全 文 ：Received 20 Oct. 2003 Accepted 18 Nov. 2003
Supported by the National Natural Science Foundation of China (39870450).
* Author for correspondence. Tel:+86 (0)21 54924111; E-mail:.
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (9): 1106-1113
Leaf Downward Curvature and Delayed Flowering Caused by
AtLH Overexpression in Arabidopsis thaliana
WU Hao, YU Lin, TANG Xiang-Rong, SHEN Rui-Juan, HE Yu-Ke*
(State Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes
of Biological Sciences, The Chinese Academy of Sciences, Shanghai 200032, China)
Abstract: AtLH gene of Arabidopsis is a BcpLH (leafy head) homolog of Chinese cabbage, which encodes
a double-stranded RNA-binding protein related to the curvature of folding leaf leading to the formation of
leafy head. In order to elucidate the regulatory function of AtLH in the development of leaf curvature, we
made a construct of 35S::AtLH and transformed it to Arabidopsis. In transgenic plants for sense-AtLH,
transcripts of AtLH gene were increased significantly in leaves and flowers, giving rise to the AtLH-
overexpressed plants in which the rosette leaves curved downward or outward in a manner of enhanced
epinastic growth. Compared with normal plants, bolting and flowering time of the transgenic plants was
significantly delayed. Moreover, the apical dominance of transgenic plants was weaker in vegetative shoots
since more axillary shoots emerged from axil of rosette leaves, while stronger in flowering shoots
because fewer cauline inflorescences were observed on the main inflorescence. In other aspects, these
transgenic plants exhibited an increase in root-stimulating response to IAA and decrease in root-inhibi-
tory reaction on ABA. It indicates that overexpression of AtLH causes downward curvature of transgenic
plants.
Key words: Arabidopsis ; AtLH ; leaf curvature ; flowering time; apical dominance
Leaves are important for the production of plant biom-
ass since they are adapted for light perception, photosyn-
thesis and gas exchange for respiration. Normally, they
develop along three-dimensional axes: the proximodistal,
transverse and adaxial-abaxial axes (Steeves and Sussex,
1989; Waites et al., 1998; Semiarti et al., 2001). Any changes
in the three axes may subsequently result in the diversity
of leaf form.
Leaves are generated from leaf organ-founder cells re-
cruited on the flanks of a small group of undifferentiated
cells, the shoot apical meristem (SAM). Curvature of leaf is
one of the most important phenomena in leaf development.
The leaf curvature can be divided into two types: down-
ward curvature (epinastic) and upward curvature
(hyponastic). The extreme sample of upward curvature is
leaf incurvature within leafy heads of Chinese cabbage
(Brassica campestris ssp. pekinensis), one of the most im-
portant vegetable crops in China and the Far East. Plant of
Chinese cabbage goes through a long period of vegetative
growth with successive changes of leaf form: rosette leaves,
folding leaves (curved upward) and head leaves (curved
inward). The leafy head, composed of whorled leaves of
incurvature, is one kind of typical edible organs of mor-
phological transformation. Initiation of leafy head is a
complicated biological process and morphological reaction
induced by environmental factors (He and Yu, 1998). Little
has been known about which genes function on leafy head
development for a long time, and the studying on the mor-
phogenetic process of leafy head was largely limited be-
cause the result of many physiological experiments could
not be explained and verified perfectly.
In order to evaluate the molecular and physiological
mechanism of leaf curvature within leafy head, BcpLH gene,
encoding a double-stranded RNA binding protein, was iso-
lated from Chinese cabbage (Yu et al., 2000). Expression
analysis showed that BcpLH gene plays a role in upward
and inward curvature of folding leaf and auxin regulates its
transcriptional expression. With the regulatory function of
BcpLH on leaf curvature of Chinese cabbage being clari-
fied further, the parallel role of homological gene in
Arabidopsis could be determined. Both Arabidopsis and
Chinese cabbage belong to Crucifer family, and they share
the similar evolutionary origin and the same developmental
phases except the formation of leafy head in which leaf
incurvature involved. Arabidopsis mutant of leaf
incurvature could be helpful for understanding the func-
tion of BcpLH on formation of leafy head in Chinese
cabbage.
WU Hao et al.: Leaf Downward Curvature and Delayed Flowering Caused by AtLH Overexpression in Arabidopsis thaliana 1107
1 Materials and Methods
1.1 Bacterial strains
Escherichia coli strain DH5a was used for all DNA re-
combinant work. Agrobacterium tumefaciens strains
LBA4404 (for vacuum infiltration) were used for generation
of Arabidopsis transgenic lines.
1.2 Plant materials
Plant materials used for transformation of sense-AtLH
are the Columbia ecotype of Arabidopsis. For selection of
transgenic plants, seeds were sterilized in ethanol and
bleach, rinsed with water, and germinated on MS (1% sugar)
agar medium containing 50 mg/L Kanamycin. Kanamycin-
resistant seedlings were transplanted into the greenhouse
and grown at 23 °C under 12 h light/12 h dark. Transgenic
plants were fertilized for at least three generations and the
seeds from each plant were harvested separately for sub-
sequent observations.
1.3 AtLH vector construction and transformation
AtLH cDNA of Arabidopsis (Columbia ecotype) was
isolated with PCR method from a mixed cDNA library as
early as in 1996. The degenerate primers, designed accord-
ing to BcpLH gene and the homolog of Arabidopsis, were
used for PCR. For the transformation experiments, a frag-
ment containing the coding sequence of AtLH cDNA was
digested with EcoRⅠ and inserted into the vector
pBluescript . Then the vector with AtLH cDNA was di-
gested with BamHⅠ and SalⅠ, and inserted directionally
into the binary vector pJR1 (Fig.1). Transformant was veri-
fied by sequencing for the constructs containing the AtLH
cDNA sequence in the sense orientation downstream of
the cauliflower mosaic virus 35S promoter. And it was trans-
formed into the A. tumefaciens strain LBA4404 by freeze-
thaw method. Vacuum infiltration method was performed to
transform Arabidopsis Columbia ecotype with sense-AtLH
gene.
1.4 Southern hybridization
Total genomic DNA was prepared from 2-week-old
Arabidopsis seedlings as described (Sambrook et al., 1989).
The full-length cDNAs for AtLH were labeled by the ran-
dom priming method as described above and used as the
probes for DNA blot analyses. The blots were hybridized
and washed under low- or high-stringency conditions.
1.5 Expression analysis
RNA samples were isolated from leaves, stems, roots
and flowers of 35S::AtLH transgenic and wild type plants.
RT-PCR was performed and 10-µL reaction mixtures were
composed of 0.4 mmol/L of each deoxynucleotide
phosphate, 7% dimethyl sulfoxide, 5 mmol/L DTT, 0.3
µmol/L of each primer, 0.2 µL Carboxydothermus
hydrogenoformans Svetlichny polymerase, and nominally
10 ng of RNA template. The amount of RNA in each sample
was carefully normalized so that RT-PCR amplification of
Histone3 resulted in a band of similar intensity on an EB
agarose gel. For Histone3 amplification, the primers were
giving a product of 400 bp. For quantitative RT-PCR, we set
up five reactions for each RNA sample and removed them
from the thermocycler at consecutive cycles. Product
amounts were assessed by electrophoresis in ethidium bro-
mide-containing agarose gels, followed by photography
and quantification with the histogram function in Adobe
Systems (Mountain View, CA) Photoshop Version 5.0.
1.6 Growth assay
Development stages such as seedling, rosette and bolt-
ing were adjudged and recorded on the time of more than
50% plants showing the appearance of next development
stage within population. Each treatment with more than 20
plants was employed. The data of leaf number, branch
Fig.1. Diagram of comparison of AtLH with BcpLH. A. Structural features of AtLH and BcpLH. B. Alignment of two double-stranded
RNA binding motifs (DSRMs) between AtLH and BcpLH. DSRM1, the first dsRNA binding protein motif; DSRM2, the second dsRNA
binding protein motif; NLS, nuclear localization site. Identical amino acids are underlined.
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041108
number were the mean of 20 individuals, and root growth
rate was calculated relative to that of wild-type plants.
2 Results
2.1 Characterization of the AtLH gene
Using degenerate primers of BcpLH gene to perform
PCR, we isolated AtLH cDNA of AtLH from mixed cDNA
library of Columbia ecotype of Arabidopsis in 1996. With
BLAST analysis, one unknown gene of Arabidopsis was
found to be completely similar to AtLH in GenBank. And
the HYL1 (hyponastic leaves 1) (AF276440) gene of
Arabidopsis (Nossen ecotype) was reported in 2000 and it
showed 99% identity with AtLH in cDNA sequence. There-
fore AtLH (derived from Columbia ecotype) and HYL1 (from
Nossen ecotype) were the same gene. Either of them is
only one copy in the Arabidopsis genome.
Compared with BcpLH in Chinese cabbage (Yu et al.,
2000), AtLH gene has extra five tandem repeats of 84 bp
downstream of coding sequence, and thereby is 420 bp
long. The fragment of AtLH coding sequence lacking of the
last 5 repeats shares 81% similarity with BcpLH coding
sequence, and two deduced protein sequences have 76%
identity (Fig.1A). Both sequences include two double-
stranded RNA binding motifs (DSRMs), each spanning 66
amino acids. Higher identities (85% and 89%) were desig-
nated respectively in the first DSRM and the second DSRM
between AtLH and BcpLH (Fig.1B).
AtLH cDNA fragment in pBluescript was digested with
BamHⅠ and SalⅠ, and inserted directionally into the bi-
nary vector pJR1 (Fig.2A). In the resultant construct, AtLH
was set under the control of CaMV35S promotor in sense
orientation. The recombinant vector was transformed into
the A. tumefaciens strain LBA4404 by freeze-thaw method.
Transgenic lines (Columbia ecotype) harboring exog-
enous sense -AtLH gene were es tab l i shed by
Agrobacterium-mediated transformation, which was per-
formed by vacuum infiltration method. Transgenic lines
were selected in the presence of 50 mg/L kanamycin and
verified by Southern hybridization (Fig.2B). Totally five
transgenic lines were identified to contain T-DNA insert,
four of which showed morphological alteration. One line
AtLH-S3 with morphological alteration was mainly studied
further.
2.2 Overexpression of AtLH in transgenic plants
The root, shoot, leaves and flower buds of Arabidopsis
were used for determining the expression level of AtLH
respectively. As shown in Fig.2C, RT-PCR analysis showed
that expression level of AtLH was equivalent in all organs
of wild type plants. Although the AtLH expression was
observed in all organs of 35S::AtLH transgenic plants, it
significantly enhanced only in leaves and flower buds
(Fig.2C). Given that CaMV35S promoter is constitutive in
expression, the expression level of AtLH gene might be the
same in different organs of 35S::AtLH plants. However, the
Fig.2. Vector structure, Southern blot and spatial expression of 35S::AtLH transgenic plants. A. Diagram of AtLH expression construct
in pJR1 binary vector. B. Southern blot of genomic DNA digested with EcoRⅠ, 1.2 kb fragment of NPTⅡ gene was used as probe. C.
AtLH expression level in different organs by RT-PCR analysis using Histone3 as internal control.
WU Hao et al.: Leaf Downward Curvature and Delayed Flowering Caused by AtLH Overexpression in Arabidopsis thaliana 1109
RT-PCR results did not accord with that. The reason for the
selective increment of AtLH expression in 35S::AtLH plants
remains to be clarified. Recently, it has been reported that
the FMV Sgt promoter, a constitutive promoter and about
2-fold stronger than the CaMV35S promoter was in the order:
root > leaf > stem（Bhattacharyya et al., 2002）of ex-
pression level in seedlings. Thus, expression level of AtLH
under CaMV35S promoter may vary in different organs of
35S::AtLH transgenic plants. In a word, the selective and
obvious increment of AtLH expression in leaves and flower
buds result from transforming exogenous sense- AtLH gene.
2.3 Modification of leaf form and inflorescence in 35S::
AtLH transgenic plants
35S::AtLH transgenic plants were compared with wild
type plants under the same growth conditions. Remarkable
morphological alterations were detected on the direction of
leaf curvature, flowering time and apical dominance (Table
1 , Fig.3B, C). The transgenic plants were characterized with
downward or/and outward curvature in rosette leaves, com-
pared with the flatten leaves in wild type plants (Fig.3C).
Moreover, those plants with more severe phenotypes also
had smaller leaves. In these cases, the top parts and mar-
gins of blades tended to bend downward.
Both axillary shoots and cauline inflorescences were
seen on Arabidopsis plants. Axillary shoots emerged from
axil of shorten shoots between rosette leaves, and the cauline
inflorescences appeared from axil of main infloresences
above cauline leaves. Axillary shoots were different from
the cauline infloresences in that the former appeared on
vegetative shoots while the latter grew on elongated repro-
ductive inflorescences. The AtLH-overexpressed plants had
more axillary shoots and fewer cauline inflorescences than
wild type plants, and even in some cases the main inflores-
cence totally disappeared (Fig.3B). In wild type plants, the
main shoot developed earlier than the axillary shoots. In
35S::AtLH plants, there were 3-5 leaves around each axil-
lary shoot before onset of bolting, and main inflorescence
and cauline inflorescences developed almost at the same
time possibly because the plant lost apical dominance at
early vegetative stage.
The axillary shoots of transgenic plants started bolting
10 d later than that of wild type plants and then began
flowering 15 d later. Either main inflorescence or cauline
inflorescences produced relatively few cauline branches
as compared with that of wild type. Thus apical dominance
of transgenic plants became stronger in flowering shoots.
2.4 Hormonal responses on 35S:: AtLH plants
35S::AtLH seeds of T3 generation were inoculated on
MS mediums containing different concentrations of IAA,
ABA, BA or 2,4-D. In the absence of exogenous hormone,
germination percentage and root growth rate of the seed-
lings were the same between 35S::AtLH transgenic lines
and wild type. On addition of 0.1 µmol/L and 1.0 µmol/L
IAA into medium, the transgenic plants were highly stimu-
lated germination of transgenic seeds and the subsequent
root growth. Roots of transgenic plants grew much faster
than that of wild type at these concentrations (Fig.4A). On
the other hand, root growths of wild type were completely
inhibited by 0.5 µmol/L ABA while roots of 35S::AtLH seed-
lings still grew. These suggested that root growth of
transgenic seedlings were less sensitive to ABA inhibition
than those of wild type plants, since relative root growth
rate (RRGR) of transgenic seedlings were significantly
higher under 0.05, 0.5 or 5.0 µmol/L ABA respectively.
Neither exogenous BA nor 2,4-D showed significant in-
fluence on root growth of transgenic plants. The growth of
sense plant shoots and that of wild-type shoots under
Fig.3. Plant phenotype of 35S::AtLH transgenic plants. A. Plant
of Columbia wild type. B. 35S::AtLH plant at flowering stage. C.
Leaves of 35S::AtLH plant (left) and wild type plant (right). D.
Inflorescences of wild type (left) and 35S::AtLH plant (right).
Table 1 Leaf curvature and inflorescence phenotype of 35S::
AtLH plants, which is described on the basis of comparison with
wild type plants
35S::AtLH Wild type
Ratio of leaf length/width 1.61 1.70
Bolting time (DAGs) 32-35 25-27
Number of branches on 2-3 1-2
shorten shoots
Number of cauline branches on 2-3 3-4
inflorescence
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041110
different concentrations of BA or 2,4-D were almost the
same.
3 Discussion
Arabidopsis AtLH and Chinese cabbage BcpLH have
the regulatory function on leaf curvature. AtLH functions
on the stature of flatten leaf while BcpLH is related to leaf
curvature (Yu et al., 2000). Our research on BcpLH gene
had been limited by lack of LH (leafy head) mutant of Chi-
nese cabbage or by failure to make the complete knock-off
of BcpLH gene. In this case, understanding of biological
function of AtLH in Arabidopsis will help us evaluate the
regulatory function of BcpLH in Chinese cabbage. AtLH
knock-off or overexpression in Arabidopsis may alter the
direction and degree of leaf curvature, and hence poses a
useful alternative way to disclose the mechanism involved
in formation of leafy head in Chinese cabbage.
AtLH, compared with BcpLH, has an additional domain
of six tandem repeats at carboxyl terminal end of protein
sequence. Such repeats were recognized as protein-pro-
tein interaction site (PPIS), a weakly homologous repeat of
protein F from S. pyogenes (24.7% identity and 55% similar-
ity in the 190-amino acid repetitive region) (Lu and Fedoroff,
2000). Both proteins have the same structural feature with
no gaps between the repeated units, and the repeats of
both proteins are rich in acidic amino acids (Asp and Glu).
Protein F is a fibronectin binding protein, and its repeat
region has been shown to be necessary for binding (Sela et
al., 1993). Lack of the repeats in Chinese cabbage means
the possible loss of the ability of protein-protein interac-
tion for BcpLH and may differentiate the function of AtLH
from BcpLH.
BcpLH gene was specifically expressed in folding leaves
of Chinese cabbage at vegetative stage (Yu et al., 2000). As
a double-stranded RNA binding protein, its function in leaf
curvature may be involved in gene interaction. Few of genes
encoding dsRNA binding proteins have been isolated from
plant, and their functions had not been identified well. For
instance, a plant protein with an RNA helicase domain and
an RNase Ⅲ-like domain was recognized through analysis
Fig.4. Root growth rate of seedlings on MS medium containing different concentratios of different hormones. Root length was measured
7 d after germination. Root growth is expressed relative to growth on MS medium without hormone. Each point represents mean of 20
seedlings. Twiled squares denote wild type seedlings; filled squares denote 35S::AtLH transgenic seedlings. Error bars indicate SD.
WU Hao et al.: Leaf Downward Curvature and Delayed Flowering Caused by AtLH Overexpression in Arabidopsis thaliana 1111
of an Arabidopsis floral mutant (Jacobsen et al., 1999). Most
biological information came from dsRNA binding proteins
of animals identified in humans, Drosophila, yeast, E. coli,
and viruses (Burd and Dreyfuss, 1994). These proteins func-
tion in developmental process by the modification of the
RNA editing, polyadenylation, signal recognition or nuclear
targeting. One of the best-characterized dsRNA binding
proteins is PKR, a dsRNA-dependent protein kinase. In-
duced by interferon treatment, and PKR mediates the anti-
viral and antiproliferative effects of interferon; it also has a
role in regulating cellular differentiation, stress response,
and apoptosis (Clemens, 1997). It is believed that dsRNA
binding proteins control many biochemical pathways by
silencing of the protein with the structure of dsRNA. As a
homolog of BcpLH, AtLH may regulate leaf curvature in
the similar way like BcpLH or in the modified way. Therefore,
it is necessary to verify biological function of AtLH in leaf
curvature.
Endogenous hormones play an important role in devel-
opment of leaf. It has been proposed a model in which the
distribution of auxin in the meristem regulates phyllotaxis
(Kuhlemeier and Reinhardt, 2001). Auxin can only accumu-
late to levels necessary for organ initiation at a characteris-
tic distance from the youngest pre-existing primordia, re-
sulting in the regular organ patterns found in nature
(Reinhardt and Kuhlemeier, 2001; 2002). In addition, auxin
influences the leaf folding (upcurvature) in Chinese cab-
bage (He and Yu, 1998). When IAA was sprayed on the
abaxial side of folding leaves, leaf curved inward. Exog-
enous auxA and auxB genes promote the process of forma-
tion of leafy head in Chinese cabbage (He et al., 1994; He
et al., 2000). On the other hand, plant sensitivity to exog-
enous hormones is one of the critical elements for genetic
performance of development. For Arabidopsis plant of wild
type, IAA has stimulating effect while ABA has inhibitory
effect on root growth. We found that AtLH overexpression
increased the stimulating effect of IAA and decreased the
ABA inhibitory effect on root growth. Since leaf curvature
is caused by difference in cell number and/or cell size be-
tween abaxial side and adaxial side of leaf, it is reasonablely
deduced that auxin-dependant proliferation of cells may be
related to the function of AtLH. The meaning of modified
sensitivity to IAA or ABA by AtLH overexpression remains
to be evaluated.
In order to understand AtLH function on the plants, we
constructed antisense vector of AtLH at the same time of
doing sense one. At the beginning we expected to know
what would happen when knocking out AtLH. But we did
not get valuable results, because antisense vector did not
work as expected (the results was not shown). Fortunately,
Lu and Fedoroff (2000) reported a Arabidopsis mutant of
leaf incurvature designated as AtLH (the same as hyl1).
The AtLH mutant was identified from an Arabidopsis Dis-
sociation (Ds) insertion mutant collection generated by
using a previously described transposon tagging system
(Fedoroff, 1993). As the leaf of AtLH mutant develops, its
abaxial side grows more than the adaxial side. This unequal
growth causes the leaf at seedling stage to curve upward,
and two sides of the blade to curve inward.
Three types of plants with differential AtLH expression
showed different direction of leaf curvature. AtLH mutant
had the leaf incurvature because AtLH gene was knocked
out, wild type plant maintained the flatten leaf when AtLH
expressed normally, and 35S::AtLH plant exhibited leaf
outcurvature because AtLH was overexpressed in leaf. The
direction of leaf curvature seems to be dependable on the
expression levels of AtLH. Increase expression of AtLH may
be an important element for morphological transformation
from incurvature to outcurvature in rosette leaves.
There also are three types of leaves in matured vegeta-
tive plant of Chinese cabbage: outcurvature (rosette leaf),
upcurvature (folding leaf) and incurvature (head leaf). The
direction of leaf curvature regulated by AtLH and its ex-
pression patterns clues in the possible function of BcpLH
on development of three types of leaves. Whether spatial
and temporal expression of BcpLH causes alternative cur-
vature of leaves in one plant is an interesting point. The
mechanisms that discriminate between AtLH and BcpLH
remain obscure. Further investigation of the molecular in-
teractions between the AtLH protein and dsRNA, as well
as of the candidate proteins that interact with AtLH, should
provide insight into the role of this gene in development
and the function of dsRNA in hormone signaling.
We found that Arabidopsis plant exhibits two types of
shoot apical dominance. Apical dominance in vegetative
shoot functions at rosette stage or earlier. In some cases, a
few of dormant axillary buds activate the growth and ap-
pear out of leaf axil among rosette leaves, and further de-
velop into axillary shoots. On the contrary, apical domi-
nance in inflorescence works in inflorescence. Secondary
and third inflorescences are called as cauline inflorescence
since they are initiated from axil of cauline leaves on flower-
ing shoots. We found that 35S::AtLH plants lost the apical
dominance on vegetative shoots and obtain strong apical
dominance on flowering shoots. From this finding, we con-
clude that AtLH play a role in timing of apical dominance of
plant.
Flowering time of 35S::AtLH plants was significantly
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041112
delayed, compared with that of the wild type plants. This
phenomenon seems to be related to apical dominance. When
apical dominance is lost, more axillary shoots grow. As a
result, the main shoot or main inflorescence grow relatively
slowly, giving rise to late flowering of the whole plant.
Leaf curvature is necessary for formation of some crop
products. As an important edible organ of Chinese cabbage,
cabbage, Brussels sprouts and heading lettuce, leafy heads
are composed of many incurved leaves with special func-
tion of nutrient storage. Floral organs such as sepal, petal,
stamen and carpel are all modified leaves, and scales of
onion. Leaf tendrils of pea are also derived from leaves (He
and Yu, 1998). These modified forms of leaves exist in cer-
tain kind of curvature. Although the genes responsible for
leaf curvature of different types may be different, the in-
volvement of LH genes in the mechanism for leaf
incurvature should be one of the most important genetic
elements.
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