CELL BIOLOGY AND MORPHOGENESIS

Promoters of two anther-specific genes confer organ-specificgene expression in a stage-specific manner in transgenic systems

Vikrant Gupta Æ Reema Khurana Æ Akhilesh K. Tyagi

Received: 17 May 2007 / Revised: 4 July 2007 / Accepted: 8 July 2007 / Published online: 28 July 2007

� Springer-Verlag 2007

Abstract Differential screening of a stage-specific cDNA

library of Indica rice has been used to identify two genes

expressed in pre-pollination stage panicles, namely OSIPA

and OSIPK coding for proteins similar to expansins/pollen

allergens and calcium-dependent protein kinases (CDPK),

respectively. Northern analysis and in situ hybridizations

indicate that OSIPA expresses exclusively in pollen while

OSIPK expresses in pollen as well as anther wall. Pro-

moters of these two anther-specific genes show the pres-

ence of various cis-acting elements (GTGA and AGAAA)

known to confer anther/pollen-specific gene expression.

Organ/tissue-specific activity and strength of their regula-

tory regions have been determined in transgenic systems,

i.e., tobacco and Arabidopsis. A unique temporal activity

of these two promoters was observed during various

developmental stages of anther/pollen. Promoter of OSIPA

is active during the late stages of pollen development and

remains active till the anthesis, whereas, OSIPK promoter

is active to a low level in developing anther till the pollen

matures. OSIPK promoter activity diminishes before

anthesis. Both promoters show a potential to target

expression of the gene of interest in developmental stage-

specific manner and can help engineer pollen-specific traits

like male-sterility in plants.

Keywords Calcium-dependent protein kinase �Expansin � Indica rice � Pollen allergen � Promoter �Transgenics

Abbreviations

CDPK Calcium-dependent protein kinase

GUS b-glucuronidase gene

OSIPA Oryza sativa Indica pollen allergen/expansin

gene

OSIPK Oryza sativa Indica calcium-dependent protein

kinase gene

PF Post-fertilization

PP Pre-pollination

Introduction

In flowering plants, life cycle alternates between the dip-

loid sporophytic and haploid gametophytic generations.

Stamen in angiosperms is associated with male reproduc-

tive processes. Microsporogenesis occurs in anther result-

ing in the production of pollen. Anther contains diploid cell

(microspore mother cell), which undergoes meiosis to

produce microspores in tetrads. These microspores separate

and their nuclei undergo an asymmetric mitosis resulting in

bicellular pollen. The tapetal cells of anther wall produce

proteins, lipids and flavonoids, which are deposited on the

Communicated by P. Kumar.

Accessions: OSIPA cDNA, AF220610; OSIPK cDNA, AF312920;

OSIPA partial gene and upstream promoter region, AY166659;

OSIPK gene-specific and upstream sequence, AY168440.

Electronic supplementary material The online version ofthis article (doi:10.1007/s00299-007-0414-8) containssupplementary material, which is available to authorized users.

V. Gupta � R. Khurana � A. K. Tyagi (&)

Interdisciplinary Centre for Plant Genomics

and Department of Plant Molecular Biology,

University of Delhi South Campus,

New Delhi 110021, India

e-mail: akhilesh@genomeindia.org

123

Plant Cell Rep (2007) 26:1919–1931

DOI 10.1007/s00299-007-0414-8

microspores and comprise outer layer, the exine, and try-

phine layers of pollen (McCormick 1993; Raghavan 1997).

All these processes are temporally and spatially regulated

and involve complex interplay of several hundred genes.

Many genes involved in anther development have been

identified (Endo et al. 2004; Lan et al. 2004; Ma 2005;

Wang et al. 2005). By following various approaches of

expression profiling, transposon tagging and functional

analysis, some genes acting in various stages of anther

development have been investigated. Certain genes show

state-specific expression within anther and have been

associated with anther cell division and differentiation

(Nonomura et al. 2003), tapetum (Jung et al. 2005; Luo

et al. 2006; Xu et al. 2006), male meiosis (Yang et al. 2003;

Kapoor and Takatsuji 2006), pollen maturation (Park et al.

2005, 2006; Zhao et al. 2006), generative cell (Durbarry

et al. 2005) and anther dehiscence (Zhu et al. 2004). Other

gene products accumulate abundantly in pollen grains

and are involved in germination or pollen tube growth

(Golovkin and Reddy 2003; Kaothien et al. 2005). A late

pollen gene, ZmMADS2 from maize, encodes a transcrip-

tion factor, which is required for anther dehiscence and

pollen maturation (Schreiber et al. 2004).

Certain genes for calcium-dependent protein kinases

(CDPK) from maize and petunia have been shown to be

having pollen-specific activity (Estruch et al. 1994; Yoon

et al. 2006). Pollen allergen-like protein encoding genes

have been characterized at the genome-wide level in rice

(Jiang et al. 2005). They include beta expansins, repre-

sented by group I pollen allergen of grasses, and may have

wall loosening activity which is characteristic to expansins

and suggested that these proteins loosen the cell walls

of the stigma and style to aid pollen tube penetration

(Cosgrove et al. 1997).

Regulation of expression of genes during the develop-

ment of anther and pollen occurs at transcriptional as well

as post-transcriptional levels. In addition to 50 upstream

regions of genes, role of 50-UTR sequences has also

emerged (Hulzink et al. 2003). Genes that express in

anther/pollen-specific manner can provide stage-specific

promoters and regulatory elements to target expression of

desirable genes in specific stages of male gametophyte

development. Various anther/pollen-specific promoters of

different plant species have been studied in transgenic

systems (van Tunen et al. 1990; Twell et al. 1991; Eyal

et al. 1995; Rogers et al. 2001; Gomez et al. 2004). A very

few anther/pollen-specific gene promoters from crops such

as rice, maize and cotton have been analyzed (Guerrero

et al. 1990; John and Petersen 1994; Tsuchiya et al. 1994;

Hamilton et al. 1998). Promoters conferring expression

specific to specialized cells of anther/pollen provide insight

into the transcriptional regulation of genes involved in

different developmental stages of male gametic cells

(Singh et al. 2003; Okada et al. 2005; Yamaji and Kyo

2006). Anther-specific promoters/regulatory elements have

biotechnological application such as induction of male-

sterility in plants. A tapetum-specific promoter has been

exploited to induce male-sterility in tobacco (Mariani et al.

1990) and cabbage (Lee et al. 2003). The promoter of an

anther-specific PsEND1 gene that expresses in epidermis,

connective, endothecium and middle layer has also been

used to produce male sterile plants in Solanaceae and

Brassicaceae (Roque et al. 2007). Recently created data-

bases of eukaryotic promoters (EPD) and plant promoter

sequences (PlantProm), as well as computational approa-

ches, aid the promoter research and analysis significantly

(Praz et al. 2002; Shahmuradov et al. 2003; Rombauts et al.

2003).

In this study, we report characterization of the regula-

tory/promoter regions of two genes OSIPA and OSIPK,

from Indica rice, found to be expressing in anther-specific

manner. OSIPA codes for a protein similar to an expansin/

pollen allergen and OSIPK shows similarity to calcium-

dependent protein kinases. Functional analysis of their

promoters in transgenic systems revealed their activity

during different stages of anther/pollen development.

These genes provide two potential promoters for targeting

expression in different stages of anther/pollen

development.

Materials and methods

Plant materials

Rice plants (Oryza sativa L. ssp. Indica cultivar Pusa

Basmati-1) were grown at standard green house conditions.

Tobacco (Nicotiana tabacum var. xanthi) and Arabidopsis

thaliana ecotype Columbia were used for transformation

studies. Tobacco plants were grown in culture room con-

ditions (25 ± 1�C under 16 h/8 h light/dark cycle) and

illuminated at 50–100 lmol quanta m�2s�1. Arabidopsis

plants were grown in cabinets in a culture room maintained

at 22�C under long day (16 h light) condition and illumi-

nated at 80 lmol quanta m�2 s�1.

Screening of cDNA/genomic libraries and sequencing

A cDNA library from pre-pollination stage panicles of rice

(Oryza sativa L. ssp. Indica cv. Pusa Basmati-1) made in

Lambda ZAP ExpressTM vector (Stratagene Cloning Sys-

tems, USA) was used. The cDNA library (5 · 104 plaques)

was differentially screened with [a�32P] dCTP labeled

cDNAs prepared from mRNAs of pre-pollinated and post-

fertilized panicles as well as from roots of 7-day-old rice

1920 Plant Cell Rep (2007) 26:1919–1931

123

seedlings according to Sambrook et al. (1989). Putative

pre-pollination stage panicle-specific clones were selected

after three rounds of differential screening. Positive clones

were used for single clone excision to obtain recombinant

pBK-CMV phagemid vector as per manufacturer’s

instruction (Stratagene, USA). OSIPA and OSIPK cDNAs

were isolated by above screening. For the isolation of

genomic clones of OSIPA and OSIPK, a genomic library

(2 · 105 plaques) of Oryza sativa L. ssp. Indica cultivar

Pusa Basmati-1, prepared in Lambda DASHTM II vector

(Stratagene Cloning Systems, USA), was screened as

described by Sambrook et al. (1989). The respective

cDNAs were radiolabeled using the Megaprime Random

Labeling Kit (Amersham Pharmacia Biotech, UK) and

a�32P dATP (BRIT, India) and used separately for library

screening. OSIPA and OSIPK cDNAs were sequenced

completely by vector-specific standard primers (T3 and T7)

and by primer walking. The gene-specific regions and

upstream regulatory regions of OSIPA and OSIPK were

sequenced by primer walking using their corresponding

genomic clones.

Southern hybridization

Fresh leaves of dark grown rice seedlings were used to

isolate total genomic DNA according to method described

by Dellaporta et al. (1983) and quantified spectrophoto-

metrically. For Southern blot hybridization, 10 lg of

genomic DNA was digested with BamHI, EcoRI, HindIII,

SacI, SalI and XbaI (Roche Molecular Biochemicals,

Germany) as per supplier’s instruction. Digested fragments

were size-separated in a 0.8% (w/v) agarose gel and

immobilized to Hybond N membrane by capillary transfer

procedure. Southern hybridization was carried out using

methods described by Sambrook et al. (1989).

Isolation of total RNA and northern hybridization

Total RNA from the plant tissue was isolated by using the

protocol described by Logemann et al. (1987) and quanti-

fied spectrophotometrically by recording OD260 using U-

2001 spectrophotometer (Hitachi, Japan). For northern

hybridization, 20 lg of total RNA was electrophoresed in

1.2% (w/v) formaldehyde-agarose gels and transferred to

Hybond N membrane (Amersham Biosciences, Piscata-

way) by capillary transfer method (Sambrook et al. 1989).

The northern hybridizations were done using OSIPA and

OSIPK cDNAs as radiolabeled probes. The cDNA frag-

ments used as probe were radiolabeled as described earlier.

Prehybridization/hybridization, washings and autoradiog-

raphy were done as described by Sambrook et al. (1989).

In situ RNA hybridization

RNA in situ hybridization was performed mainly as

described by Duck (1994). Panicles that had just emerged

from the boot leaf were excised from rice plants and a

small cut was made at the upper part to allow the fixative to

enter inside the spikelet. Rice spikelets were fixed in

paraformaldehyde fixative (100 mM phosphate buffer, pH

7.0 containing 4% paraformaldehyde and 0.25% glutaral-

dehyde). Fixed material was subjected to dehydration fol-

lowed by graded tert-butanol series to 100% tertiary

butanol. Samples were then embedded in paraffin (Sigma

Aldrich). Embedded tissue was sliced to 8 lm sections

which were mounted on to poly-L-lysine-coated slides and

incubated at 42�C for 1 h. De-waxation of the section was

done by treating the slides with xylene. Sections were then

hydrated in descending series of ethanol (90, 70, 50, and

30%). These sections were washed 4 times with DEPC-

treated MQ water. Sections were treated with proteinase K

(2 lg/ml in 100 mM Tris–HCl, pH 7.5; 50 mM EDTA;

37�C for 30 min) followed by two washes with phosphate

buffered saline (PBS). Sections were again dehydrated in

ascending series of ethanol for 2 min each. One microgram

of linearized plasmid was used to prepare sense or anti-

sense probe, using DIG-UTP RNA labeling mix and SP6 or

T7 RNA polymerase (Roche Molecular Biochemicals,

Germany) at 37�C for 2 h. After labeling reaction was

complete, DNase I treatment was given to remove the

plasmid DNA. Finally, riboprobe was precipitated, dried

and dissolved in DEPC treated water. Hybridization was

carried out as described by Duck (1994). After hybridiza-

tion, sections were washed thrice in 0.1· SSC at 60�C.

Detection of hybridization was performed using the

Digoxigenin Nucleic Acid Detection Kit (Roche Molecular

Biochemicals, Germany), according to the manufacturer’s

instructions at 30�C overnight in the dark. Sections were

dehydrated as described earlier, mounted using DPX mount

and photographed.

Cloning for promoter constructs

Regulatory regions of 1,887 and 1,547 bp upstream to the

translation initiation site of OSIPA and OSIPK, respec-

tively, were PCR amplified from cloned genomic DNA

representing these genes. Oligonucleotide primer pairs,

50-ATACCGTCGACCTCGGCATC-30 and 50-GATCCC

GGGGTCGCTTTTATTTG-30 were used for the amplifi-

cation of OSIPA promoter region while 50-ATAGTCGAC

CACCTTAGGTGTGTATTGGAGG-30 and 50-ATACCC

GGGTCTTCTTCTTCTTCACCGCC-30 were used for

amplifying OSIPK regulatory region. The underlined

region in the primers denotes SalI and SmaI sites which

Plant Cell Rep (2007) 26:1919–1931 1921

123

were added to facilitate directional cloning of the amplified

fragment. The PCR reaction for amplifying OSIPA pro-

moter region was performed under the conditions: 94�C

(5 min)/[94�C (30 s)/40�C (30 s)/72�C (1 min)] · 3

cycles/[94�C (30 s)/58�C (30 s)/72�C (1 min)] · 25

cycles/72�C (7 min)/4�C (?). The PCR reaction to

amplify the OSIPK regulatory region was performed under

the conditions: 94�C (5 min)/[94�C (30 s)/60�C (30 s)/

72�C (1 min)] · 25 cycles/72�C (7 min)/4�C (?). The

corresponding amplified products were digested with SalI

and SmaI restriction enzymes and ligated to the binary

vector pBI101 (Jefferson et al. 1987), linearized with the

same enzymes (SalI and SmaI), upstream to the promoter-

less b-glucuronidase (GUS) gene.

Plant transformation

The binary vector constructs were introduced into tobacco

and Arabidopsis plants by Agrobacterium tumefaciens

(AGL1)-mediated gene transfer. Tobacco leaf discs were

used as an explant for transformation by a method descri-

bed by Jani et al. (2004). Transformation of Arabidopsis

was done by floral dip method (Clough and Bent 1998).

Analysis of GUS activity in transgenic tobacco

and Arabidopsis plants

Histochemical and fluorometric assays of GUS gene

expression in different organs of transgenic tobacco and

Arabidopsis plants were carried out according to Jefferson

(1987). Buds of various lengths (6 ± 2, 11 ± 2, 14 ± 2,

17 ± 2, 25 ± 2, 34 ± 2 and 38 ± 2 mm/open flower), hav-

ing anthers at different developmental stages (Koltunow

et al. 1990), from transgenic as well as wild type tobacco

plants were plucked carefully and anther, gynoecium, petal

and sepal were dissected. Root, stem, leaf, flower without

anthers, anthers and silique from transgenic and wild type

Arabidopsis plants were also dissected. Fluorometric GUS

assay was done for 6 lg protein, after 15 h of incubation

with the substrate (4-methylumbelliferyl b-D-glucuronide)

in dark, using a DyNA QuantTM 200 fluorometer (Hoefer

Pharmacia Biotech Inc., California, USA).

Results

Expression pattern of anther-specific OSIPA

and OSIPK

To identify genes and their promoters for anther-specific

expression, several putative pre-pollination stage panicle-

specific cDNAs from Indica rice were isolated initially by

performing differential screening of a cDNA library. All

cDNAs were tested for organ-specificity by performing

northern analysis using total RNA from different organs, e.

g. root, stem, leaf, pre-pollination (PP) and post-fertilization

(PF) stage panicle, and rachis of mature rice plant, as well as

root and shoot of young rice seedlings. Two cDNAs showing

pre-pollination stage panicle-specific expression (Fig. 1a)

were sequenced and designated as OSIPA (1,106 bp) and

OSIPK (2,138 bp) based on the similarity (up to 90%) to

other genes encoding expansins/pollen allergen proteins and

OSIPA

Mat

ure

stem

Youn

gle

afM

atur

ele

afR

achi

s

PPpa

nicl

e

Youn

gro

otM

atur

ero

ot

PFpa

ncile

OSIPK

rRNA

Sense Antisense

OSIPA

OSIPK

po

aw

po

aw

po

aw

po

aw

A B

C D

(B)

(A)

Fig. 1 a Panicle-specific expression of OSIPA and OSIPK. RNA was

isolated from leaves and roots of young 7-day-old seedlings as well as

from mature leaves, stem and mature roots of 3-month-old field

grown plants. Rachis, pre-pollination (PP) and post-fertilization (PF)

stage panicles were taken from flowering rice plants. Twenty

microgram of RNA was blotted and probed with radiolabeled OSIPAor OSIPK cDNA. Lower panel shows ethidium bromide-stained

rRNA indicating equivalent loading and quality of RNA. b Anther/

pollen-specific expression of OSIPA and OSIPK. In situ hybridization

of transverse sections of fixed rice spikelet with OSIPA or OSIPKin vitro transcribed RNA as probe was done. The spikelets were

sampled from panicles that had just emerged from the boot leaf. Blue

colour was observed exclusively in pollen in the sections of rice

spikelet, probed with antisense OSIPA RNA. OSIPK expression was

observed in pollen as well as anther wall in the sections of rice

spikelet, when probed with antisense OSIPK RNA. No signal was

observed in sections probed with any of the two sense RNAs. awAnther wall, po pollen. Scale bars 200 lm

1922 Plant Cell Rep (2007) 26:1919–1931

123

calcium-dependent protein kinases, respectively. To further

confirm the organ/cell specificity of OSIPA and OSIPK

expression within the rice floret, in situ hybridization was

performed using DIG (Digoxigenin) labeled antisense RNA

as a probe. Transverse section of pre-pollination stage rice

spikelets were made from the panicle that had just emerged

from the boot leaf. OSIPA antisense probe produced a strong

hybridization signal exclusively and specifically in pollen,

while OSIPK antisense probe showed strong hybridization

signal in pollen and weak expression in anther wall (Fig. 1

b). The sections probed with labeled OSIPA and OSIPK

sense RNA did not show any hybridization signal above the

background levels (Fig. 1b).

Regulatory regions of OSIPA and OSIPK

Southern hybridizations of genomic DNA with cDNA,

carried out under stringent conditions, indicate that both

OSIPA as well as OSIPK are single copy genes in the rice

genome. As shown in Supplementary Fig. 1, the genomic

DNA digested with restriction enzyme with no site within

cDNA gave single band and those with one site inside

cDNA gave two bands. Analysis of anther/pollen-speci-

ficity of OSIPA and OSIPK entails functional character-

ization of their promoters. To isolate regulatory regions of

OSIPA and OSIPK, a rice genomic library prepared in

Lambda DASHTM II was screened and DNA from positive

clones was analyzed by Southern hybridization, with

cDNA as probe. A 2.4 kb (SalI–EcoRI) DNA fragment

that hybridized to OSIPA cDNA probe was sequenced and

found to contain partial gene and upstream promoter

region. On the other hand, two DNA fragments (XbaI) of

3.6 and 8.0 kb hybridized to labeled OSIPK cDNA. The

3.6 kb XbaI genomic fragment contained partial gene-

specific region as well as upstream regulatory region while

8.0 kb fragment contained remaining gene-specific 30-end

region of OSIPK. The promoter-containing fragment

(3.6 kb) of OSIPK was completely sequenced while only

gene-specific region of 8.0-kb fragment was sequenced.

All the sequences were assembled to 5,207 bp in length.

The transcription start sites of these two cDNAs were also

mapped in order to demarcate the promoter region (data

not shown). The promoter region and 50 UTR along with

ATG initiation codon for OSIPA and OSIPK are shown in

Fig. 2.

The sequenced region of OSIPA and OSIPK included

1,823 and 1,372 bp regions upstream to transcription start

sites, respectively (Fig. 2). A putative TATA box is present

32 bp upstream to the transcription start site for OSIPA

(Fig. 2a). Upstream regions were used to search for the

presence of regulatory motifs using PlantCARE and

PLACE (Lescot et al. 2002; Higo et al. 1999). Various cis-

acting regulatory elements that are known to be involved in

(A) (B)

Fig. 2 Nucleotide sequence of promoter region and 50 UTR of

OSIPA a and OSIPK b gene. ATG at the end of both sequences

represents initiation codon. A putative TATA box is boxed in a. The

extension of cDNA by primer extension is indicated in italicized

capital letters. The first base of transcript (+1) and binding sites for

primers used for amplification of promoter regions are underlined.

The GTGA and AGAAA sequences in the regulatory region, which

are similar to the conserved sequence motifs in the promoter regions

of some other anther/pollen-specific genes, are indicated in bold and

italicized lower case letters

Plant Cell Rep (2007) 26:1919–1931 1923

123

anther/pollen-specific expression were detected in the

promoter region of both the genes (Fig. 2). Two such

motifs, GTGANTG10 (GTGA) and POLLEN1LeLAT52

(AGAAA), are present in multiple in each promoter.

Activity of OSIPA and OSIPK promoters in transgenic

plants

The 1,887 bp regulatory region of OSIPA, from �1,823 to

+64 bp, and 1,547 bp promoter region of OSIPK, from

�1,328 to +219 bp, were cloned in pBI101 to create

P-OSIPA-pBI101 and P-OSIPK-pBI101, recombinant plant

transformation binary vectors (Fig. 3a, b). P-OSIPA-pBI101

and P-OSIPK-pBI101 constructs would drive b-glucuroni-

dase (GUS) gene expression under the control of OSIPA and

OSIPK promoters, respectively, in transformed plants. The

promoter-GUS constructs, P-OSIPA-pBI101 and P-OSIPK-

pBI101, were mobilized into A. tumefaciens strain AGL1,

separately and used to perform genetic transformation of

tobacco and Arabidopsis plants via leaf disc and floral dip

methods, respectively. Out of several transgenic tobacco

plants obtained, lines PA8-1, PA10-1, PA18-1 and PA44-1

of P-OSIPA-pBI101, and lines PK2-1, PK12-1, PK19-1,

PK22-1 and PK24-1 of P-OSIPK-pBI101 transformants

were analyzed for specific promoter activity by fluorometric

Wild type plant Transgenic plants harboring

Petal

Sepal

Gynoecium

DissectedAnther

Pollen

pBI101 P-OSIPK-P-OSIPA- pBI101

O

A

B

C

D

E

F

G

H

I

J

K

L

M

N

poaw

aw

aw

po

po

po po

gusAnptII

caS

Io c

EI

RtsP

I

RB LBpnos

GT

A

laS

I am

SI

tsP

Ini

HI II

d

n iH

IId c a

SI I

niH

IId

P-OSIPA

1887 bp

P-OSIPA-pBI101

ret

ocE

IR o c

EI

R

gusAnptII

caS

Ioc

EI

RtsP

I

RB LBpnos

GT

A

l aS

I am

SI

t sP

Ini

HIII

dP-OSIPK

1547 bp

P-OSIPK-pBI101

ret

(A)

(B)

(C)

Fig. 3 Schematic diagrams of

P-OSIPA-pBI101 a and P-OSIPK-pBI101 b constructs

used for analysis of promoter

(regulatory region) from OSIPAand OSIPK genes, respectively.

The upstream regulatory regions

were PCR amplified using

respective primer pairs

containing SalI and SmaI

restriction sites in forward and

reverse primers, respectively, to

facilitate directional cloning.

The amplified products were

cloned separately in binary

vector pBI101 at the same sites

(SalI and SmaI). RB and LB

indicate right and left borders of

T-DNA, respectively. c Shows

specificity of OSIPA and OSIPKpromoter driven GUS activity in

different floral organs of wild

type and transgenic tobacco

plants. OSIPA promoter showed

pollen-specific while OSIPKpromoter showed anther-

specific activity in transgenic

tobacco plants. No GUS

expression was observed in any

organ of wild type plant. awAnther wall, po pollen. Scalebars in E, J and O 200 lm

1924 Plant Cell Rep (2007) 26:1919–1931

123

analysis. Several transgenic lines from tobacco as well as

Arabidopsis for P-OSIPA-pBI101 and P-OSIPK-pBI101

constructs were also analyzed for GUS expression by his-

tochemical assay.

Histochemical analysis

All the P-OSIPA-pBI101 transgenic tobacco plants ana-

lyzed showed GUS activity exclusively in pollen (line

PA10-1, Fig. 3). Expression was not seen in petal, sepal,

gynoecium and anther wall. All the P-OSIPK-pBI101

transgenic tobacco plants showed GUS activity in anthers

(line PK12-1, Fig. 3), the whole anther including the

developing pollen stained blue in histochemical assay.

Within the flower, no expression was seen in petal, sepal,

and gynoecium (Fig. 3). GUS activity was not visible in

vegetative organs of transgenic plants (data not shown).

The untransformed wild type plant did not show GUS

activity in any of the organs tested (Fig. 3). Furthermore, a

detailed analysis of temporal GUS expression during dif-

ferent developmental stages of pollen was done. Activity

was evaluated in pollen grains of developing buds of dif-

ferent sizes, i.e., 6 ± 2, 11 ± 2, 14 ± 2, 17 ± 2, 25 ± 2,

34 ± 2 and 38 ± 2 mm to open flower. These buds/flowers

and their anthers/pollen are at different developmental

states representing stage �4 to 1, stage 2, stage 3, stage 4,

stage 6 to 7, stage 8 and stage 12, respectively, as defined

by Koltunow et al. (1990). The expression of GUS in P-

OSIPA-pBI101 harboring tobacco plants became visible in

pollen of the anthers of 14 ± 2 to 17 ± 2 mm bud (stage 3–

stage 4) and continued in pollen even after the dehiscence

of the anther (38 ± 2 mm bud to open flower, i.e., stage

12). The expression of GUS in pollen increases as the

flower matures and maximum expression is seen in the

pollen of dehisced anther (Fig. 4). In the same way, the

activity was monitored in pollen from anthers at different

developmental stages of transgenic tobacco plants harbor-

ing P-OSIPK-pBI101. In all P-OSIPK-pBI101 transgenic

tobacco plants analyzed, the expression became evident in

the anthers of 11 ± 2 to 14 ± 2 mm bud (stage 2–stage 3)

and continues till 25 ± 2 to 34 ± 2 mm flower/bud (stage

6–stage 8). The GUS activity was seen till the pollen

matures and no expression was observed in pollen col-

lected after the dehiscence of the anther (Fig. 4). Lack of

expression of GUS in the pollen of dissected anthers of

various stages from a wild type plant is also shown in

Fig. 4. Similar organ-specific histochemical expression

patterns of GUS for both the promoter constructs were also

observed in all the lines (T1 plants) of transgenic Arabid-

opsis plants (Fig. 5). It may be noted that intensity of GUS-

specific blue color was low in Arabidopsis as compared to

tobacco.

Fluorometric analysis

GUS fluorometric assays were performed to determine the

strength of OSIPA and OSIPK promoters by measuring the

specific GUS activity in tobacco. T1 generation tobacco

plants containing OSIPA or OSIPK promoter constructs

were raised and maintained till flowering. The floral buds/

flowers of different stages were dissected and anther,

gynoecium, petal and sepal were separated. Protein was

extracted from the dissected organs and assayed for

enzyme (GUS) specific activity. Three T1 plants for each

transgenic line of tobacco, transformed with each promoter

construct, were used for fluorometric assays. Anthers of all

6±2 mm

11±2 mm

14±2 mm

17± 2 mm

25±2 mm

34 ±2 mm

38±2 mm/ open flower

Pollen of wild type plant

Pollen of transgenic tobacco plants

Flower/bud size

P-OSIPA- pBI101 P-OSIPK-pBI101

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

Fig. 4 Evaluation of OSIPA and OSIPK promoter driven GUS

activity in different stages of pollen development. GUS expression

was evaluated in the pollen of developing buds/flowers of different

sizes sampled from transgenic and wild type tobacco plants. OSIPApromoter was found to be active in late stages of pollen development

while OSIPK promoter was active in early stages of pollen

development. Scale bar 200 lm

Plant Cell Rep (2007) 26:1919–1931 1925

123

four lines of tobacco (PA8-1, PA10-1, PA18-1 and PA44-

1) transformed with P-OSIPA-pBI101 showed increasing

trend of GUS activity and very high specific activity was

seen in anthers with mature pollen (Fig. 6). The expression

of GUS became evident from anthers at 14 ± 2 to

17 ± 2 mm bud length (stage 3–stage 4) and maximum

expression observed in dehisced anthers, which agreed

with the qualitative histochemical analysis (Fig. 6).

Transgenic (T1) tobacco plants of all lines harboring

OSIPK promoter construct (P-OSIPK-pBI101) showed

specific GUS activity in developing anthers until pollen

matures (up to 34 ± 2 mm bud i.e., stage 8). At first, the

activity increases and shows a decline subsequently.

During the late stages of development, the promoter

becomes inactive. The activity at any stage was low (>10

folds) when compared to plants transformed with P-OSI-

PA-pBI101 indicating that this promoter is not as strong as

OSIPA promoter in heterologous dicot system (Fig. 7).

Other floral organs such as gynoecium, petal and sepal of

transgenic tobacco plants containing P-OSIPA-pBI101 and

P-OSIPK-pBI101 constructs, did not show GUS activity

above the levels detected in same tissues of control plants

(Supplementary Figs. 2 and 3). Activity levels in vegetative

tissues (root and leaves) of transgenic tobacco were also

measured and were not significantly above the values

obtained from the same tissues of wild type plants (Sup-

plementary Figs. 2 and 3).

Discussion

Development of reproductive organs in grasses is an

important and challenging area of research (Bommert et al.

2005). Genes expressing in stage-specific manner and their

promoters are primary targets for such analysis. In an effort

to gain insight into gene activity during the development of

anthers in monocots, activity of the promoters of two

anther/pollen-specific genes identified from Indica rice has

been assessed in transgenic systems.

OSIPA and OSIPK express in anther/pollen of rice

Anther- and pollen-specific expression of OSIPA and

OSIPK, respectively, in rice is evident from northern as

well as in situ hybridizations. Group I pollen allergens

comprise major allergens from grasses that are structurally

related to expansins of plant cell wall (Cosgrove et al.

1997). OSIPA shows similarity to beta expansins or group I

pollen allergens. Beta-expansins (ZEA M 1 isoforms) from

maize pollen induced extension of a variety of grass cell

walls (Li et al. 2003). Calcium-dependent protein kinases

from plants have been found to participate in several plant

processes (Ludwig et al. 2004). Estruch et al. (1994) were

first to clone a calcium-dependent protein kinase (CDPK)

gene from maize specifically expressing in mature pollen.

Anther-specific OSIPK, isolated in this report, showed

significant similarity to several CDPKs at the protein level.

Thus, single copy genes OSIPA and OSIPK isolated in this

study are members of multi-gene families (Asano et al.

2005; Jiang et al. 2005). The transcription of OSIPK starts

at A, 243 bp upstream of ATG, but no perfect TATA box

was found to be located upstream of the transcription start

site of OSIPK. This gene may belong to the category of

many animal and plant genes known to be TATA-less

(Padbury et al. 1995; Carrari et al. 2001).

A

B

C

D

E

G

H

F

I

J

K

L

M

N

O

P

Q

R

Gynoecium

Silique

Anther

Stem

Leaf

Root

Wild type plant

Transgenic plants harboring

P-OSIPA-pBI101 P-OSIPK-pBI101

po

awpo

aw

po

aw

Fig. 5 Evaluation of OSIPA and OSIPK promoter activity in

transgenic Arabidopsis plants. The activity of GUS in different

organs of wild type and transgenic Arabidopsis plants transformed

with P-OSIPA-pBI101 or P-OSIPK-pBI101 is shown. OSIPA pro-

moter showed pollen-specific while OSIPK regulatory region showed

anther-specific activity. Control plant did not show histochemical

GUS staining in any of the organ tested. aw Anther wall, po pollen

1926 Plant Cell Rep (2007) 26:1919–1931

123

OSIPA and OSIPK promoters show known

pollen-specific regulatory elements

Search of sequences upstream to the transcription start site

(+1) of OSIPA and OSIPK for conserved cis-acting regu-

latory elements showed prevalence of two pollen-specific

elements, GTGA and AGAAA. In the pollen-specific

LAT56 and LAT59 promoters, a common cis-acting regu-

latory element (LAT 56/59 box) was identified at the

similar relative positions (�103 to �94 bp in LAT56 and

�114 to �105 in LAT59) having a core sequence of GTGA

(Twell et al. 1991). The late pollen gene G10 (LAT56

homologue) of tobacco possesses five GTGA motifs. Out

of these five, the one present at �96 bp position is at the

same relative position as the GTGA present in LAT56 and

LAT59 promoters (Rogers et al. 2001). Any mutation in

this box reduces pollen-specific promoter activity. In

OSIPA promoter, the GTGA core sequences are located at

-10.00

10.00

30.00

50.00

70.00

90.00

110.00

130.00

150.00

170.00

190.00

210.00

230.00

250.00

270.00

290.00

310.00

330.00

350.00

)re

htn

A(d

ub

mm

2±

6

)re

htn

A(d

ub

mm

2±

11

)re

htn

A(d

ub

mm

2±

41

)re

htn

A(d

ub

mm

2±

71

)re

htn

A(d

ub

mm

2±

52

)re

htn

A(d

ub

mm

2±

43

)re

htn

A(d

ub

mm

2±

83

Size of floral bud/flower

)rh/

nietor

pg

m/U

M4fol

om

n(ytivitca

cificeps

SU

G

PA8-1

PA10-1

PA18-1

PA44-1

Fig. 6 GUS fluorometric assay

for evaluation of OSIPApromoter strength in four

transgenic tobacco plants. The

anthers of developing floral

buds/flowers of different sizes

were dissected and protein

extracted. Six-microgram

protein from each was used for

enzyme assay. Mean of the

specific GUS activity (in nmol

of 4 MU/mg protein/h) for each

dissected anther of three plants

of four independent transgenic

lines is shown. Standard errorbars are also shown. The value

was depicted after subtracting

any background specific GUS

activity observed in dissected

anthers of wild type tobacco

from the activity observed in

anthers of transgenic tobacco

plants

0.00

2.00

4.00

6.00

8.00

10.00

)rehtnA(

d ubm

m2±6

) rehtnA(

dubm

m2±11

)r ehtnA(

dubm

m2 ±41

)reh tnA(

du bm

m2±71

) rehtnA(

dubm

m2 ±52

)rehtnA(

dubm

m2 ±43

)rehtnA (

du bm

m2 ±83

Size of floral bud/flower

)rh/

nietor

pg

m/U

M4f

olo

mn(

yt ivitcacif ic e

psS

UG

PK2-1

PK12-1

PK19-1

PK22-1

PK24-1

Fig. 7 Quantitative

(fluorometric) GUS assay for

evaluation of OSIPK promoter

strength in five transgenic

tobacco plants. Other details are

similar to Fig. 6

Plant Cell Rep (2007) 26:1919–1931 1927

123

�661, �668, �1,317 and �1,376 bp. The pollen-specific

LAT52 promoter consists of three independent activator

domains (A, B and C). Each domain is capable of acti-

vating a minimal promoter in a pollen-specific manner.

Within the domain C, the activity was dependent on two

regulatory elements, AGAAA and TCCACCATA (Bate

and Twell 1998). OSIPA promoter has AGAAA sequence

at �424 and �1,425 bp positions and these may have an

important role in imparting the pollen-specificity to OSIPA

promoter. In OSIPK promoter, a consensus GTGA core

sequence was present at �115, �548, �738, �778, �808,

�837 and �1,220 bp positions. Another pollen-specific

regulatory element AGAAA was present at �267, �585

and �1,128 bp positions. Thus, promoters of both the

anther/pollen-specific genes reported here contain some of

the known cis-regulatory elements required for pollen-

specific expression. However, functionality of these

elements and the presence of novel elements can be

determined only by mutation and gain-of-function analysis.

Activity of OSIPA and OSIPK promoters

is anther/pollen-specific

Pollen-specific promoters fused to a reporter gene have

been used to elucidate regulatory elements (Twell et al.

1991; Twell 1992; John and Petersen 1994; Tsuchiya et al.

1994; Hamilton et al. 1998; Rogers et al. 2001). By mon-

itoring the localization of GUS reporter gene activity, the

developmental regulation of a gene can also be studied.

Transgenic Arabidopsis plant containing 50 flanking

sequences (533 bp) of a tubulin (TUA1) fused to b-glucu-

ronidase (GUS) coding region showed the localization of

GUS in post-mitotic pollen grains (Carpenter et al. 1992).

ACT1 gene is most strongly expressed in pollen. Its pro-

moter drove the reporter gene expression in pollen and

ovules of transgenic tobacco and rice, strongly (Vitale et al.

2003). Singh et al. (2003) studied the 0.8 kb promoter

sequence of a generative cell-specific gene, LGC1 and

found it to be sufficient to regulate the expression of

reporter gene in a cell-specific manner. Transgenic tobacco

plants carrying LGC1-DT/A (LGC1 promoter-diptheria

toxin A chain) construct showed sterile and aborted pollen

(Singh et al. 2003). The specificity and strength of OSIPA

and OSIPK promoters were evaluated in heterologous plant

systems, i.e., tobacco and Arabidopsis, by raising

transgenics.

To characterize the activity of OSIPA promoter, the

region between �1,823 to + 64 bp was cloned upstream to

GUS, and the resulting construct was transformed into

tobacco and Arabidopsis. Transgenic plants of tobacco and

Arabidopsis showed pollen-specific GUS activity as

revealed by both histochemical and fluorometric assays.

The development of anther/pollen is tightly regulated with

respect to time and correlates with floral bud size (Koltu-

now et al. 1990). Transgenic tobacco lines analyzed in

detail were found to show GUS expression starting from

stage 3/4 of pollen development in 14 ± 2 to 17 ± 2 mm

floral bud and it continued even after the anthesis. As the

flower matures, the expression keeps on increasing and

maximum expression is observed in pollen after the

dehiscence of flower. During stage 3 to stage 4 of floral

bud, microspores have already separated, tapetum shrunken

and the pollen grains begin to form (Koltunow et al. 1990).

The activity of OSIPA promoter indicates a role of this

gene in late stages of pollen development. OSIPA gene, as

indicated by its promoter activity, is a late expressing gene,

and its product may have significant role in the extension or

growth of pollen wall or pollen tube. A pollen-specific gene

LLP-PG from lily, related to pollen allergens, is expressed

in mature pollen (Chiang et al. 2006). Other late genes

analyzed from anther/pollen include tomato LAT56 and

LAT59 encoding pectate lyases involved in pollen tube

growth (Wing et al. 1990), ZM13 involved in the pollen

tube growth (Hamilton et al. 1989; Hanson et al. 1989) and

a pollen-specific gene PEX1 of maize containing extensin-

like domain and a putative globular domain at the N-ter-

minus (Rubinstein et al. 1995). The tobacco G10 gene

(homolog of tomato LAT56) encodes pectate lyases and is

maximally expressed in mature pollen. The 1,190 bp pro-

moter region fused to GUS preferentially expressed in

developing anthers and mature pollen (Rogers et al. 2001).

Another late pollen gene, ZmMADS2 from maize, encodes

a transcription factor responsible for the dehiscence of the

anther and pollen maturation. It is expressed in endothe-

cium and connective tissue of anther, a day before dehis-

cence and mature pollen, just after the dehiscence

(Schreiber et al. 2004). We speculate that OSIPA gene

product accumulates in the maturing pollen and has simi-

larity to expansins, which might have specialized role

during pollen germination and pollen tube growth.

The promoter-GUS construct of OSIPK (containing a

region from �1,328 to +219 bp) showed anther-specific

expression in transgenic tobacco and Arabidopsis plants.

The extensive study of OSIPK promoter-GUS construct in

transgenic tobacco lines revealed that the expression of

GUS reporter gene started in the anthers (including pollen)

of 11 ± 2 to 14 ± 2 mm floral bud (stage 2–stage 3) and

continued till the maturity of the flower. At the stage 2 of

flower development, microspores in the developing anthers

separate (Koltunow et al. 1990). As the flower opened and

anthers dehisced, no activity was seen in anthers (pollen as

well as other anther tissues). The anther-specific activity of

the OSIPK promoter indicates a probable role of this cal-

cium-dependent protein kinase in early development of

anther/pollen. Several dicot and monocot genes have been

1928 Plant Cell Rep (2007) 26:1919–1931

123

predicted to be involved in early development and function

of anther/pollen grains. The APG gene from Arabidopsis

expresses in the tapetum, between microspore release and

tapetal dissolution stages, in anther wall and stomium

(Roberts et al. 1993). Similarly, the BP4 gene family in

Brassica is activated in anther during early development of

microspores (Albani et al. 1990). BP10 of Brassica napus

comprises a small pollen-specific gene family that is

maximally expressed in early binucleate microspores. A

396 bp fragment of BP10 promoter is sufficient to direct a

strong and correct temporal and spatial expression of GUS

in the pollen of transgenic tobacco plants (Albani et al.

1992). A MYB-related gene from tobacco expresses in

tapetum, stomium of anther wall and pollen grains (Yang

et al. 2001). Estruch et al. (1994) cloned a calcium-

dependent protein kinase (CDPK) from mature maize

pollen, which is specific to a particular cell type. PiCDPK1

and PiCDPK2 from petunia have distinct functions in

regulating pollen tube polarity and growth (Yoon et al.

2006). BRA R1 codes for a Ca+2 binding protein specifi-

cally expressed in anthers of Brassica rapa. The promoter–

reporter gene fusion revealed the male gametophyte-spe-

cific promoter activity in tobacco, Arabidopsis and Bras-

sica napus (Okada et al. 2000). Since CDPKs have an

important role in various physiological and developmental

processes, OSIPK is a candidate gene to study such pro-

cesses responsible for the anther/pollen development.

In conclusion, we have reported the anther/pollen-spe-

cific promoter activities of two Indica rice genes encoding

an expansin/allergen and a calcium-dependent protein

kinase. Both these genes show anther/pollen-specific

expression in rice and further work would unravel their

specific function. The upstream regulatory sequences have

been found to possess some basic features controlling gene

expression in male reproductive tissue. The redundancy in

distribution of anther/pollen-specific elements in these

promoters entails further investigation to specify the role of

individual element. This study identifies two promoters that

confer anther-specific gene expression in a developmental

stage-specific manner. These rice gene promoters are

important tools for controlling gene expression in anthers

of different plants and for biotechnological application.

Genes such as OSIPA and OSIPK, expressing specifically

in tissues of sexual organs (anthers or ovules) are of par-

ticular interest in breeding programs for developing hybrid

seeds and parthenocarpic seedless fruits. Their promoters

in combination with cytotoxic genes can be exploited to

engineer traits like male sterility in plants and help in cell/

tissue-specific ablation for investigating functions at cel-

lular level.

Acknowledgments We would like to thank Dr Promila Gupta for

her help during in situ localization experiments. We are also grateful

to Dr Ambika Gupta for helping and assisting in fluorometric assays.

This work was supported by Department of Biotechnology and Uni-

versity Grants Commission, Government of India.

References

Albani D, Robert LS, Donaldson PA, Altosaar I, Arnison PG,

Fabijanski SF (1990) Characterization of a pollen-specific gene

family from Brassica napus which is activated during early

microspore development. Plant Mol Biol 15:605–622

Albani D, Sardana R, Robert LS, Altosaar I, Arnison PG, Fabijanski

SF (1992) A Brassica napus gene family which shows sequence

similarity to ascorbate oxidase is expressed in developing pollen.

Molecular characterization and analysis of promoter activity in

transgenic tobacco plants. Plant J 2:331–342

Asano T, Tanaka N, Yang G, Hayashi N, Komatsu S (2005) Genome-

wide identification of the rice calcium-dependent protein kinase

and its closely related kinase gene families: comprehensive

analysis of the CDPKs gene family in rice. Plant Cell Physiol

46:356–366

Bate N, Twell D (1998) Functional architecture of a late pollen

promoter: pollen-specific transcription is developmentally regu-

lated by multiple stage-specific and co-dependent activator

elements. Plant Mol Biol 37:859–869

Bommert P, Satoh-Nagasawa N, Jackson D, Hirano H-Y (2005)

Genetics and evolution of inflorescence and flower development

in grasses. Plant Cell Physiol 46:69–78

Carpenter JL, Ploense SE, Snustad DP, Silflow CD (1992) Preferen-

tial expression of an a-tubulin gene of Arabidopsis in pollen.

Plant Cell 4:557–571

Carrari F, Frankel N, Lijavetzky D, Benech-Arnold R, Sanchez R,

Iusem ND (2001) The TATA-less promoter of VP1, a plant gene

controlling seed germination. DNA Seq 12:107–114

Chiang JY, Balic N, Hsu SW, Yang CY, Ko CW, Hsu YF, Swoboda I,

Wang CS (2006) A pollen-specific polygalacturonase from lily is

related to major grass pollen allergens. Plant Physiol Biochem

44:743–751

Clough SJ, Bent AF (1998) Floral dip: a simplified method for

Agrobacterium-mediated transformation of Arabidopsis thali-ana. Plant J 16:735–743

Cosgrove DJ, Bedinger P, Durachko DM (1997) Group I allergens of

grass pollen as cell wall-loosening agents. Proc Natl Acad Sci

USA 94:6559–6564

Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA miniprepa-

ration: version II. Plant Mol Biol Rep 1:19–21

Duck NB (1994) RNA in situ hybridization in plants. In: Gelvin SB,

Schilperoort RA (eds) Plant molecular biology manual, vol G1.

Kluwer, Belgium, pp 1–13

Durbarry A, Vizir I, Twell D (2005) Male germ line development

in Arabidopsis. duo pollen mutants reveal gametophytic

regulators of generative cell cycle progression. Plant Physiol

137:297–307

Endo M, Tsuchiya T, Saito H, Matsubara H, Hakozaki H, Masuko H,

Kamada M, Higashitani A, Takahashi H, Fukuda H, Demura T,

Watanabe M (2004) Identification and molecular characteriza-

tion of novel anther-specific genes in Oryza sativa L. by using

cDNA microarray. Genes Genet Syst 79:213–226

Estruch JJ, Kadwell S, Merlin E, Crossland L (1994) Cloning and

characterization of a maize pollen-specific calcium-dependent

calmodulin-independent protein kinase. Proc Natl Acad Sci USA

91:8837–8841

Eyal Y, Curie C, McCormick S (1995) Pollen specificity elements

reside in 30 bp of the proximal promoters of two pollen-

expressed genes. Plant Cell 7:373–384

Plant Cell Rep (2007) 26:1919–1931 1929

123

Golovkin M, Reddy ASN (2003) A calmodulin-binding protein from

Arabidopsis has an essential role in pollen germination. Proc

Natl Acad Sci USA 100:10558–10563

Gomez MD, Beltran J-P, Canas LA (2004) The pea END1 promoter

drives anther-specific gene expression in different plant species.

Planta 219:967–981

Guerrero FD, Crossland L, Smutzer GS, Hamilton DA, Mascarenhas

JP (1990) Promoter sequences from a maize pollen-specific gene

direct tissue-specific transcription in tobacco. Mol Gen Genet

224:161–168

Hamilton DA, Bashe DM, Stinson JR, Mascarenhas JP (1989)

Characterization of a pollen-specific genomic clone from maize.

Sex Plant Reprod 2:208–212

Hamilton DA, Schwarz YH, Mascarenhas JP (1998) A monocot

pollen-specific promoter contains separable pollen-specific and

quantitative elements. Plant Mol Biol 38:663–669

Hanson DD, Hamilton DA, Travis JL, Bashe DM, Mascarenhas JP

(1989) Characterization of a pollen-specific cDNA clone from

Zea mays and its expression. Plant Cell 1:173–179

Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting

regulatory DNA elements (PLACE) database: 1999. Nucleic

Acids Res 27:297–300

Hulzink RJM, Weerdesteyn H, Croes AF, Gerats T, van Herpen

MMA, van Helden J (2003) In silico identification of putative

regulatory sequence elements in the 50-untranslated region of

genes that are expressed during male gametogenesis. Plant

Physiol 132:75–83

Jani D, Singh NK, Bhattacharya S, Meena LS, Singh Y, Upadhyay

SN, Sharma AK, Tyagi AK (2004) Studies on the immunogenic

potential of plant-expressed cholera toxin B subunit. Plant Cell

Rep 22:471–477

Jefferson RA (1987) Assaying chimeric genes in plants: the GUS

gene fusion system. Plant Mol Biol Rep 5:387–405

Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusion: b-

glucuronidase as a sensitive and versatile gene fusion marker in

higher plants. EMBO J 6:3901–3907

Jiang S-Y, Jasmin PXH, Ting YY, Ramachandran S (2005) Genome-

wide identification and molecular characterization of Ole-e-I,

Allerg-1 and Allerg-2 domain-containing pollen-allergen-like

genes in Oryza sativa. DNA Res 12:167–179

John ME, Petersen MW (1994) Cotton (Gossypium hirsutum L.)

pollen-specific polygalacturonase mRNA: tissue and temporal

specificity of its promoter in transgenic tobacco. Plant Mol Biol

26:1989–1993

Jung K-H, Han M-J, Lee Y-S, Kim Y-W, Hwang I, Kim M-J, Kim Y-

K, Nahm BH, An G (2005) Rice Undeveloped Tapetum 1 is a

major regulator of early tapetum development. Plant Cell

17:2705–2722

Kaothien P, Ok SH, Shuai B, Wengier D, Cotter R, Kelley D,

Kiriakopolos S, Muschietti J, McCormick S (2005) Kinase

partner protein interacts with the LePRK1 and LePRK2 receptor

kinases and plays a role in polarized pollen tube growth. Plant J

42:492–503

Kapoor S, Takatsuji H (2006) Silencing of an anther-specific zinc-

finger gene, MEZ1, causes aberrant meiosis and pollen abortion

in petunia. Plant Mol Biol 61:415–430

Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB

(1990) Different temporal and spatial gene expression patterns

occur during anther development. Plant Cell 2:1201–1224

Lan LF, Chen W, Lai Y, Suo J, Kong Z, Li C, Lu Y, Zhang Y, Zhao

X, Zhang X, Han B, Cheng J, Xue YB (2004) Monitoring of

gene expression profiles and isolation of candidate genes

involved in pollination and fertilization in rice (Oryza sativaL.) with a 10 K cDNA microarray. Plant Mol Biol 54:471–487

Lee Y-H, Chung K-H, Kim H-U, Jin Y-M, Kim H-I, Park B-S (2003)

Induction of male sterile cabbage using a tapetum-specific

promoter from Brassica campestris L. ssp. pekinensis. Plant Cell

Rep 22:268–273

Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y,

Rouze P, Rombauts S (2002) PlantCARE, a database of plant

cis-acting regulatory elements and a portal to tools for in silico

analysis of promoter sequences. Nucleic Acids Res 30:325–327

Li L-C, Bedinger PA, Volk C, Jones AD, Cosgrove DJ (2003)

Purification and characterization of four b-expansins (Zea m 1

isoforms) from maize pollen. Plant Physiol 132:2073–2085

Logemann J, Schell J, Willmitzer L (1987) Improved method for the

isolation of RNA from plant tissues. Anal Biochem 163:16–20

Ludwig AA, Romeis T, Jones JDG (2004) CDPK-mediated signaling

pathways: specificity and cross-talk. J Exp Bot 55:181–188

Luo H, Lee J-Y, Hu Q, Nelson-Vasilchik K, Eitas TK, Lickwar C,

Kausch AP, Chandlee JM, Hodges TK (2006) RTS, a rice anther-

specific gene is required for male fertility and its promoter

sequence directs tissue-specific gene expression in different plant

species. Plant Mol Biol 62:397–408

Ma H (2005) Molecular genetic analyses of microsporogenesis and

microgametogenesis in flowering plants. Annu Rev Plant Biol

56:393–434

Mariani C, De Beuckeleer M, Truettner J, Leemans J, Goldberg RB

(1990) Induction of male sterility in plants by a chimaeric

ribonuclease gene. Nature 347:737–741

McCormick S (1993) Male gametophyte development. Plant Cell

5:1265–1275

Nonomura K-I, Miyoshi K, Eiguchi M, Suzuki T, Miyao A, Hirochika

H, Kurata N (2003) The MSP1 gene is necessary to restrict the

number of cells entering into male and female sporogenesis and

to initiate anther wall formation in rice. Plant Cell 15:1728–1739

Okada T, Sasaki Y, Ohta R, Onozuka N, Toriyama K (2000)

Expression of Bra r1 gene in transgenic tobacco and Bra r1promoter activity in pollen of various plant species. Plant Cell

Physiol 41:757–766

Okada T, Bhalla PL, Singh MB (2005) Transcriptional activity of

male gamete-specific histone gcH3 promoter in sperm cells of

Lilium longiflorum. Plant Cell Physiol 46:797–802

Padbury JF, Tseng Y-T, Waschek JA (1995) Transcription initiation

is localized to a TATA-less region in the ovine b1 adrenergic

receptor gene. Biochem Biophys Res Commun 211:254–261

Park BS, Kim JS, Kim SH, Park YD (2005) Characterization of a

pollen-preferential gene, BAN102, from Chinese cabbage. Plant

Cell Rep 24:663–670

Park J-I, Hakozaki H, Endo M, Takada Y, Ito H, Uchida M, Okabe T,

Watanabe M (2006) Molecular characterization of mature

pollen-specific genes encoding novel small cysteine-rich proteins

in rice (Oryza sativa L.). Plant Cell Rep 25:466–474

Praz V, Perier R, Bonnard C, Bucher P (2002) The eukaryotic

promoter database, EPD: new entry types and links to gene

expression data. Nucleic Acids Res 30:322–324

Raghavan V (1997) Molecular embryology of flowering plants.

Cambridge University Press, Cambridge

Roberts MR, Foster GD, Blundell RP, Robinson SW, Kumar A,

Draper J, Scott R (1993) Gametophytic and sporophytic

expression of an anther-specific Arabidopsis thaliana gene.

Plant J 3:111–120

Rogers HJ, Bate N, Combe J, Sullivan J, Sweetman J, Swan C,

Lonsdale DM, Twell D (2001) Functional analysis of cis-

regulatory elements within the promoter of the tobacco late

pollen gene g10. Plant Mol Biol 45:577–585

Rombauts S, Florquin K, Lescot M, Marchal K, Rouze P, van de Peer

Y (2003) Computational approaches to identify promoters and

cis-regulatory elements in plant genomes. Plant Physiol

132:1162–1176

Roque E, Gomez MD, Ellul P, Wallbraun M, Madueno F, Beltran J-P,

Canas LA (2007) The PsEND1 promoter: a novel tool to produce

1930 Plant Cell Rep (2007) 26:1919–1931

123

genetically engineered male-sterile plants by early anther

ablation. Plant Cell Rep 26:313–325

Rubinstein AL, Broadwater AH, Lowrey KB, Bedinger PA (1995)

Pex1, a pollen-specific gene with an extensin-like domain. Proc

Natl Acad Sci USA 92:3086–3090

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A

laboratory manual, 2nd edn. Cold Spring Harbor Laboratory

Press, Cold Spring Harbor

Schreiber DN, Bantin J, Dresselhaus T (2004) The MADS box

transcription factor ZmMADS2 is required for anther and pollen

maturation in maize and accumulates in apoptotic bodies during

anther dehiscence. Plant Physiol 134:1069–1079

Shahmuradov IA, Gammerman AJ, Hancock JM, Bramley PM,

Solovyev VV (2003) PlantProm: a database of plant promoter

sequences. Nucleic Acids Res 31:114–117

Singh M, Bhalla PL, Xu H, Singh MB (2003) Isolation and

characterization of a flowering plant male gametic cell-specific

promoter. FEBS Lett 542:47–52

Tsuchiya T, Toriyama K, Ejiri S, Hinata K (1994) Molecular

characterization of rice genes specifically expressed in the anther

tapetum. Plant Mol Biol 26:1737–1746

Twell D (1992) Use of a nuclear-targeted b-glucuronidase fusion

protein to demonstrate vegetative cell-specific gene expression

in developing pollen. Plant J 2:887–892

Twell D, Yamaguchi J, Wing RA, Ushiba J, McCormick S (1991)

Promoter analysis of genes that are coordinately expressed

during pollen development reveals pollen-specific enhancer

sequences and shared regulatory elements. Genes Dev 5:496–

507

van Tunen AJ, Mur LA, Brouns GS, Rienstra J-D, Koes RE, Mol

JNM (1990) Pollen- and anther-specific chi promoters from

petunia: tandem promoter regulation of the chiA gene. Plant Cell

2:393–401

Vitale A, Wu RJ, Cheng Z, Meagher RB (2003) Multiple conserved 50

elements are required for high-level pollen expression of the

Arabidopsis reproductive actin ACT1. Plant Mol Biol 52:1135–

1151

Wang Z, Liang Y, Li C, Xu Y, Lan L, Zhao D, Chen C, Xu Z, Xue Y,

Chong K (2005) Microarray analysis of gene expression

involved in anther development in rice (Oryza sativa L.). Plant

Mol Biol 58:721–737

Wing RA, Yamaguchi J, Larabell SK, Ursin VM, McCormick S

(1990) Molecular and genetic characterization of two pollen-

expressed genes that have sequence similarity to pectate lyases

of the plant pathogen Erwinia. Plant Mol Biol 14:17–28

Xu SX, Liu GS, Chen RD (2006) Characterization of an anther- and

tapetum-specific gene and its highly specific promoter isolated

from tomato. Plant Cell Rep 25:231–240

Yamaji N, Kyo M (2006) Two promoters conferring active gene

expression in vegetative nuclei of tobacco immature pollen

undergoing embryonic dedifferentiation. Plant Cell Rep 25:749–

757

Yang S, Sweetman JP, Amirsadeghi S, Barghchi M, Huttly AK,

Chung W-I, Twell D (2001) Novel anther-specific myb genes

from tobacco as putative regulators of phenylalanine ammonia-

lyase expression. Plant Physiol 126:1738–1753

Yang X, Makaroff CA, Ma H (2003) The Arabidopsis MALEMEIOCYTE DEATH1 gene encodes a PHD-finger protein that is

required for male meiosis. Plant Cell 15:1281–1295

Yoon GM, Dowd PE, Gilroy S, McCubbin AG (2006) Calcium-

dependent protein kinase isoforms in Petunia have distinct

functions in pollen tube growth, including regulating polarity.

Plant Cell 18:867–878

Zhao Y, Zhao Q, Ao G, Yu J (2006) Characterization and functional

analysis of a pollen-specific gene st901 in Solanum tuberosum.

Planta 224:405–412

Zhu Q-H, Ramm K, Shivakkumar R, Dennis ES, Upadhyaya NM

(2004) The ANTHER INDEHISCENCE1 gene encoding a single

MYB domain protein is involved in anther development in rice.

Plant Physiol 135:1514–1525

Plant Cell Rep (2007) 26:1919–1931 1931

123