
Short
Short
Short
Semi-dwarf
Semi-dwarf Short
Semi-dwarf
Semi-dwarf
Wrapped
Wrapped
Big grain
Big grain
Semi-dwarf Short
EL, extremely low; M, medium; L, low; N, normal.
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269

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five showed reduced vegetative growth (Fig. 3). Among the three
closely related genes, HDA702 and HDA710 showed a very similar
expression pattern (Fig. 1). Chung et al. [11] have isolated two T-
DNA insertion alleles of HDA702/HDAC1, but failed to obtain homo-
zygous mutants, suggesting that the gene may be essential for
plant growth. However, in the heterozygous insertion plants, root
growth was affected [11]. The vegetative growth of HDA710 RNAi
plants was reduced, which may be due to poorly developed roots
(Fig. 3). The similar expression pattern and the down-regulation
phenotypes of HDA702/HDAC1 and HDA710 suggest that both genes
may play a similar but important role in root and vegetative
growth. Furthermore, the expression levels of the two genes may
be crucial for plant development, as heterozygous mutant plants
of HDA702/HDAC1 displays already phenotypes and no HDA702/
HDAC1 knock-down or only slightly reduced HDA710-expression
plants could be obtained by amiRNAs (Fig. 2).
Loss-of-function and over-expression studies suggest that
AtHD1/HDA19 is a global regulator of gene expression in develop-
ment and stress responses [3–6]. Loss-of-function mutants of this
gene produce a large range of abnormalities including seedling
development, plant height, floral organ number and morphology,
reduced fertility and fruit morphology. The floral/seed phenotypes
of HDA703-down-regulation plants and vegetative effects of
HDA710 RNAi shown in this study, together with the HDA702/
HDAC1 mutant phenotype [11], suggest that the AtHD1/HDA19-like
function has been diversified and shared by the three genes in rice.
Down-regulation of HDA704 and HTD702 affected plant height and
flag leaf morphology
In the HDA704 RNAi transgenic populations, several lines dis-
played a dwarf or semi-dwarf phenotype (Fig. 4). Remarkably,
the flag leaf (formed during the heading stage) was severely
twisted (Fig. 4). These phenotypic plants showed a reduced expres-
sion of HDA704 (Fig. 4).
Rice genome contains at least 2 HD2 genes (Table 1). Over-
expressionof
HDT701
and
HTD702
HDT701 did not produce any obvious phenotype. However,
HDT702 RNAi plants displayed severely narrowed leaves (Fig. 4).
In addition, the diameter of transgenic stems was also reduced
(Fig. 4). The observations suggest that HDT702 might be involved
in cell division or growth in rice. In leaves, the HDT702 promoter
(1857 bp upstream from the ATG codon) was found to be most
active in vascular bundles (Fig. 4). Arabidopsis HDT1 (AtHDT1)
and HDT2 (AtHDT2) are found to play a role in leaf polarity deter-
mination [27]. Down-regulation of these genes in asymmetric
leaves2 (as2) mutants caused the formation of abaxialized and
filamentous leaves [27]. The narrow leaf phenotype of HDT702
RNAi and its expression pattern in the leaf vascular system sug-
gests that the gene may have a function in rice leaf development,
which is likely to be different from that of the Arabidopsis homo-
log. Neighbour-joining tree analysis of Arabidopsis, rice and maize
HD2 genes clustered the members within the species and dicot
and monocot sequences are separated into two distinct clades
[1], suggesting that monocots and dicots had different HD2 ances-
tors and that monocot HD2 members may have been evolved to
gain different functions from the dicot homologs. Therefore, the
developmental functions of individual rice HDAC genes seem to
be divergent from their closely related homologs in Arabidopsis.
The data produced in this work provide a basis for further studies
aiming to understand the function of histone acetylation/decaty-
lation homeostasis in rice development and responses to environ-
mental conditions.
ordown-regulationof
Acknowledgments
We thank Weibo Xie and Lei Wang for microarray data analy-
sis; Professors Caiguo Xu and Xianghua Li for field management
and technical aids and Professor Qifa Zhang for support. This
work was supported by grants from the Chinese Ministry of
Science and Technologyand the
Foundation.
National Natural Science
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2009.07.162.
HDA704
rRNA
rRNA
WT1234567891011
A
0.6
0.3
0.9
1.2
1.5
B
0
WT R2R3 R9R10
C
WT R2WTR9
R2
D
abc
de
Fig. 4. Down-regulation of HDA704 by RNAi affected plant height and flag leaf
development. (A). Expression analysis of HDA704 RNAi T0 plants by RNA gel blot
hybridization. rRNA was revealed as loading controls. (B). Real time RT-PCR analysis
of transgenic lines showing decreased HDA704 expression revealed by RNA gel blot
hybridization. The PCR signals were normalized with that of actin1 transcripts.
Transcript levels from wild type were set at 1. Data are means ± SD (n = 3 biological
repeats). (C). Comparison between wild type (left) and two HDA704 RNAi transgenic
lines (right) (left and middle panels) and twisted flag leaves (right panel, white
arrow) observed in these lines. (D). HDT702 RNAi transgenic plants and HDT702
promoter activity in leaves. a: Comparison between wild type (left) and a HDT702
RNAi plant (right) at heading stage. b: Comparison between wild type (left) and a
HDT702 RNAi (right) leaves. c: Comparison between wild type (left) and a HDT702
RNAi (right) stems. d and e: GFP detection in leaves transformed by HDA702p-GFP.
270
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