全 文 ：Received 23 Oct. 2003 Accepted 18 Dec. 2003
Supported by the State Key Basic Research and Development Plan of China (G19990116).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (2): 224-229
Analysis of Transgenic Tobacco with Overexpression of
Arabidopsis WUSCHEL Gene
LI Jun-Hua1, 2, XU Yun-Yuan1, CHONG Kang1*, WANG Hui2
(1. Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany,
The Chinese Academy of Sciences, Beijing 100093, China;
2. College of Agronomy, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100, China)
Abstract: The Arabidopsis WUSCHEL (WUS) gene plays a key role in the specification of the stem cells
in the shoot apical meristem (SAM). A cDNA of WUS has been amplified with the RT-PCR approach from
Arabidopsis. The plant overexpression vector was constructed. It was driven by a dual enhanced CaMV35S
promoter. The construct was transformed into tobacco (Nicotiana tabacum L.) via Agrobacterium mediation.
Dramatic phenotypic changes appeared in the WUS overexpression transgenic plants. Aberrant cell
divisions and ectopic organogenesis could be found in almost every aerial parts of the transgenic tobacco
except the meristems and the inner two floral whorls. The data showed a highly conserved function of WUS
in tobacco, and suggested that WUS is involved in organogenesis. The leaves were malformed, which
strongly matched those only described previously for plants grown in the presence of polar auxin transport
inhibitors. It suggested a possible function of WUS in leaf development. These results provide useful
information for functional analysis of WUS and important biotechnological implication as well.
Key words: WUSCHEL; Nicotiana tabacum ; overexpression; phenotypic analysis
Post-embryonic development in higher plants is char-
acterized by continuous and repetitive formation of new
structures and organs, which is different from most ani-
mals (Bowman and Eshed, 2000; Clark, 2001; Weigel and
Jürgens, 2002). The derivatives of a shoot apical meristem
(SAM) give rise to all the organs of the aerial parts of the
plant except cotyledons. Genetic analysis in Arabidopsis
has identified a central regulator of SAM, the WUSCHEL
(WUS) gene. Shoot meristems of wus mutant terminate pre-
maturely after producing only a few leaves, and flowers of
the mutant are formed occasionally but lack carpel and
most stamens. So WUS is required to keep the pool of stem
cells (Laux et al., 1996). WUS encodes a homeodomain
protein, which functions as a transcriptional regulator
(Mayer et al., 1998).
The observation of the constitutively overexpression
of WUS in Arabidopsis is difficult, as it would preclude
recovery of the seedlings (Schoof et al., 2000), an alter-
ation is the use of the inducible system (Zuo et al., 2002).
Here we constitutively overexpressed WUS in tobacco
under the drive of the dual enhanced CaMV35S promoter.
The transgene caused dramatic phenotypic changes,
which provided useful information for functional analysis
of WUS.
1 Materials and Methods
1.1 Plant and bacteria
Nicotiana tabacum L. cv. W38 and Arabidopsis
thaliana L. Wassilewskija-2 ecotype, as well as bacteria of
Agrobacterium tumefaciens strain GV3101 (pMP90) (Koncz
and Schell, 1986) were used in this study.
1.2 Construction of overexpression vector
Total RNA was extracted using the TRIZOL kit (Gibco
BRL, USA) from aerial parts of Arabidopsis plants. First-
strand cDNAs were synthesized by reverse transcription
kit (TaKaRa, Japan), open reading frame (ORF) of WUS
was amplified using primers P1 (5-TTCTGGTACCATGGA-
GCCGCCACAGCATCAG-3) and P2 (5-TCTTGGAGCTCC-
TAGTTCAGACGTAGCTCAAG-3), which were designed
according to the sequence information of WUS (Mayer et
al., 1998). The PCR products were cloned into pGEM-T
vector (Promega, USA) and sequenced.
A dual enhanced CaMV35S promoter was inserted be-
tween HindⅢ and KpnⅠsites of the pBIB-KAN plasmid
(Becker et al., 1992) to produce vector pKAN-35S kindly
provided by Dr. LI. The WUS cDNA was digested with
KpnⅠ and SacⅠ and cloned between KpnⅠ and SstⅠ
sites in binary vector pKAN-35S to create the
LI Jun-Hua et al.: Analysis of Transgenic Tobacco with Overexpression of Arabidopsis WUSCHEL Gene 225
overexpression vector pBKB. The constructs were exam-
ined by PCR and KpnⅠ/SacⅠ double digestion. Plasmid
extraction, digestion, electrophoresis, ligation and Escheri-
chia coli transformation were according to Sambrook et
al. (1989).
1.3 Plant transformation and identification
pBKB and pKAN-35S were in t roduced by
electroporation into Agrobacterium strain GV3101. Trans-
formation of tobacco leaf discs was performed as described
previously (Horsch et al., 1985). The transformed plants
were selected by kanamycin. Transgenic plants were trans-
ferred into greenhouse at about 25 ℃ under natural light.
The positive lines were identified with tissue PCR as
described by Klimyuk et al. (1993). Level of WUS expres-
sion was detected by RT-PCR. Total RNA from leaf, stem
and flower was obtained respectively with the same method
mentioned above. After quantification one microgramme
of RNA was used in every RT-PCR reaction with One Step
RNA PCR kit (TaKaRa, Japan) and primers P1 and P2.
1.4 Phenotypic analysis
For conventional scanning electron microscopy (SEM),
fresh materials were prepared as described by Chen et al.
(2000), and examined with Hitachi S-2460 scanning elec-
tron microscope (Japan). The images were photographed
on Lucky 120 films.
Other photographs were taken with Sony DSC-F707
digital still camera (Japan).
2 Results
2.1 WUS cloning and transformations
A cDNA of WUS gene was obtained with the RT-PCR
method, which was identified as 899 bp in length with a
879 bp ORF. The construct of pBKB was confirmed by
PCR and KpnⅠ/SacⅠdouble digestion (data not shown).
The transgenic plants screened by kanamycin were identi-
fied with PCR amplication (Fig.1A), and target fragments
were obtained from transformed but not from control plants.
It showed that the target gene had been integrated into
the genome of transformed tobacco. The transcripts were
detected further with RT-PCR (Fig.1B). Again the target
fragments were obtained from each parts of transgenic
plant detected but not the control plant.
2.2 Phenotypic characterization of transgenic tobacco
plants
Eight independent lines of plants with ectopic WUS
gene were obtained, they displayed a wide range of al-
tered phenotype as early as at the in vitro regeneration
stage. When the seeds were inseminated directly in soil,
almost all of the T1 progeny of transgenic plant showed
severe defects and did not develop beyond the seedling
stage. Some wild-type seedlings developed normally in T1
plants. This suggested that transgenic plants is lethal at
young seedling stage. To overcome this difficulty, the
transgenic seeds were germinated and cultured on MS
medium till the seedlings had 4 leaves and the roots devel-
oped well. A higher viability of T1 plants was obtained by
this method and their phenotype was consistent with T0
plants. The higher viability of T0 and T1 plants by in vitro
culture could be due to the transgene having more defec-
tive effects on the seedlings and the adult shoots having
a better tolerance.
After planted in soil for three months, ectopic lateral
outgrowths appeared on the laminas, the stems, and in the
leaf axils of WUS overexpressing plants (Fig.2D, F, H).
During flowering phase, ectopic outgrowths also appeared
on the receptacles and even the corollas (Fig.2J, L). It
should be noted that some of the outgrowths could de-
velop into shoot meristems or flower buds (Fig.2G, H, J).
The transgenic plants had flowers with shorter filaments
and styli, and the stigmas are smaller than that of wild-
type, but none ectopic outgrowth could be found (Fig.2K,
L).
The alterrance of leaves was also obvious. From the
third or fourth leaf, the young leaves showed reduced ex-
pansion and upright position, subsequently, the laminas
showed curled phenotypes and rolled up at their fringes,
and the leaf vein pattern was also altered (Fig.2C). Some-
times conjointed leaves were formed, and trumpet-shaped
Fig.1. Identification of transgenic plants. A. PCR assay of
regenerated palnts from transgenic calluses. Lanes 1-4, transgenic
plants harboring exogenous WUSCHEL (WUS); Lane 5, wild-
type tobacco genomic DNA as template (negative control); Lane
6, pBKB plasmid DNA was used as template (positive control);
Lane 7, DNA marker. B. Detection of 35S::WUS transcripts by
RT-PCR. Lane 1, pBKB plasmid (positive control); Lanes 2, 4
and 6, leaf, stem and flower of wild-type tobacco respectively
(negative control); Lanes 3, 5 and 7, leaf, stem and flower of one
of the PCR positive plants, respectively.
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004226
LI Jun-Hua et al.: Analysis of Transgenic Tobacco with Overexpression of Arabidopsis WUSCHEL Gene 227
leaves were seen at in vitro stage (data not shown).
To character the ectopic cells described above, the leaf
outgrowths were observed by SEM. Results showed that
the outgrowths were leaf primordia-like, with small and
dense cells resembled the meristematic cells (Fig.3). The
meristems of the plants were examined by histological sec-
tions and no evident histological differences were observed
between the wild-type and transgenic plants.
3 Discussion
3.1 Overexpression of WUS in tobacco leads to ectopic
organogenesis
Owing to the results above, WUS overexpression is
sufficient to promote aberrant cell divisions and ectopic
organogenesis de novo in differentiated tissue in tobacco
(Fig.2D-L). However , it is reported that WUS
overexpression only induces aberrent cell divisions and
embryonic cell clusters but not organogenesis in
Arabidopsis (Gallois et al., 2002; Zuo et al., 2002). We
consider the difference in the phenotypic effect of WUS in
tobacco and Arabidopsis reported is due to an enough
period of overproduction of WUS protein in tobacco, but
not a difference in the molecular function of the WUS gene
that maintains SAM activity as reported (Noriko et al.,
2003), because the similar multiple shoots phenotype have
been observed in Arabidopsis (unpublished data). This
result suggested a new definition of the function of WUS
in organ formation, that is, WUS is involved in
organogenesis. This effect of WUS could also have impor-
tant biotechnological implications for vegetative propa-
gation from differentiated cells.
3.2 WUS and meristem cells
Several observations suggested that the size of the stem
cell population in the SAM and the floral meristem of
Arabidopsis are regulated by a negative feedback loop
between WUS and CLAVATA3 (CLV3), the stem cell marker
gene. In this loop, WUS activates the expression of CLV3,
and CLV3 repress WUS expression (Brand et al., 2000;
Fig.2. Phenotypic characterization of tobacco plants overexpressing the Arabidopsis WUSCHEL (WUS) gene. A. Gross morphology
of wild-type tobacco plant (the left one) and WUS overexpression transgenic plants (the right one). B. Leaf of the wild-type plants. C.
Leaf of WUS transgenic plants, the lamina curled up at fringes and leaf vein pattern altered. D. Leaf surface of a transgenic line with
outgrowths. E. Wild-type internote with only one axillary bud per axil. F. Internote of a WUS overexpression transgenic tobacco with
ectopic outgrowths on the stem and the leaf axils. G. Stem of a transgenic tobacco with a ectopic shoot meristem. H. Leaf axil of a
transgenic line with additional shoot meristems besides the axillary bud, the insert is the higher-magnification image of the ectopic buds.
I. Rachis of wild-type plants. J. Rachis of a transgenic line with ectopic columned outgrowths and flower buds on the receptacles, the
insert is a epiclinal flower. K. Mature wide-type flowers, the right one has been partly moved. L. flowers of a transgenic line, the right
one has been partly moved, with columned outgrowths on the receptacles and the corollas. Bars = 0.5 m (A), 10 cm (B,C), 2 cm (D-L)
and 0.5 cm (H inset).
Fig.3. Scanning electron micrograph of the leaf epidermis. A. Fully-grown wild-type epidermis. B. Leaf primordium-like outgrowths on
the leaf of transgenic line with small and dense cells. Bars = 1 900 µm.
←
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004228
Schoof et al., 2000). A initially similar self regulating cir-
cuitry is established between WUS and AGAMOUS (AG),
the floral homeotic gene which plays a key role in floral
meristem termination and specifies organ identity in whorls
3 (stamens) and 4 (carpels) (Bowman et al., 1989).
The transgenic plants have abnormal flowers with
shorter filaments, styli and smaller stigmas, which indi-
cated an unclear effect of transgene on these two organs,
but no ectopic outgrowth was seen (Fig.2L). Histological
section analysis of transgenic plant showed that the cells
of the meristem still positioned properly (data not shown).
Therefore, in spite of the widespread expression directed
by the 35S promoter, it seems that WUS can not promote
excess cell division or organogenesis in the shoot apical
and floral meristems. Similar meristematic phenotype was
reported when WUS was overexpressed ectopically in
Arabidopsis under the drive of inducible or meristem-spe-
cific promoters (Schoof et al., 2000; Lenhard et al., 2001;
Lohmann et al., 2001; Zuo et al., 2002). It was known that
the level of WUS expression was increased in mutants clv3
and ag because of loss of its suppressor. In these mutants,
the enlarged shoot apical and floral meristems in clv3 and
indeterminate flowers with pepals in ag mutant formed
(Bowman et al., 1989; Yanosky et al., 1990; Clark et al.,
1995). One conceivable interpretation for this difference is
that the similar repressors of WUS exist and functioning in
tobacco, as CLV3 and AG in Arabidopsis, and this kind of
suppression is strong. This is also proof of the high con-
servation of the function of WUS in tobacco.
3.3 WUS and auxin
The malformed leaves showed by the WUS-
overexpressing plants matched those only described pre-
viously for plants grown in the presence of polar auxin
transport inhibitors (Liu et al., 1993; Sieburth, 1999).
Therefore, these changes probably result specifically from
the loss of auxin polar transport or the decrease of auxin
synthesis level due to widespread expression of WUS. This
observation suggested that WUS may function non-cell-
autonomously in leaf development, and auxin is mediated
here. Previous study suggested that WUS regulates stem
cells and integument initiation in the chalaza by a non-cell-
autonomous way, but the mechanism is unknown (Mayer
et al., 1998; Gross-Hardt et al., 2002), our results provide a
possible hint for this research.
In summary, overexpression of the Arabidopsis WUS
gene leads to aberrant cell divisions and ectopic organo-
genesis in tobacco, and these changes can not be found
in the meristems and the inner two floral whorls, suggest-
ing a new definition of the function of WUS in organ
formation and a high conservation of its function in
tobacco. The malformation of leaves indicated that WUS
might function in leaf development and auxin polar trans-
port may contribute to it.
Acknowledgements: The authors are grateful to Dr. LI
Jia (Department of Botany and Microbiology, University
of Oklahoma) for his kind gift of DKAN-35S and Dr. CHEN
Chang-Bin (Department of Biology, Pennsylvania State
University) for his valuable suggestion and comments.
References:
Becker D, Kemper E, Schell J, Masterson R. 1992. New plant
binary vectors with selectable markers located proximal to the
left T-DNA border. Plant Mol Biol, 20:1195–1197.
Bowman J L, Smyth D R, Meyerowitz E M. 1989. Genes direct-
ing flower development in Arabidopsis. Plant Cell, 1:37–52.
Bowman J L, Eshed Y. 2000. Formation and maintenance of the
shoot apical meristem. Trends Plant Sci, 5:110–115.
Brand U, Fletcher J C, Hobe M, Meyerowitz E M, Simon R.
2000. Dependence of stem cell fate in Arabidopsis on a feed-
back loop regulated by CLV3 activity. Science, 289:617–619.
Chen C B, Wang S P, Huang H. 2000. LEUNIG has multiple
functions in gynoecium development in Arabidopsis. Genesis,
26:42–54.
Clark S E, Running M P, Meyerowitz E M. 1995. CLAVATA3 is
a specific regulator of shoot and floral meristem development
affecting the same processes as CLAVATA1. Development,
121:2057–2067.
Clark S E. 2001. Cell signalling at the shoot meristem. Nat Rev, 2:
276–284.
Gallois J L, Woodward C, Reddy G V, Sablowski R. 2002. Com-
bined SHOOT MERISTEMLESS and WUSCHEL trigger ec-
topic organogenesis in Arabidopsis. Development, 129:3207–
3217.
Gross-Hardt R, Lenhard M, Laux T. 2002. WUSCHEL signaling
functions in interregional communication during Arabidopsis
ovule development. Gen Dev, 16:1129–1138.
Horsch R B, Fry J E, Hoffman N L, Eichholts D, Rogers S G,
Fraley R T. 1985. A simple method for transferring genes into
plants. Science, 227:1229–1231.
Klimyuk V I, Carroll B J, Thomas C M, Jones J D G. 1993. Alkali
treatment for rapid preparation of plant material for reliable
PCR analysis. Plant J, 3:493–494.
Koncz C, Schell J. 1986. The promoter of TL-DNA gene 5 con-
trols the tissue-specific expression of chimaeric genes carried
by a novel types of Agrobacterium binary vector. Mol Gen
Genet, 204:383–396.
Laux T, Mayer K F X, Berger J, Jürgens G. 1996. The WUSCHEL
gene is required for shoot and floral meristem integrity in
LI Jun-Hua et al.: Analysis of Transgenic Tobacco with Overexpression of Arabidopsis WUSCHEL Gene 229
(Managing editor: ZHAO Li-Hui)
Arabidopsis. Development, 122:87–96.
Lenhard M, Bohnert A, Jürgens G, Laux T. 2001. Termination of
stem cell maintenance in Arabidopsis floral meristems by in-
teractions between WUSCHEL and AGAMOUS. Cell, 105:
805–814.
Lenhard M, Jürgens G, Laux T. 2002. The WUSCHEL and
SHOOTMERISTEMLESS genes fulfil complementary roles
in Arabidopsis shoot meristem regulation. Development, 129:
3195–3206.
Liu C M, Xu Z H, Chua N H. 1993. Auxin polar transport is
essential for the establishment of bilateral symmetry during
early plant embryogenesis. Plant Cell, 5:621–630.
Lohmann J, Hong R, Hobe M, Busch M, Parcy F, Simon R,
Weigel D. 2001. A molecular link between stem cell regulation
and floral patterning in Arabidopsis. Cell, 105:793–803.
Mayer K F X, Schoof H, Haecker A, Lenhard M, Jürgens G, Laux
T. 1998. Role of WUSCHEL in regulating stem cell fate in the
Arabidopsis shoot meristem. Cell, 95:805–815.
Noriko K, Hiroshi N, Atsushi M, Yutaka S, Makoto M. 2003.
Isolation and characterization of a rice WUSCHEL-type
homeobox gene that is specifically expressed in the central
cells of a quiescent center in the root apical meristem. Plant J,
35:429–441.
Sambrook L, Fritsch E F, Maniatis T. 1989. Molecular Cloning: a
Laboratory Manual. 2nd ed. New York: Cold Spring Harbor
Laboratory Press.
Schoof H, Lenhard M, Haecker A, Mayer K F M, Jürgens G,
Laux T. 2000. The stem cell population of Arabidopsis shoot
meristems is maintained by a regulatory loop between the
CLAVATA and WUSCHEL genes. Cell, 100:635–644.
Sieburth L E. 1999. Auxin is required for leaf vein pattern in
Arabidopsis. Plant Physiol, 121:1179–1190.
Weigel D, Jürgens G. 2002. Stem cells that make stems. Nature,
415:751–754.
Xu Y-Y , Chong K , Xu Z-H , Tan K-H . 2002. The practical
technique of in situ hybridization with RNA probe. Chin Bull
Bot , 19:234-238 (in Chinese with English abstract)
Yanofsky M F, Ma H, Bowman J L, Drews G N, Feldmann K A,
Meyerowitz E M. 1990. The protein encoded by the
Arabidopsis homeotic gene agamous resembles transcription
factors. Nature, 346:35–39.
Zuo J, Niu Q W, Frugis G, Chua N H. 2002. The WUSCHEL gene
promotes vegetative-to-embryonic transition in Arabidopsis.
Plant J, 30:349–59.