Activation tagging, an efficient tool for functional analysisof the rice genome
Shuyan Wan Æ Jinxia Wu Æ Zhiguo Zhang Æ Xuehui Sun Æ Yaci Lv ÆCi Gao Æ Yingda Ning Æ Jun Ma Æ Yupeng Guo Æ Qian Zhang ÆXia Zheng Æ Caiying Zhang Æ Zhiying Ma Æ Tiegang Lu
Received: 5 February 2007 / Accepted: 17 September 2008 / Published online: 2 October 2008
� Springer Science+Business Media B.V. 2008
Abstract Over the past 6 years, we have generated about
50,000 individual transgenic rice plants by an Agrobacte-
rium-mediated transformation approach with the pER38
activation tagging vector. The vector contains tandemly
arranged double 35S enhancers next to the right border of
T-DNA. Expression analysis by reverse transcription-PCR
indicates that the activation efficiency is high if the genes
are located within 7 kb of the inserted double 35S
enhancers. Comparative field phenotyping of part of the
activation tagging and enhancer trapping populations in
two generations (6,000 and 6,400 lines, respectively, in the
T0 generation, and 36,000 and 32,000 lines, respectively, in
the T1 generation) identified about four hundred dominant
mutants. Characterization of a dominant mutant with a
large leaf angle (M107) suggests that this mutant pheno-
type is caused by enhanced expression of CYP724B1/D11.
The activation tagging pool described in this paper is a
valuable alternative tool for functional analysis of the rice
genome.
Keywords Activation tagging � Dominant mutation �Enhancer trapping � Flanking sequence � Rice
(Oryza sativa L.) � T-DNA insertion
Introduction
The most direct approach for functional gene analysis is to
find a correlation between phenotype and genotype in a
specific mutant. There are several types of chemical,
physical and biological methods for creating mutants, the
most widely used are ethyl methanesulfonate treatment,
fast neutron irradiation, T-DNA and transposon insertion.
Correlations between phenotype and genotype in T-DNA
and transposon mutants can be easily identified. However,
these simple approaches usually cause loss-of-function
mutations, which have several disadvantages. T-DNA and
transposon insertion approaches are not applicable to dis-
secting the function of redundant genes. It is also difficult
for dissecting the function of genes involved in multiple
stages of the life cycle whose loss-of-function mutations
usually resulted in early embryogenic or gametophytic
lethality (Jeong et al. 2002; Weigel et al. 2000). T-DNA
and transposon insertion mutant collections have been
established worldwide for a number of plant species and
have been used to analyze gene function by forward and
reverse approaches, especially in Arabidopsis, the model
dicot (Azpiroz-Leehan and Feldmann 1997; Krysan et al.
1999; Parinov et al. 1999; Parinov and Sundaresan 2000;
Sessions et al. 2002; Speulman et al. 1999; Sussman et al.
2000; Tissier et al. 1999), and rice, the model monocot
(Hirochika 2001; Hirochika et al. 2004; Hsing et al. 2007;
Jeon et al. 2000; Miyao et al. 2003; Piffanelli et al. 2007;
Sallaud et al. 2003, 2004; Wu et al. 2003; Yang et al.
2004).
Gene activation has been used for functional analysis
of redundant genes required in multiple important life
process. A histidine kinase homolog, CKI1 that is involved
in cytokinin signal transduction has been identified by
means of gene activation (Kakimoto 1996). Systematic
Shuyan Wan, Jinxia Wu and Zhiguo Zhang contributed equally to this
work.
S. Wan � J. Wu � Z. Zhang � X. Sun � Y. Lv � C. Gao � Y. Ning �J. Ma � Y. Guo � Q. Zhang � X. Zheng � T. Lu (&)
Biotechnology Research Institute/National Key Facility for Gene
Resources and Gene Improvement, Chinese Academy of
Agricultural Sciences, Beijing 100081, China
e-mail: [email protected]
Y. Lv � C. Gao � Y. Ning � C. Zhang � Z. Ma
College of Agronomy, Hebei Agriculture University,
Hebei Baoding 071001, China
123
Plant Mol Biol (2009) 69:69–80
DOI 10.1007/s11103-008-9406-5
gene activation tagging systems have been established in
Arabidopsis using vectors carrying four copies of 35S
enhancers in the T-DNA left border region (Nakazawa
et al. 2003; Weigel et al. 2000). Activation tagging sys-
tems using only an enhancer(s) is thought to induce
endogenous gene expression. Thus, the phenotypic chan-
ges that resulted from the increased gene expression most
likely reflect the normal role of the activated gene. Other
activation tagging systems also have been established in
Arabidopsis using the transposon Ac/Ds system or En-I
system, which carries CaMV 35S enhancers (Marsch-
Martinez et al. 2002; Wilson et al. 1996). Significant
progress in establishing activation-tagged populations has
been made in other species, such as rice (Hsing et al.
2007; Jeong et al. 2002, 2006; Mori et al. 2007), tomato
(Mathews et al. 2003) and barley (Ayliffe et al. 2007).
CaMV 35S enhancer activation tagging has been used to
identify a number of novel functional genes in Arabid-
opsis (Borevitz et al. 2000; Graaff et al. 2000; Li et al.
2001, 2002; Neff et al. 1999), rice (Mori et al. 2007;
Hsing et al. 2007), tomato (Mathews et al. 2003) and
poplar tree (Busov et al. 2003). Tissue- and organ-specific
transcriptional activation of genes immediately adjacent to
the inserted enhancers has been confirmed using reverse
transcription-PCR analysis in the respective transgenic
lines (Jeong et al. 2002).
Gene activation tagging efficiency was analyzed based
on results from gain-of-function mutant screens. Weigel
et al. (2000) obtained more than 30 dominant mutants with
obvious phenotype changes from a population of 49,000
(0.08%). Wilson et al. (1996) found four dominant mutants
in a Ds activation tagging population of 1,100 (0.36%). In
the En-I activation tagging system, Marsch-Martinez et al.
(2002) found 31 dominant mutants after screening 2,900
lines (1.07%). To estimate activation tagging efficiency,
Jeong et al. (2002) randomly selected 10 genes, which
ranged from 1.5 to 4.3 kb upstream or downstream from
the CaMV 35S enhancers. Expression was significantly
enhanced in four of them (Jeong et al. 2002). In other
words, the gene expression activation efficiency is about
40%; the activation tagging efficiency, however, has yet to
be determined based on visible phenotype changes. Here,
we report the generation of an activation tagging popula-
tion of 50,000 lines in rice by Agrobacterium-mediated
transformation using pER38 vector carrying double CaMV
35S enhancers. Field phenotyping was carried out for large
activation tagging and enhancer trapping pools in two
generations, and gene expression was analyzed in selected
pER38 lines. Identification and characterization of a
dominant mutant, M107, is also described. M107 has a
large leaf angle phenotype that may be caused by enhanced
expression of CYP724B1/D11 via the tandem 35S
enhancers in pER38 vector.
Experimental procedures
Binary transformation vectors
The pER38 activation tagging vector was kindly provided
by Dr Eric van der Graaff (Institute of Molecular Plant
Sciences, Leiden University, Netherlands). Double CaMV
35S enhancers were located tandemly at the right border of
the T-DNA vector. The selection markers for the pER38
vector are kanamycin resistance (NPTII) in bacteria and
hygromycin resistance (HPT) in plants. The pFX-E
enhancer trapping vector was kindly provided by Dr.
Andrzej Kilian of CAMBIA (Center for the Application of
Molecular Biology to International Agriculture, Australia).
The selection markers for the pFX-E vector are chloram-
phenicol resistance in bacteria and hygromycin resistance
in plants. The functional regions of the two plasmids are
shown in Fig. 1a and b.
Rice transformation and growth of transgenic plants
The activation tagging and enhancer trapping vectors were
introduced into the Agrobacterium tumefaciens strain
EHA105 using the heat shock method. Single clones were
selected and authenticated by PCR as well as by restriction
enzyme digestion. Validated clones were cultured at 28�C
in darkness in LB liquid medium containing selection
antibiotics. Actively growing agrobacteria were suspended
in glycerol at a final concentration of 15% (v/v) and stored
at -80�C. The agrobacteria were cultured on solid AB
medium at 22�C in darkness for 5 days, then collected and
LB
HPT PUC
RB
p35S DE
(A)
(B)
1kb
Gal4/Vp16EGFP BoGUS
LB RB
CAT-1 intron6XUASCAT-1 intron
HPT
PolyA 35S
35S PolyA
Fig. 1 Functional regions of T-DNA used to generate enhancer
trapping and activation tagging populations. a pER38 T-DNA. LB:
left border; RB: right border; DE: double CaMV 35S enhancers; HPTCassette: CaMV 35S promoter, HPT coding sequence and NOS
terminator; PUC: pUC9 vector complete sequence. b pFX-E T-DNA.
CAT-1: catalase-1 gene; GAL4/VP16: a fusion gene of the yeast
transcriptional activator Gal4 DNA-binding domain with the Herpes
simplex virus Vp16 activation domain; 69 UAS: upstream activator
sequence with six repeats; BoGUS: Bacillus OZ glucuronidase gene;
EGFP: enhanced green fluorescent protein gene
70 Plant Mol Biol (2009) 69:69–80
123
suspended in AAM medium (Hiei et al. 1994) when used
for transformation experiments. The OD600 values of the
agrobacteria suspensions used for transformation were
between 0.10 and 0.15.
Transformation of the Nipponbare rice variety (Oryza
sativa spp. japonica) was carried out according to the
protocol established in our laboratory (Yang et al. 2004).
When regenerated T0 plants with the pER38 and pFX-E
vectors were about 15 cm tall, they were transferred to
an experimental paddy field designed specifically for
transgenic plants during a suitable planting season
(15 cm between plants, 30 cm between rows). T1 seeds
were germinated in a greenhouse. When plants were
15 cm tall, they were transplanted to the paddy field as
described for the T0 plants. Minimum of 20 plants were
planted for each T1 line. The activation tagging and
enhancer trapping plants were planted in an alternating
manner, 20 lines for each plot. For further observation
and confirmation of dominate mutations, all the T2 plants
are planted in a single-seed-descent manner. Transgenic
plant plots were surrounded with plots of wild-type
Nipponbare plants in the paddy field to ensure that
growth conditions were identical. All the plants were
managed according to standard watering and fertilizing
protocols (Xia 2006).
DNA extraction and PCR analysis of transgenic plants
Young leaves from more than 200 randomly selected T0
plants (hygromycin resistant, 100 enhancer trapping lines
and 100 activation tagging lines) were collected for DNA
extraction using a modified CTAB method (Murray and
Thompson 1980). DNA samples were quantified using a
DU 800 spectrophotometer (Beckman, Fullerton, CA,
USA) and verified by gel electrophoresis. Putative trans-
genic plants containing pER38 and pFX-E vectors were
confirmed by PCR analysis (Takara kit, Dalian, China)
using primers designed to amplify a 900-bp fragment of the
hygromycin-resistant gene, HPT. Primer sequences were
50-AAG TTC GAC AGC GTC TCC GAC-30 and 50-TCT
ACA CAG CCA TCG GTC CAG-30.
Southern blot analysis and T-DNA/Tos17 copy number
estimation from transgenic plants
To estimate T-DNA or Tos17 copy number, 5 lg DNA
for each line was digested with HindIII or XbaI. More
than 100 pER38 lines and pFX-E lines each were tested.
Digested DNA was loaded onto 0.8% agarose gels for
electrophoresis, transferred to a nylon membrane (Hybond
N?; Amersham Pharmacia Biotech, Piscataway, NJ,
USA) and hybridized with [a-32P]dATP-labeled probes
derived from HPT or Tos17 sequences to reveal the copy
number of T-DNA or newly transposed Tos17 insertions,
respectively (Primer-A-Gene labeling kit, Promega,
Madison, WI, USA). Southern blots were carried out
according to the Amersham-Pharmacia protocol. Because
HPT is located outside the HindIII-cut region in both the
pFX-E and pER38 vectors, and there is only one XbaI
cutting site in the entire Tos17 sequence, the number of
hybridized bands reflects T-DNA or Tos17 insertion copy
number.
Amplification of T-DNA flanking sequences
T-DNA left border flanking regions were rescued using
PCR walking according to the method described by Peng
et al. (2005) except that the plant samples were ground
using a Geno Grinder 2000 (SPEX CertiPrep, Methucen,
NJ, USA). This method consists of three steps: digestion
of genomic DNA using blunt end restriction enzyme and
ligation of an asymmetrical adaptor, PCR amplification
using primers specific to the T-DNA and the adaptor,
and successive PCR using two nested specific primers.
PCR products were purified using a gel extraction kit
(Qiagen, Valencia, CA, USA), and recovered DNA
samples were used for direct sequencing according to the
protocol for the Bigdye Terminator v3.1 Cycle Sequenc-
ing kit (ABI 3730xl, Applied Biosystems, Foster City,
CA, USA). LB2 (see below) was used as the sequencing
primer.
The adaptor (ADAR, 50 mM) was prepared by heating a
mixture of equal volumes of complementary oligonucleo-
tides, ADAR1 (100 mM, 50-CTA ATA CGA GTC ACT
ATA GCG CTC GAG CGG CCG CCG GGG AGG T-30)and ADAR2 (100 mM, 50-P-ACC TCC CC- NH2-30), to
80�C for 10 min, and then allowing the mixture to cool
gradually to room temperature for annealing. The specific
primers for the adaptor were APR1 (20 mM, 50-GGA TCC
TAA TAC GAG TCA CTA TAG CGC-30) and APR2
(20 mM, 50-CTA TAG CGC TCG AGC GGC-30). The
T-DNA left region-specific primers for the pER38 vector
were PLB1 (20 mM, 50-CTG TGT TCT TGA TGC AGT
TAG TCC TG-30) and PLB2 (20 mM, 50-CGT CTT GAT
GAG ACC TGC TGC-30); the distance between the PLB2
binding site and the left border was about 124 bp. The
T-DNA left region-specific primers for the pFX-E vector
were LB1 (20 mM, 50-CGA TGG CTG TGT AGA AGT
ACT CGC-30) and LB2 (20 mM, 50-GTT CCT ATA GGG
TTT CGC TCA TGT GTT G-30); the distance from the
LB2 binding site to the T-DNA left border was about
180 bp. T-DNA insertion locations could be identified by
NCBI BLAST homology searches of the rice genome
database (http://www.ncbi.nlm.nih.gov/Blast/) using the
rescued flanking sequences.
Plant Mol Biol (2009) 69:69–80 71
123
Semi-quantitative RT-PCR analysis of activation
of gene expression
Genes within 7 kb of the double CaMV 35S enhancers
were selected from the flanking sequence database of the
pER38 pool. Single-copy insertion lines were selected
based on Southern blot results. DNA was extracted from
individual 2-week-old plants using the modified CTAB
method. PCR amplification using combinations of the
PLB2 primer and two gene-specific primers that flank the
T-DNA insertion was performed to identify wild-type,
hemizygous, and homozygous plants.
Primers for semi-quantitative RT-PCR were designed
using DNAMAN software (version 6, Lynnon Biosoft,
Quebec, Canada). To minimize the effects of possible
DNA contamination, one of the two gene-specific primers
was located across two exons, so minor DNA, if there was
any, would not be amplified.
Total RNA was extracted using Trizol (Invitrogen,
Carlsbad, CA, USA) from wild-type and homozygous
transgenic plants (both roots and shoots were included) in
the segregating population. RNA was quantified using a
UV spectrophotometer (Beckman, DU 800). First-strand
cDNA was synthesized by reverse transcription using a
cDNA synthesis kit (Takara, Dalian, China) in 20 ll con-
taining 1 lg total RNA, 10 ng oligo(dT)14 primer, 2.5 mM
dNTPs, 1 ll AMV and 0.5 ll RNAsin. The PCR reaction
was performed in 20 ll containing a 1/20 aliquot of the
cDNA reaction, 0.5 lM gene-specific primers, 10 mM
dNTPs, 1 U rTaq DNA polymerase, and 2 ll of 109
reaction buffer. The reaction protocol was as follows:
denaturation at 94�C for 3 min followed by 25 cycles of
94�C for 30 s, 60�C for 45 s, and 72�C for 1 min, and a
final step at 72�C for 10 min. A 1-ll aliquot of the reaction
was loaded on a 1.0% agarose gel (regular, BIOWEST,
Spanish) and analyzed by electrophoresis. PCR products
were extracted using a gel extraction kit (Qiagen, Valencia,
CA, USA) after gel analysis and sequenced with the ori-
ginal PCR primers to verify that the products were correct.
Genes with significantly increased expression levels in
the respective pER38 lines were selected for further study
of tissue-specific activation of gene expression by double
CaMV 35S enhancers. Shoot and root RNA was extracted
from wild-type and homozygous transgenic plants in the
segregating population.
Co-segregation analysis of the dominant mutant M107
Co-segregation analysis of the dominant mutant M107 was
performed by PCR using two gene-specific primers flank-
ing the insertion site (P1 and P2) and another T-DNA right-
border primer (P3). PCR reactions were carried out in 20 ll
containing 20 ng plant DNA, 109 PCR buffer, 0.2 mM
dNTP, 0.5 U rTaq polymerase, and 1 lM of primers. DNA
was denatured at 95�C for 4 min, followed by 35 cycles of
94�C for 1 min, 58�C for 1 min, and 72�C for 2 min. The
primers for genotyping were 50-GTA AGC TAG CAC
CGC CTG G-30 (P1), 50-CAA AAA AAC GCC CTG CCC
C-30 (P2), and 50-GAT ACA GTC TCA GAA GAC
CAG-30 (P3).
Results
Generation and growth of transgenic rice plants
Using the pER38 vector, we generated about 50,000 indi-
vidual transgenic lines. We also generated a large
population of enhancer trapping lines using an Agrobac-
terium-mediated transformation approach with pFX-E as
reported by Yang et al. (2004). Greater than 95% of
hygromycin-resistant plants contained at least one copy of
T-DNA, as confirmed by PCR (data not shown). Trans-
formation protocols, particularly the time course of tissue
culture and transformation process, were exactly the same
during the generation of pER38 and pFX-E populations.
Southern blot hybridization of about 100 independent lines
for each population yielded T-DNA copy numbers of 2.85
and 2.76 per line on average in the pFX-E and pER38
populations, respectively. There are two copies of endog-
enous retrotransposon Tos17 in the genome of japonica
variety Nipponbare that can be activated by tissue culture
and the transformation process (Hirochika et al. 1996).
Using the same transformation protocol, the newly trans-
posed Tos17 copy numbers in pFX-E and pER38
populations were quite similar—2.15 and 2.10 per line on
average, respectively. Representative results are shown in
Fig. 2.
Field phenotyping of activation tagging and enhancer
trapping populations
Phenotyping was performed carefully for plants in different
growth and developmental stages using the same criteria
for both pER38 and pFX-E T0 and T1 generations. By
comparing plants in the same segregating population,
mutants with visible phenotype changes were identified,
recorded and labeled. Young leaves of mutants were col-
lected and stored at -80�C for DNA isolation and
subsequent flanking sequence amplification as described
above under ‘Amplification of T-DNA flanking sequences’.
The appropriate environment and growth conditions are
extremely critical for rice mutant phenotyping. The donor
material we used for generating mutant pools was Nip-
ponbare, which grows well in the northern part of China,
but not in the southern part. Rice expresses mutant
72 Plant Mol Biol (2009) 69:69–80
123
phenotypes better in natural field conditions than in the
greenhouse. At least two types of mutant phenotypes,
curled leaf and sterility, appeared at high frequencies in the
paddy field but were hardly identified in the greenhouse.
About 6,000 T0 generation pER38 lines and 6,400 T0
generation pFX-E lines, and 36,000 T1 generation pER38
lines and 32,000 T1 generations pFX-E lines (minimum 20
plants per line for T1 generation), were planted in the
experimental paddy field designed specifically for trans-
genic plants. These plants were characterized carefully
throughout their life cycle, from the seedling stage to the
completely mature stage. The most frequently observed
mutant phenotype in the T0 generation was dwarfism,
which accounted for 0.31% of mutations in the pER38
population and 0.20% in the pFX-E population. The
number of dwarfed mutants increased in the T1 generation,
reaching 1.67 and 1.61% in the pER38 and pFX-E popu-
lations, respectively. In the T0 generation, we identified
127 mutant lines with obvious phenotype changes from the
6,000 pER38 lines studied and 78 mutant lines from the
6,400 pFX-E lines; the respective mutation frequencies
were 2.12 and 1.21% (mutant lines versus total lines
planted), respectively. In the T1 generation, we identified
2,312 mutant lines with obvious phenotype changes in the
36,000 pER38 lines studied and 1,734 mutant lines in the
32,000 pFX-E lines. The results obtained from field phe-
notyping are shown in Table 1.
Mutants with obvious phenotype changes accounted for
6.42 and 5.43% of the total in our activation tagging and
enhancer trapping populations, respectively. These mutant
Fig. 2 Detection of T-DNA and Tos17 copy numbers in the T0
generation of pFX-E (lane 1 to lane 20) and pER38 (lane 21 to lane
40) lines. Nipponbare (lane 41) was used as the control. a Detection
of T-DNA copy number using an HPT probe. b Detection of newly
transposed Tos17 copy number using the Tos17 probe
Table 1 Characterization of different mutant types in pER38 and pFX-E populations
Classification
of mutant
T0 generation T1 generation
6,000 lines pER38 6,400 pFX-E
lines
36,000 pER38 lines (minimum
20 plants per line)
32,000 pFX-E lines (minimum
20 plants per line)
Mutant lines % Mutant lines % Mutant lines % Mutant lines %
Albino – – – – 673 1.77 410 1.31
Dwarf 18 0.31 12 0.20 600 1.67 518 1.61
High 20 0.33 12 0.20 102 0.28 51 0.14
Sterile 10 0.17 8 0.13 225 0.63 148 0.46
Tillering 10 0.17 10 0.17 148 0.41 258 0.63
Early maturing 30 0.50 12 0.20 154 0.43 166 0.52
Late maturing 12 0.20 8 0.13 191 0.53 80 0.25
Curled leaf 15 0.25 8 0.13 109 0.30 31 0.01
Lesion mimic 12 0.20 8 0.13 110 0.31 72 0.22
Sum 127 2.12 78 1.21 2,312 6.42 1,734 5.42
Plant Mol Biol (2009) 69:69–80 73
123
phenotypes are stable and reproducible in different years
(data not shown). The mutation frequencies of our popu-
lations described in this paper are much lower than that in
TRIM population (about 20%) (Chern et al. 2007). These
differences may be due to firstly, the phenotypes we
focused in mutant screening are much less than Chern et al.
in TRIM mutant screening (11 categories and 65 catego-
ries). Secondly, plant materials, vectors and protocols used
for transformation and phenotyping in our populations are
different from those in TRIM population. More than
186,500 lines (including redundant lines) of the pER38
activation tagging and the pFX-E enhancer trapping pop-
ulations have been distributed to the researchers for mutant
screening (especially conditional screening) in more than
10 laboratories in China.
Typical dominant mutant phenotypes
More than 400 dominant mutants were identified in the
pER38 T0 generation. Phenotype changes included steril-
ity, dwarfism, overgrowth, early flowing, late flowering,
light green leaf, leaf angle, lesion mimicking, radical
growth, curled leaf, early senescence, late senescence, and
over tillering etc. Some of the dominant mutants, about
20% of the total, were sterile and could not set seeds.
Typical dominant mutants in the pER38 population are
shown in Fig. 3.
Comparative characterization of T-DNA insertion
and integration in pER38 and pFX-E populations
The typical flanking sequence rescued by PCR walking was
a fusion of T-DNA and rice sequences. We selected 1645
pER38 lines and 1732 pFX-E lines for comparative char-
acterization of the T-DNA insertion and integration pattern.
We have amplified 1261 and 1533 PCR products and
obtained 985 and 1293 sequences for the pER38 and pFX-
E lines, respectively. The detailed characteristics of the left
border integration patterns are shown in Table 2.
The notable differences of T-DNA left border integra-
tion between the two populations include left border
recombination and read-through frequencies, which
occurred at much lower frequencies in the pER38 vector
(10.4%) than in the pFX-E vector (39.1%). The efficiency
of mutant lines with both T-DNA and rice DNA sequences
was much greater in the pER38 population (83.1%) than in
the pFX-E population (39.2%).
To understand the distribution pattern of T-DNA inser-
tions in the genome, 506 successful pER38 and 493
successful pFX-E integrations with both T-DNA left border
and rice genome DNA sequences were randomly selected
and analyzed. Similar T-DNA distribution patterns were
found in the two populations, with nearly 70% of inserts
located at genic regions (Table 3).
Confirmation of enhanced gene expression
in the pER38 population
The most direct way to reveal gene activation is to study
gene expression in the pER38 population with molecular
approaches. To examine enhanced gene expression by
activation tagging, we selected 10 individual transgenic
lines from the T1 generation with a single-copy T-DNA
insertion (data not shown) located in an intragenic region.
The enhancer elements in these lines inserted between 1.1
and 6.9 kb upstream or downstream of start codon of the
nearest open reading frame for the genes. Candidate gene
expression in wild-type and homozygous 2-week-old
transgenic seedlings carrying the pER38 vector in one
segregating population was examined using semi-quanti-
tative RT-PCR. Twelve candidate genes were tested, and
seven RT-PCR products were obtained. The PCR products
were confirmed by sequencing to be the gene we expected
(data not shown). Expression levels for five of the seven
genes examined were significantly enhanced in pER38
plants relative to wild-type plants (Fig. 4). Expression
levels of two other genes were comparable with wild-type
plants (data not shown). We did not obtain RT-PCR
products from the other five genes in the respective trans-
genic plants, suggesting that they were not transcribed at
the seedling stage or they were transcribed conditionally.
Taken together, the double CaMV 35S enhancers in the
pER38 vector act in the same fashion as the tetra CaMV
35S enhancers in the pSKI015 vector and acutely enhance
expression of nearby genes.
Tissue-specific, rather than ectopic, gene activation by
the double CaMV 35S enhancers of the pER38 vector was
also studied in transgenic plants. Shoots and roots were
collected from wild-type and transgenic plants carrying
homozygous pER38 sequences in the segregating popula-
tion. Total RNA was extracted and used for semi-
quantitative RT-PCR analysis. LOC_Os04g01290 in line D
(Fig. 4), which was predominately expressed in wild-type
roots, was selected for further study. Enhanced expression
of the LOC_Os04g01290 gene was found in roots of
homozygous transgenic plants carrying the pER38 vector,
indicating that double CaMV 35S enhancers can enhance
gene expression in a tissue-specific manner (Fig. 5).
Identification and characterization of a dominant
mutant caused by activation tagging
To demonstrate the utility of the activation tagging mutant
population, a mutant line named M107 was selected for
further study because of its dominant nature. The M107
74 Plant Mol Biol (2009) 69:69–80
123
Fig. 3 Typical dominant
mutants in pER38 population.
CK: Wild-type Nipponbare; A:
severe dwarf mutant; B: semi-
dwarf mutant; C: early
flowering mutant; D: light-green
leaf mutant; E: lesion mimic
mutant; F: dwarf and early
flowering mutant; G: radical
growth mutant; H: curled leaf
mutant; I: early senescence
mutant
Table 2 Comparative characterization of T-DNA left border integration patterns in pER38 and pFX-E lines
T-DNA integration patterns pER38 population pFX-E population
No. of sequences
rescued
% No. of sequences
rescued
%
Partial deleted left border with rice DNA sequences 690 70.0 340 27.3
Complete left border with rice DNA sequences 25 2.5 24 1.9
Partial deleted left border with both filler DNA and rice
DNA sequences
104 10.6 129 10.0
Partial deleted left border with filler DNA only 64 6.5 294 22.7
Left border recombination 47 4.8 198 15.3
Left border read-through 55 5.6 308 23.8
Total 985 100 1,293 100
Plant Mol Biol (2009) 69:69–80 75
123
mutant had a larger leaf angle phenotype during mature
stages than the wild-type (135� in the mutant versus 10.0�in the wild-type) (Fig. 6a). Segregation analysis of the
heterozygous T0 selfing population showed a 3:1 ratio of
M107 mutants to wild-type (78 mutants to 30 wild-type,
P \ 0.01), indicating that the M107 mutant phenotype is
caused by a single dominant gene mutation. Using the
PCR-walking method, a T-DNA flanking sequence was
rescued. A BLAST search using the flanking sequence
against the NCBI rice database showed that the T-DNA
double 35S enhancer was about 6.9 kb from the start codon
of LOC_Os04g39430 gene (Fig. 6b). Co-segregation
analysis showed that all plants carrying the defined T-DNA
insertion had a mutant phenotype, whereas plants that did
Table 3 T-DNA distribution in
the genomes of pER38 and
pFX-E populations using the
TIGR annotation system
Distribution of T-DNA inserts pER38 vector pFX-E vector
No. of sequences % No. of sequences %
Exon region 82 16.2 105 21.3
Intron region 85 16.8 92 18.7
30 UTR (500 bp) 92 18.2 37 7.5
50 UTR (500 bp) 90 17.8 100 20.3
Repeat sequences 32 6.3 39 7.9
Intergenic region 125 24.7 120 24.3
Total 506 100 493 100
Line C
Line No. Location of T-DNA integration RT-PCR Putative genes
Line A
Line E
T/T
W/W
-1.1kb TAG(2.9kb) LOC_Os04g45900
LOC_Os02g55560
LOC_Os08g30490
LOC_Os04g01290
ATG
RBLB
Line D
Line B-2.9kb
1kb
ATG
RBLB
3.8kb
TGA(2.6kb)ATG
RBLB
TGA(3.4kb)
ATG TAA(509bp)
RB LB
-1.9kb
LOC_Os04g39430TGA(3.8kb)ATG
RBLB
-6.9kb
T/T
W/W
T/T
T/T
T/T
W/W
W/W
W/W
Actin
Actin
Actin
Actin
Actin
Fig. 4 Enhanced gene
expression in transgenic plants
by the double CaMV 35S
enhancers in the pER38 vector.
T/T: homozygous transgenic
plant with single T-DNA insert;
W/W: segregated wild-type
plant. A, B, C, D and E: lines
with enhanced expression of
genes in seedlings;
corresponding gene accession
numbers in the TIGR database
are LOC_Os04g45900 (Metal-
nicotianamine transporter);
LOC_Os02g55560
(Phosphatase 2C protein);
LOC_Os08g30490 (Expressed
protein); LOC_Os04g01290
(Putative eukaryotic translation
IF3) and LOC_Os04g39430
(Cytochrome P450 family
protein)
76 Plant Mol Biol (2009) 69:69–80
123
not carry the defined T-DNA insertion had a wild-type
phenotype (Fig. 6c). Semi-quantitative RT-PCR analysis
confirmed that LOC_Os04g39430 gene expression was
dramatically increased in the M107 mutant plants but
remained the same as non-transgenic Nipponbare plants in
segregating wild-type plants (Fig. 4). A protein homology
search against the NCBI database showed that the
LOC_Os04g39430 gene encodes the cytochrome P450
family protein, CYP724B1, also termed D11, that is
involved in C-22 hydroxylation (the rate-limiting step in
brassinosteroid biosynthesis pathway). CYP724B1 is
functionally redundant with CYP90B2/OsDWARF4
(Sakamoto et al. 2006). A loss-of-function mutant of
CYP90B2/OsDWARF4 shows erect leaves in mature
stages, a short second internode, and reduced grain length
(Tanabe et al. 2005). To analyze the activity of exoge-
nously applied bioactive brassinosteroids, a lamina joint
inclination test (i.e., the degree of bending between the rice
leaf sheath and blade) was conducted. Two d11 alleles,
d11-1 and d11-2, showed a hypersensitive response to
brassinolide treatment (Tanabe et al. 2005). Over-expres-
sion of OsDWARF4, which has the highest sequence
similarity to CYP724B1/D11, recaptures the large leaf
angle phenotype (Sakamoto et al. 2006). Taken together,
this suggests that over-expression of the LOC_Os04g39430
gene (CYP724B1/D11), via the upstream double 35S
enhancers near the right border of the T-DNA, is respon-
sible for the M107 mutant phenotype. This example
demonstrates that the activation tagging population
described herein could be a valuable alternative tool for
functional analysis of the rice genome.
Discussion
The pER38 activation tagging vector used in this study
Activation tagging has been successfully applied to
uncover the function of novel genes in plant development
(Borevitz et al. 2000; Graaff et al. 2000; Li et al. 2001,
2002; Neff et al. 1999), especially redundant genes whose
loss-of-function mutations produce no visible phenotype
changes and genes required for multiple stages in the life
cycle whose loss-of-function mutations are lethal (Weigel
et al. 2000). Establishment of activation tagging popula-
tions in plants has mainly focused on Arabidopsis; there are
only few reports published on other plant species such as
tomato (Mathews et al. 2003) and barley (Ayliffe et al.
2007).
Rice is one of the most important crop plants because it
feeds more than 60% of the world’s population. Breeding,
genetics and genomics of rice have been extensively
studied. Sequencing of the entire rice genome has revealed
a large preponderance of gene duplication. For example,
the redundancy for the rice receptor-like kinase gene
family is about 40% (Shiu et al. 2004; Sun et al. 2004).
Single mutants, double mutants and even triple mutants are
necessary to elucidate the function(s) of these genes in
simple insertion populations, which is time consuming and
cost inefficient. Jeong et al. (2006) reported 47,932
Line D
T W
LOC_Os04g01290
Actin
Fig. 5 Tissue-specific activation of LOC_Os04g01290 gene expres-
sion in root tissue from the pER38 line D. T: Transgenic plant with
homozygous pER38 sequence; W: segregated wild-type plant
(C)
Phenotype
P1/P3
P1/P2
Line E
LOC_Os04g39430 RBLB
-6.9kb
1k
P1
P3
He
He
Ho
P2
Genotype W He
He
He
He
He
He
He
W W WHo
Ho
Ho
Ho
M M M W MMMMM MM W W WM M M M
31 2 4 1713119 5175 8 12 166 10 14 18
(B)
(A)
TGA(3.8kb)ATG
Fig. 6 Identification and characterization of the dominant M107
mutant. a Phenotype of the M107 mutant showing an enlarged leaf
angle. T/T: homozygous mutant; W/W: wild-type Nipponbare. bDiagram showing T-DNA insertion site on the rice genome. ATG:
start codon; TGA: termination codon; RB: right border; LB: left
border; P1, P2 and P3 are primers used for genotyping (their
sequences are described in ‘‘Experimental procedures’’). Nine exons
are indicated by the boxes. c Co-segregation analysis of M107
genotype and phenotype from a segregating population. All plants
with T-DNA insertion showed enlarged leaf angle, indicating that the
dominant mutation is caused by T-DNA insertion. Arabic numbers
represent different plants tested; P1/P2: PCR reactions using P1 and
P2 primers; P1/P3: PCR reactions using P1 and P3 primers; He:
hemizygous; Ho: homozygous; W: wild-type; M: mutant
Plant Mol Biol (2009) 69:69–80 77
123
activation tagging lines with tetra CaMV 35S enhancers
generated by Agrobacterium-mediated transformation.
Establishment of additional activation tagging populations
provides a valuable resource for functional analysis of rice
genome (Hsing et al. 2007; Mori et al. 2007). CaMV 35
enhancers and promoters have been widely used for gene
over-expression in both dicots and monocots. Most of the
activation tagging populations contain vectors with tetra
CaMV 35S enhancers, but the 35S enhancers in these
vectors are unstable and undergo progressive loss with
storage at 4�C (Weigel et al. 2000). We have assessed the
stability of the 35S enhancer in the pSKI015 vector by
overnight growing agrobacteria cells stored at -80�C
before culture. We frequently observed four products that
differ by about 500 bp when we use specific primers to
amplify tetra 35S enhancers, indicating that the plasmid
DNA we used was a mixture of mono, double, triple and
tetra 35S enhancer vectors (data not shown). In contrast,
the pER38 activation tagging vector is very stable at 4�C
(Graaff et al. 2000, 2002) and room temperature. We have
recovered pER38 complete plasmid DNA from agrobac-
teria stored at 4�C for over a year (data not shown).
There was not much difference in the activation tagging
efficiency and enhancement of gene expression between
double 35S enhancers in pER38 and tetra 35S enhancers in
pSKI015 (Graaff, personal communications). The Leafy
Petiole mutant was isolated from pool of 550 transgenic
lines with an activation tagging vector containing double
CaMV 35S enhancers. The mutant phenotype was caused
by activation of two tandemly arranged nearby genes,
VASCULAR TISSUE SIZA (VTS) and Leafy Petiole (LEP).
Activation of highly efficient gene expression was also
observed in our rice activation tagging lines containing
T-DNA with double CaMV 35S enhancers (five genes were
over-expressed among seven RT-PCR positive lines with
enhancers located within 6.9 kb of the gene of interest).
Dominant mutants found in the pER38 and pFX-E T0
generation
There are several reports of gene activation where the first
ATG of the gene of interest was several kb away from the
insertion sites of the CaMV 35S enhancers (Jeong et al.
2002; Weigel et al. 2000). Genes located up to 8.2 kb away
from the enhancer sequence are activated in an Arabidopsis
activation tagging population (Ichikawa et al. 2003). Hsing
et al. (2007) reported that expression of genes within
genetic distances of 12.5 kb was enhanced in their rice
activation tagging population. In zebrafish, genes could be
activated by enhancers 15 kb away from the coding region.
In two surprising cases, the genes sox11b and otx1 acti-
vated insertions at distances between 32 and 132 kb from
the coding region (Ellingsen et al. 2005). Our present study
identified 127 mutant lines with obvious phenotype chan-
ges in the 6,000 pER38 lines studied and 78 mutant lines in
the 6,400 pFX-E lines studied (T0 generation). The
respective frequencies of dominant mutations were 2.12
and 1.21% in the T0 generation of the two populations
(mutant lines versus total lines planted). Dominant muta-
tions may be caused by enhanced gene expression of
double CaMV 35S enhancers. 35S promoters or enhancers
in the HPT cassettes of both pER38 and pFX-E vectors
may also promote dominant mutations in the T0 generation.
The 35S promoter or enhancer was located in regions less
than 1 kb from the left border and 6 kb from the right
border of T-DNA in the pER38 vector, and less than 2.5 kb
from the left border and 6 kb from the right border of
T-DNA in the pFX-E vector. The distances were definitely
within the range required for gene activation described in
the literature (Ellingsen et al. 2005; Hsing et al. 2007;
Jeong et al. 2002; Weigel et al. 2000). Other sources of
dominant mutations are somaclone variations that occur at
the chromosomal level, such as chromosome breakage
(Chen and Sun 1994). The distance from the gene(s) of
interest to the enhancer(s) affects gene activation effi-
ciency, as does the structure of the chromosomes in which
the gene and enhancer are located (Rubtsov et al. 2006).
Advantages and strategies for using an activation
tagging population to uncover novel gene functions
in rice
There are several advantages for activation tagging-induced
gain-of-function mutations over simple insertion-induced
loss-of-function mutations. First, activation tagging is a
very efficient way to study the function of redundant genes.
Functional analysis of genes using a loss-of-function
approach is time consuming and cost inefficient. More than
4,000 genes in Arabidopsis chromosomes are found in a
tandem repeat manner, with two or more copies, and the
number in rice is even greater. For example, nearly 40% of
receptor-like kinase genes are redundant in rice (Shiu et al.
2004; Sun et al. 2004). Second, activation tagging is an
ideal approach to study the function of genes whose loss-of-
function mutations are lethal. Third, activation tagging
usually induces agronomically beneficial traits for crop
improvement. Fourth, activation tagging-induced gain-of-
function mutations, especially conditional mutants such as
stress-responsive mutants, are easy to identify using for-
ward genetics approaches because of the dominant nature of
these mutations. Corresponding genes could be further
identified by gene expression analysis.
There are both gain-of-function and loss-of-function
mutations in our pER38 pool. Mutations caused by simple
insertion of T-DNA and Tos17 are also valuable resources
and could be used directly to amplify the flanking
78 Plant Mol Biol (2009) 69:69–80
123
sequences and identify functional genes. Large numbers of
loss-of-function mutations exist naturally, whereas activa-
tion tagging mutants can only be produced in the
laboratory. Therefore, activation tagging mutations are a
valuable complementary tool to classical loss-of-function
mutations. Genes within 7 kb to CaMV 35S enhancers can
be first selected from the flanking sequence database of
dominant mutants and then subjected to expression analy-
sis, providing the opportunity to link the mutant phenotype
with gene expression. A similar approach has been adopted
in Arabidopsis (Ichikawa et al. 2003).
Acknowledgements This work was supported in part by the National
High-Tech Research and Development Project, China Rice Functional
Genomics (project number: 2001AA225051, 2006AA10A101) and an
award to excellent researchers of the Chinese Academy of Agricultural
Science. Yupeng Guo is a visiting Ph.D. student from Lanzhou
University.
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