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: tiegang@caas.net.cn

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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