ORIGINAL PAPER

Characterization of a pollen-preferential gene OSIAGPfrom rice (Oryza sativa L. subspecies indica) codingfor an arabinogalactan protein homologue, and analysisof its promoter activity during pollen developmentand pollen tube growth

Saurabh Anand Æ Akhilesh K. Tyagi

Received: 4 June 2009 / Accepted: 19 August 2009 / Published online: 22 September 2009

� Springer Science+Business Media B.V. 2009

Abstract During differential screening of inflores-

cence-specific cDNA libraries from Oryza sativa

indica, an arabinogalactan protein (OSIAGP) cDNA

(586 bp) expressing preferentially in the inflores-

cence has been isolated. It encodes an arabinogalac-

tan protein of 59 amino acids (6.4 kDa) with a

transmembrane domain and a secretory domain at

the N terminus. The protein shows homology with

AGP23 from Arabidopsis, and its homologue in

japonica rice is located on chromosome 6. OSIAGP

transcripts also accumulate in shoots and roots of rice

seedling grown in the dark, but light represses

expression of the gene. Analysis of a genomic clone

of OSIAGP revealed that its promoter contains

several pollen-specificity and light-regulatory ele-

ments. The promoter confers pollen-preferential

activity on gus, starting from the release of microsp-

ores to anther dehiscence in transgenic tobacco, and

is also active during pollen tube growth. Analysis

of pollen preferential activity of the promoter in

the transgenic rice system revealed that even the

*300 bp fragment has activity in pollen and the

anther wall and further deletion down to *100 bp

completely abolishes this activity, which is consistent

with in-silico analysis of the promoter. Arabinoga-

lactan proteins have been shown to be involved in the

cell elongation process. The homology of OSIAGP

with AGP23 and the fact that seedling growth in the

dark and pollen tube growth are events based on cell

elongation strengthen the possibility of OSIAGP

performing a similar function.

Keywords Arabinogalactan protein �Pollen-preferential gene � Pollen tube �Rice � Tobacco � Transgenic

Introduction

The development of a flowering plant involves well-

coordinated and regulated expression of a large

number of genes. The molecular mechanisms of

flowering time regulation and flower development are

being explored in different plant systems. In past

few years, the genome-wide analysis of Arabidopsis

(a dicot) has helped in gathering information about

genes involved in the regulation of flowering.

Extrapolation of the same in a cereal like rice has

been facilitated by the availability of its genome

sequence (Izawa et al. 2003). The flower of rice bears

Electronic supplementary material The online version ofthis article (doi:10.1007/s11248-009-9319-3) containssupplementary material, which is available to authorized users.

S. Anand � 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

Present Address:A. K. Tyagi

National Institute of Plant Genome Research, Aruna Asaf

Ali Marg, New Delhi 110067, India

123

Transgenic Res (2010) 19:385–397

DOI 10.1007/s11248-009-9319-3

both male and female reproductive organs. The genes

expressing in anthers include tapetum and pollen-

specific genes (Ma 2005). The genes expressing in

pollen have been categorized in early and late genes

(Raghavan 1997). Early expressing genes play a role

in cell division during pollen development, whereas

the late expressing genes are involved in the forma-

tion of cytoskeletal elements during pollen germina-

tion and pollen tube growth. The complement of the

pollen transcriptome and the proteome of anthers

belonging to different stages of development in

Arabidopsis and rice have been analysed (Becker

et al. 2003; Honys and Twell 2003; Kerim et al. 2003;

Wang et al. 2005; Hobo et al. 2008; Suwabe et al.

2008). To a large extent, anther and pollen-specific

expression of genes has been correlated with their

promoter activity (Gupta et al. 2007 and references

cited therein).

Arabinogalactan protein (AGP) is an umbrella

term applied to a diverse class of cell-surface

glycoproteins in plants, which are equivalent to

proteoglycans of animals (Seifert and Roberts

2007). The tissue-specific and cell-specific localiza-

tion of these molecules has been extensively studied

(Majewska-Sawka and Nothnagel 2000; Showalter

2001). These are a family of highly glycosylated

hydroxyproline-rich glycoproteins (HRGPs). An

AGP molecule consists of a hydroxyproline-rich core

protein rich in Hyp, Ala, Ser, Thr, and Gly, decorated

with arabinose and galactose-rich polysaccharide

units, and it reacts with a synthetic chemical reagent

called Yariv reagent (Nothnagel 1997). However,

these generalizations cannot be applied to all AGPs,

because there are a few which are hydroxyproline

poor, lightly glycosylated, and largely unreactive

with Yariv reagent (Showalter 2001). Biochemical

and immunolocalization studies using antibodies

against AGP epitopes revealed that these molecules

are localized in the plasma membrane and the cell

wall, and are also secreted outside the cell in a

soluble form. AGPs might act as modulators or

coreceptors of apoplastic morphogens and their

amphiphilic nature makes them a prime candidate

for mediator between cell wall, plasma membrane,

and cytoplasm (Seifert and Roberts 2007). These

molecules have been shown to function in many

important biological processes including develop-

ment, signal transduction, reproduction, somatic

embryogenesis, root growth and regeneration, cell

elongation, and cell death (Rumyantseva 2006; Jones

et al. 2006; Qin et al. 2007). Despite two decades of

research on AGPs, the exact biological role of these

molecules is not known.

The use of genomic and proteomic tools has led to

detailed analysis of AGP-encoding genes in Arabid-

opsis. The abundance and regulated expression of a

particular AGP in the stigma, stylar transmitting

tissue, and pollen grains, indicates a role of these

proteins in plant reproduction (Du et al. 1996; Wu

et al. 2000; Coimbra et al. 2009). A purified tobacco

AGP, specifically expressing in stylar transmitting

tissue, has been shown to enhance pollen tube growth

in vitro and to attract pollen tubes in semi-in-vivo

systems (Cheung et al. 1995). Transgenic tobacco

plants engineered for down regulation of stylar

transmitting tissue AGP show reduced pollen tube

growth in the style and reduced seed setting com-

pared with wild-type (Cheung et al. 1995). In another

experiment, Yariv reagent was added to lily pollen

tubes and shown to inhibit their growth and produce a

balloon-like morphology at the end of the pollen

tubes (Roy et al. 1998).

We report here the molecular characterization and

promoter activity of a rice pollen-preferential gene,

OSIAGP. The deduced gene and protein sequence

show homology with an arabinogalactan protein

encoding gene AGP23 from Arabidopsis. An effort

has been made to correlate the expression and

promoter activity data with the probable function of

OSIAGP.

Materials and methods

Plant materials

Rice (Oryza sativa subsp. indica var. Pusa Basmati 1)

seeds were obtained from the Indian Agricultural

Research Institute, New Delhi, and the Indian Agri-

cultural Research Institute Substation, Karnal. Nico-

tiana tabacum var. Xanthi seeds were obtained from

the International Centre for Genetic Engineering and

Biotechnology, New Delhi.

The rice seeds were surface-sterilized using 0.1%

mercuric chloride (HgCl2) for 15 min and washed

with autoclaved water. Overnight-soaked seeds were

sprinkled on a 5 or 6 layered (each 1 cm thick) cotton

bed saturated with water. Seedlings were grown

386 Transgenic Res (2010) 19:385–397

123

either in the dark or under a 16/8 h light/dark cycle

where light was provided by fluorescent tubes

(Philips India Champion 40 W/54) at a fluence of

50–100 lmol m-2 s-1. Tobacco seeds (Nicotiana

tabacum var. Xanthi) were surface-sterilized with

freshly prepared 70% ethanol for 45 s and then in 2%

(v/v) sodium hypochlorite solution. The culture room

was maintained at 25 ± 1�C under a 16/8 h light/

dark cycle, and each rack was illuminated with light

(50–100 lmol m-2 s-1) provided by three white

fluorescent tubes (Philips Champion 40 W/54) and

one yellow fluorescent tube (Philips Trulight 36 W/82).

Transgenic plants were grown under standard con-

tainment green house conditions.

In-silico analysis of promoter sequence

To determine conserved cis-acting promoter ele-

ments, databases hosted at PLACE (Higo et al. 1999)

and PlantCARE (Lescot et al. 2002) were used.

Total RNA isolation and northern analysis

Total RNA from the rice tissues was isolated in

accordance with the procedure described by Loge-

mann et al. (1987), with minor modifications, and

quantified by recording the OD260 using a U-2001

spectrophotometer (Hitachi, Japan). For northern

hybridization, 15 lg total RNA was electrophoresed

in 1.2% (w/v) formaldehyde-agarose gels and trans-

ferred on to a Hybond-N membrane (Amersham

Biosciences, Piscataway, USA) by the capillary

transfer method (Sambrook et al. 1989). The northern

hybridization was performed using radiolabelled

OSIAGP cDNA as probe. Prehybridization, hybrid-

ization, washing, and autoradiography were per-

formed as described by Sambrook et al. (1989).

cDNA/Genomic library screening

The cDNA and genomic libraries from indica rice

were used to isolate the OSIAGP gene, as described

(Gupta et al. 2007). Plaques were purified to homo-

geneity and used for isolating phage DNA following

Santos (1991). OSIAGP genomic clone was finally

confirmed by Southern hybridization with phage

DNA. OSIAGP cDNA and promoter region were

completely sequenced using gene-specific primer.

Primer extension

Primer extension analysis was performed to define

the 50 end of the OSIAGP transcript. The primer with

sequence 50 aggcaatcttcttcatc 30 was designed from

the 50 untranslated region (UTR) region *100 bp

downstream of the 50 end of the cDNA. The primer

was labelled at the 50 end by incorporating

c-p32dATP. The 50 end-labelled oligonucleotides

were incubated with the total RNA from pre-pollina-

tion stage inflorescence. The primer-RNA mixture

was cleaned and subsequently reverse transcribed

using M-MuLV reverse transcriptase at 37�C for

90 min. The extended product was cleaned and run

alongside a sequencing reaction performed with the

same primer and the plasmid containing the OSIAGP

gene and the upstream regulatory region on a 6%

polyacrylamide gel.

Cloning for OSIAGP promoter and deletion

constructs

The regulatory region of OSIAGP upstream of the

NotI site was cloned in pBI101 and pCAMBIA1391Z

for tobacco and rice transformation, respectively. The

1.5 kb NotI fragment was end-filled using Klenow

enzyme and cloned at the SmaI site in pBSK?blue-

script. Its orientation was checked by restriction

digestion of clones with asymmetrically placed sites.

The fragment was released using BamHI and EcoRI

enzymes and cloned at same sites in pBI101 and

pCAMBIA1391Z. The promoter deletion construct in

pCAMBIA1391Z (PD1 with promoter fragment of

1104 bp starting at position -1019 bp, PD0.7 with

promoter fragment of 706 bp starting at position

-621 bp, and PD0.3 with promoter fragment of

385 bp starting at position -300 bp) were made by

using restriction sites already present in the OSIAGP

promoter. PD0.1 (159 bp starting at position -85 bp)

was created by PCR amplification. Primer pairs,

50-tggaagcttgctcacaaataaattaacca-30 and 50-taaggatccg

gccgctagagacga-30, were used for amplification and

HindIII and BamHI sites were added to facilitate

directional cloning of the amplified fragment.

The amplified fragment was digested with BamHI

and HindIII restriction enzymes and ligated to

pCAMBIA1391Z, linearized with the same enzymes.

Sequencing was performed to confirm cloned

fragments.

Transgenic Res (2010) 19:385–397 387

123

Tobacco transformation

Agrobacterium tumefaciens strain AGL1 was used

for transformation of tobacco plants with OSIAGP

promoter constructs. Tobacco leaf discs (1 cm2 in

size) were placed on MSO medium containing 1 mg/l

BAP and 0.1 mg/l NAA. Cocultivation of leaf

explants was performed with Agrobacterium culture

for 2 days. Subsequently, the explants were washed

with autoclaved Millipore water and placed on

selection plates containing kanamycin 200 mg/l

and cefatoxime 250 mg/l. Regenerating calli were

subcultured in culture bottles with MSO medium

supplemented with kanamycin 200 mg/l and

cefatoxime 250 mg/l for rooting. Regenerated puta-

tive transgenic plants were transferred to pots in a

culture room before transfer to a greenhouse.

Rice transformation

For transformation of rice, the procedure described

by Mohanty et al. (1999) was used, with some

modifications. Overnight-soaked seeds were placed

on 2MS medium for 21 days. After 21 days of

callusing, the calli were broken into 3-4 mm diameter

fragments and placed on 2MSCA medium for four

days. On the same day, growth of Agrobacterium

containing different OSIAGP constructs were set up

in YEM medium supplemented with 100 mg/l rif-

ampicin and 50 mg/l kanamycin followed by incu-

bation at 26 ± 2�C and 200 rpm for two days. It was

subcultured again in fresh YEM medium. The

secondary culture was pelleted and resuspended in

AA medium to a density of 3–4 9 109 cells/ml. Four-

day-old subcultured embryogenic calli were dipped in

bacterial suspension for 30 min. Excess bacterial

suspension was decanted, and the calli were blotted

on an autoclaved tissue-paper pad and placed on

2MSAS medium and incubated in the dark at

precisely 26 ± 2�C for four days. On fourth day,

the calli were washed 3–4 times with sterile RO water

containing 250 mg/l cefotaxime and blotted on an

autoclaved tissue-paper pad and placed on

2MSCHCA plates at 26 ± 2�C for 15 days. The

hygromycin-resistant white calli were cleaned with

sterile forceps and placed on fresh 2MSCHCA plates

for 15 days. Subculturing was repeated twice after

15-day intervals. White proliferating hygromycin-

resistant calli were transferred to regeneration

(MSRNH) medium and incubated in a culture room

at 26 ± 2�C and with a 16/8 h light/dark cycle of

100–125 lmol m-2 s-1. After a week, green regen-

erating calli with shoots were transferred to MSBH

medium in glass culture tubes for rooting. Plantlets in

tubes were incubated under the same conditions of

light and temperature. Rooted plantlets were trans-

ferred to Yoshida medium (Yoshida et al. 1976) for

hardening in glass culture tubes under culture room

conditions. After one week of incubation in Yoshida

medium, the putative transgenic plants were trans-

ferred to a greenhouse operating at 26–28�C with a

16/8 h light/dark cycle of 100–125 lmol m-2 s-1

and 70–75% relative humidity.

PCR analysis of transgenic plants

Transgenic tobacco and rice plants harboring the

OSIAGP promoter and its deletions with the gus gene

were analysed by amplifying the gus gene by

performing PCR using the Extract-N-Amp PCR kit

(Sigma–Aldrich, USA). About 1.8 kb of gus was

amplified using forward primer, 50 atccgtccgtcc

gtagaaac 30 and reverse primer, 50 catacctgttcaccga

cgacg 30.

Histochemical and fluorimetric analysis of GUS

Histochemical analysis of GUS activity was carried

out according to Chaudhury et al. (1995). Small

pieces of plant materials were incubated in GUS

histochemical buffer (50 mM sodium phosphate, pH

7.0, 50 mM EDTA, pH 8.0, 0.5 mM K3Fe(CN)6,

0.5 mM K4Fe(CN)6, 0.1% Triton X-100, and 1 mM

X-Gluc that was initially dissolved in DMSO) at

37�C. The GUS activity was measured by the

procedure described by Jefferson et al. (1987), with

some modifications (Chaudhury et al. 1993). Tissues

were homogenized in extraction buffer (50 mM

sodium phosphate buffer, pH 7.0, 10 mM disodium

EDTA, 0.1% Triton X-100, 0.1% N-lauroylsarcosine

and 10 mM b-mercaptoethanol). Protein estimation

was performed with Bradford reagent. Protein sam-

ples were added to the GUS assay buffer containing

MUG. Relative fluorescence was measured with a

DNA Quant 200 fluorimeter (Hoefer Pharmacia

Biotech, USA). Finally the specific activity of GUS

was expressed in picomoles of 4-MU (mg pro-

tein)-1h-1. For all constructs, 4–7 transgenic lines

388 Transgenic Res (2010) 19:385–397

123

were analysed, with up to three replicates for each

line. Representative data for each line have been

given.

In-vitro germination of transgenic pollen

Pollen grains from transgenic tobacco plants harbor-

ing the OSIAGP promoter fused to the gus gene were

germinated on medium (sucrose 10%, boric acid

100 mg/l, calcium nitrate 300 mg/l, magnesium sul-

phate 200 mg/l, potassium nitrate 100 mg/l, casein

hydrolysate 1 mg/ml, and 1% agar as solidifying

agent) after Brewbaker and Kwack (1963). The

pollen grains were sprinkled on autoclaved medium

and plates were incubated in the dark at room

temperature for 2–24 h.

Results

Structural and expression analysis of OSIAGP

The cDNA for OSIAGP is 586 bp long with a 19 bp

poly A tail. The GC content of the cDNA is 50.27%

when poly A is excluded from the sequence and a

unique NotI site is present 60 bp downstream of the

50 end (Fig. 1a). The cDNA was translated in silico,

in all three open reading frames, using Generunner

(Hastings Software, USA). The positions of the start

and stop codons are 95 and 274 bp downstream of the

50 end of the cDNA, respectively. The open reading

frame codes for a polypeptide of 59 amino acids with

a predicted molecular weight of 6.4 kDa. In-silico

analysis of the encoded protein using pSORT reveals

it to possess a ten-amino-acids-long secretory domain

at the N terminus. Further, the polypeptide sequence

may have two hydrophobic domains with the

potential to traverse a membrane (Fig. 1b). The

polypeptide is also rich in a-helix, as predicted by

various software. OSIAGP showed significant homol-

ogy with putative AGP23 (AT3G57690) (Fig. 1b). Its

homologue is present on chromosome 6 in japonica

rice.

Expression analysis from different organs, i.e.

root, stem, leaf, pre-pollination (PP) and post-fertil-

ization (PF) stage inflorescence, rachis of the mature

rice plant, and root and shoot of young rice seedlings,

revealed that the maximum level of expression is

seen in pre-pollination stage inflorescence tissue

(Fig. 2a). A very low level of transcript is detected

in mature root sample and the transcript level was

below the detection limit in other organs. Among the

flower parts, i.e. lemma, palea, anther, gynoecium,

anther wall, and pollen, the maximum level of

expression for OSIAGP was seen in pollen, followed

by anther and anther wall (Fig. 2b). Because arabi-

nogalactan genes are known to be involved in cell

elongation and the OSIAGP promoter contains light

regulatory elements, its expression was also analysed

in dark/light-grown seedlings. OSIAGP mRNA was

found to accumulate in both shoot and root of dark-

grown seedlings and the expression level increased

from the 5th to the 9th day before showing a decline

(Fig. 2c, d). Interestingly, expression of the gene in

light-grown seedling tissues was negligible.

50 End mapping of the OSIAGP transcript

In order to map the 50 end of the OSIAGP transcript,

primer extension was performed by using a primer (50

aggcaatcttcttcatc 30, Tm 53.3�C) from the 50 UTR

region, *100 bp downstream of the 50 end of the

OSIAGP cDNA. A clear sharp band of the extended

product corresponding to base T was seen. Therefore,

the transcription start site (?1) was mapped to base

A, 18 bases upstream of the first base of the OSIAGP

cDNA. A TATA box was found 28 bases upstream of

the transcription start site. The actual length of

OSIAGP full cDNA turned out to be 586 bp including

poly A tail (Fig. S1).

The OSIAGP promoter shows pollen-preferential

activity in transgenic tobacco

The OSIAGP promoter harbors cis elements

(GTGANTG10, GTGA, and AGAAA) conferring

pollen-specificity and GT1, IBOX, and IBOXCORE

conferring light regulation (Fig. 1a). The organ-

specificity of the OSIAGP promoter was evaluated

for transgenic tobacco plants, transformed with

OSIAGPpBI101 construct, by staining different tis-

sues with GUS staining buffer. An untransformed

plant was taken as a non-transgenic control. The blue

colour for GUS was visible in the pollen grains of the

anthers of the transgenic plants. Anthers had to be cut

open in order to stain the anther and blue coloured

pollen grains were visible at the bottom of the

microcentrifuge tube. In fluorimetric GUS analysis,

Transgenic Res (2010) 19:385–397 389

123

Fig. 1 a The nucleotide sequence of the genomic clone of

OSIAGP gene. Letters in lower case show the sequence of the

upstream region. Bold letters in lower case show the 50 and 30

UTRs whereas letters in bold and upper case show the open

reading frame. The first 18 bases shown in brown have been

found to be part of the 50 UTR after primer extension-based

mapping of the transcription start site. Amino acids of the

deduced polypeptide have been written under the respective

codons. Start codon and the stop codon have been shown in

red. The TATA box has been located 28 bp upstream of the

transcription start site. Consensus of putative upstream cis-

acting elements have been shown in bold, italics, underlined,

and with different colours. b Comparison of deduced amino

acid sequences from OSIAGP with OSJAGP and AGP23

390 Transgenic Res (2010) 19:385–397

123

seven different transgenic plants (confirmed by PCR

analysis) and a control plant were used. Maximum

level of activity was seen in anthers. Although calyx,

corolla, gynoecium, and root show a significant level

of GUS (though manyfold less than the anther), in

leaf the level of GUS was almost equal to that in the

control (Fig. 3a). These results match the observa-

tions made after histochemical staining (data not

shown).

The promoter shows temporal regulation

of activity after pollen meiosis

GUS activity driven by the OSIAGP promoter was

quantified from anthers of the flower buds, belonging

to different size groups and developmental stages, of

transgenic tobacco plants, already confirmed by PCR

analysis (Fig. 3b). Fluorimetric analysis was per-

formed with anthers of flower buds from different

size groups. Flower buds from four different trans-

genic plants and one control plant were used for

analysis. The fluorimetry shows that the level of GUS

increases with increase in bud size (Fig. 3b).

In order to study the temporal expression of gus in

the process of pollen development in flower buds,

buds belonging to different size groups were stained

with GUS staining buffer. Various flower buds were

cut open to help the buffer penetrate inside. There is

no expression of GUS in sporogenous tissue and in

the tetrad stage of pollen development. The blue

colour, however, is evident after pollen meiosis and

the level of expression increases as the bud size

increases, the maximum being in the mature flower

(Fig. 4a). Thus, the OSIAGP promoter shows variable

temporal activity during flower development in a

heterologous system.

The OSIAGP promoter is active in pollen tubes

In order to study the activity of the promoter after

germination of the pollen grains, transgenic and

untransformed control pollen grains were germinated

on in-vitro germination medium. Staining of germi-

nated pollen with GUS buffer revealed the expression

of GUS in the pollen tubes of transgenic pollen

germinated for 2–24 h. It was localized to the body of

Pal

eaL

emm

aG

ynoe

cium

Ant

her

Ant

her

wal

lP

olle

n

OSIAGP

rRNAM

atu r

e st

em

Yo u

ng s

h oo t

Ma t

ure

leaf

Ra c

h is

PP

Pan

icle

PF

Pan

icle

Mat

u re

root

You

ng r

oot

OSIAGP

rRNA

~550 bp

ROOT

LIGHT DARK

5D 7D 9D 13D

5D 7D 9D 13D

11D

11D

LIGHT DARK

5D 7D 9D 13D

5D 7D 9D 13D

11D

11D

SHOOT

OSIAGP

rRNA

OSIAGP

rRNA

A

C D

B

Fig. 2 Organ-specific expression and light regulation of

OSIAGP. a RNA isolated from leaves, roots, stem, rachis,

and pre-pollination (PP) and post-fertilization (PF) stage

panicles of mature flowering rice plants, and from root and

shoot of young (7-day-old) seedlings was used. b RNA isolated

from palea, lemma, gynoecium, anther, anther wall, and pollen

was used. c Accumulation of transcripts of OSIAGP in shoots

and roots (d) of dark-grown samples of rice. RNA was isolated

from shoots and roots harvested from rice seedlings grown in

light and dark after various days (D) of germination. Northern

blots were probed with cDNA of OSIAGP. The lower panels in

a–d show ethidium bromide-stained rRNA indicating equiva-

lent loading and quality of RNA samples

Transgenic Res (2010) 19:385–397 391

123

the pollen and the small pollen tube formed after 2 h

of germination. After 24 h of germination, blue

colour was observed in the contents throughout the

pollen tube, being more intense towards the growing

end of pollen tube. There was no blue colour in the

pollen tubes of the control pollen grains (Fig. 4b).

Analysis of the OSIAGP promoter

and its deletions in transgenic rice

Organ-specificity of the OSIAGP promoter was also

evaluated for transgenic rice plants, transformed with

the PD1 construct and confirmed by PCR analysis, by

staining different tissues with GUS staining buffer.

Various organs showed very low activity compared

with pollen grains and results of fluorimetric analysis

(Fig. S2) were similar to those from histochemical

staining (data not shown). The OSIAGP promoter

shows pollen-preferential expression and its deletions

showed reduced expression of GUS (Figs. S2, 5). The

intense blue colour was seen in the pollen and

the anther wall of the transgenic plants. However, the

expression of GUS was abolished in the pollen and

anther wall of the transgenic rice plants harboring the

smallest deletion named PD0.1 (Fig. 5). The loss of

pollen specificity observed in smallest promoter

deletion correlates well with in-silico analysis of its

promoter, i.e. no known cis element conferring pollen

specificity is present in this region. It may, however,

be noted that PD0.3 shows pollen/anther-specific

activity although the region between -300 and

-85 bp does not contain typical anther/pollen-

specificity regulatory elements. This may be because

of a novel, yet to be identified, regulatory element(s).

Discussion

Characterization of genes expressing in a stage-

specific manner and their regulatory regions has been

a task of utmost importance to understanding the

complexities of flower development. In this investiga-

tion, an effort has been made to characterize an

arabinogalactan protein-encoding gene and study its

promoter activity in homologous and heterologous

transgenic systems. The rice gene OSIAGP (Accession

no. AF 358652) was isolated from the inflorescence on

the basis of screening of cDNA libraries and its

different expression profiles in various organs of Oryza

sativa ssp. indica var. Pusa Basmati 1. OSIAGP protein

has a secretory domain at its N terminus and in-silico

analysis revealed it to be a secretory and transmem-

brane protein. When databases were searched using

OSIAGP nucleotide and protein sequences, they

showed significant homology with a pollen-preferen-

tial gene, AGP23, encoding for arabinogalactan

Spec

ific

Act

ivit

y(P

icom

oles

of 4

MU

/mg

prot

ein/

h)

0

50000

100000

150000

200000

250000

Lea

f

Roo

t

Ant

her

Cal

yx

Cor

olla

Gyn

oeci

um

Plant Organs

0.7-

1.0

cm

1.7-

2.0

cm

0.3-

0.6

cm

1.2-

1.5

cm

2.5-

2.8

cm

3.5-

3.8

cm

(Pic

omol

esof

4M

U /m

g pr

otei

n /h

)

B0 B1

B2

B3 B4

Bud Size

0.3-

0.6

cm

0.7-

1.0

cm

1.2-

1.5

cm

1.7-

2.0

cm

0

50000

100000150000

200000250000

300000

2.5-

2.8

cm

B5

3.5-

3.8

cm

Spec

ific

Act

ivit

y

B0 B1 B2 B3 B4 B5

A

B

Fig. 3 GUS-specific activity from different organs and flower

buds of different sizes of transgenic tobacco plants transformed

with OSIAGPpBI101 containing the OSIAGP promoter.

a Individual transgenic lines have been represented by different

bars in the same order. Control has been represented by the

first bar in each case. b Flower buds belonging to different size

groups and developmental stages from transgenic tobacco

plants transformed with OSIAGPpBI101 construct. The lowerpanel of b shows GUS-specific activity of anthers of the flower

buds from B0 to B5 shown in the upper panel

392 Transgenic Res (2010) 19:385–397

123

protein from Arabidopsis (AT3G57690). The eight

amino acids of the secretory domain at the N terminus

are 100% conserved among OSIAGP, OSJAGP, and

AGP23. The AGP23 gene is 369 bp long and encodes a

61-amino-acid protein with homology (score bit 611,

e value e-174) with other arabinogalactan proteins

from Arabidopsis thaliana.

Arabinogalactan proteins are a family of exten-

sively glycosylated hydroxyproline-rich glycopro-

teins (HRGPs) playing many important biological

roles (Seifert and Roberts 2007). These are cell

surface molecules with 1–10% protein, typically rich

in hydroxyproline/proline (Hyp/Pro), alanine (Ala),

serine (Ser), and threonine (Thr) (Schultz et al. 2002).

OSIAGP of 59 amino acids has an abundance of

alanine residues (19/59, i.e. 32.2%), whereas serine

(6/59), proline (4/59), and threonine (3/59) have

average occurrence. In all, these four amino acids

represent [50% of total amino acids of the deduced

protein from OSIAGP. AGPs have been classified on

the basis of structure and one of the category named

‘‘classical types’’ has a secretory domain at the N

terminal region of the protein. Thus OSIAGP could

be regarded as another member of the classical type

arabinogalactan proteins. Several anther-expressing

proteins possess, in their amino terminal end, a signal

peptide signifying their secretory nature, e.g. ZMC-13

(Hamilton et al. 1989; Hanson et al. 1989), PS-1 (Zou

et al. 1994), TPC-90 (Turcich et al. 1994), BP-19 and

BP-10 (Albani et al. 1991, 1992).

Many genes expressing in pollen grains have been

characterized from different plants (Gupta et al. 2007;

Hobo et al. 2008; Suwabe et al. 2008). Few of these

express specifically in pollen and the others show

0.7-1.0 cm

1.2-1.5 cm 1.7-2.0 cm 2.5-2.8 cm 3.5-3.8 cm

0.3-0.6 cmSporogenous mass Tetrad stage

4 h

24 h

Control Transgenic

A

B

Fig. 4 OSIAGP promoter-

driven GUS activity during

pollen development in

transgenic tobacco. a No

GUS activity is seen in

sporogenous mass and

tetrad stage of pollen

development. Pollen of

transgenic tobacco stained

after GUS histochemical

assay was from flower buds

of different sizes shown in

Fig. 3b. b In-vitro

germination of pollen grains

from transgenic tobacco

transformed with

OSIAGPpBI101 construct.

The gus activity is evident

in pollen tubes of transgenic

pollen upon in-vitro

germination for 4 and 24 h

on Brewbaker and Kwack

(1963) medium

Transgenic Res (2010) 19:385–397 393

123

Anther Wall

Pollen

0

5000

10000

15000

20000

25000

P ∆ 1 P∆ 0.7 P∆ 0.3 P∆ 0.1

Deletions of OSIAGP promoter

Spec

ific

Act

ivit

y(P

icom

oles

of 4

MU

/mg

prot

ein/

h)

C

P∆ 0.1P∆ 0.3NTA

B

Fig. 5 Organ-preferential gus activity in transgenic rice under

control of OSIAGP promoter deletions. a Histochemical

analysis of GUS expression driven by deletions of the OSIAGPpromoter (PD0.3 and PD0.1) in pollen and anther wall of

transgenic rice. NT non-transgenic rice; T transgenic rice.

b GUS fluorimetric analysis of leaf samples from transgenic

rice plants containing four deletions, namely PD1.0, PD0.7,

PD0.3, and PD0.1 of the OSIAGP promoter. An untransformed

rice plant was used as a control (c)

394 Transgenic Res (2010) 19:385–397

123

expression in other organs also. OSIAGP, with high

expression in pre-pollination stage inflorescence, falls

in the category of pollen-preferential gene. A cis

element named GTGANTG10, conferring pollen

specificity, was found in the OSIAGP promoter at

five positions; -463, -472, -778, -888, and

-1326 bp (upstream to the transcription start site).

In the pollen-specific LAT56 and LAT59 promoters, a

common cis-acting regulatory element (LAT56/59

box) was identified at the same relative positions

having a core sequence of GTGA (Twell et al. 1991).

The late pollen gene G10 promoter of tobacco

possesses five such GTGA motifs, one of which, at

-96 bp, is in the same relative position as the GTGA

found in the LAT56 and LAT59 promoters (Rogers

et al. 2001). Mutation in this box results in many-fold

reduction in the pollen-specific activity of the

promoter. Another cis element with a consensus of

AGAAA, known to confer pollen specificity, is

POLLEN1LELAT52. This element is one of the

two co-dependent regulatory elements in the tomato

LAT52 gene. The sequence AGAAA is present at

-1226 bp in the OSIAGP promoter. Some AGP-

encoding genes have been shown to express in pollen

(during development, germination, and tube growth)

and other reproductive and vegetative tissues of the

plant. Examples include STA 39-3 and STA39-4 from

Brassica (Gerster et al. 1996). AGP was shown to

localize in anther, pollen, and pollen tubes of

Nicotiana tabacum (Qin et al. 2007). The arabinoga-

lactan proteins have already been shown to be

important in cell elongation, a phenomenon associ-

ated with growth of pollen tubes, root hairs, and the

seedlings in dark. LeAGP-1, an AGP encoding gene,

was shown to be expressed during seedling develop-

ment in the tomato and likely to function in root

growth, more specifically in cell elongation, cell

proliferation, root hair formation, and water uptake

(Willats and Knox 1996; Lu et al. 2001). The

OSIAGP gene is also shown to be expressing in

early development of rice seedlings. But transcripts

of the gene accumulate only in the dark and the

pattern was almost identical both for shoots and roots.

Analysis of the OSIAGP promoter sequence also

revealed the presence of cis elements associated with

light regulation, namely GT1, I BOX, and I BOX-

CORE (Terzaghi and Cashmore 1995; Zhou 1999).

Pollen development requires timely expression of

genes in both gametophytic and sporophytic tissues.

Transcripts of ‘‘early genes’’, which encode products

involved in the synthesis of cytoskeletal elements,

become active soon after meiosis but for a short

period only and start declining by the interphase

stage. On the other hand, the ‘‘late genes’’, encoding

products involved in cell-wall formation, pollen

maturation, pollen germination, pollen-tube growth,

and pollen recognition, represented by the pollen-

specific genes, are expressed after pollen mitosis and

continue to accumulate as pollen matures, i.e. show

maximum expression in mature flower (Raghavan

1997). Examples of late pollen genes include LAT-51,

LAT-52, LAT-58, and LAT-59 from tomato (Twell

et al. 1989; Ursin et al. 1989; Wing et al. 1989), P-2

from O. organensis (Brown and Crouch 1990), BCP-

1 from B. campestris (Theerakulpisut et al. 1991),

and BP-10, STA39-3, STA39-4, and STA44-4 from

B. napus (Albani et al. 1992; Roberts et al. 1993;

Gerster et al. 1996). BcMF8 is an example of

classical arabinogalactan protein-encoding gene from

Brassica compestris ssp. chinensis showing pollen-

specific expression (Huang et al. 2008). The OSIAGP

promoter shows activity after the microspores have

been separated from callose wall; thus it could be

categorized as a late pollen-expressing gene with

maximum level of expression in pollen grains from

mature flowers. It was also active during germination

and tube growth. Many genes expressing in germi-

nating pollen and pollen tube growth have been

investigated in different systems. Genes encoding for

calcium-dependent calmodulin-independent protein

kinase (CDPK) and receptor-like serine-threonine

protein kinase (PRK1) have been found to be

indispensable for the process of germination and

pollen tube growth, because their antisense leads to

inhibition of germination (Estruch et al. 1994; Mu

et al. 1994). AtAGP 6 and 11 have been shown to be

required for stamen and pollen function in Arabid-

opsis. Pollen grains failed to develop normally and

eventually collapsed in agp6agp11-null mutants,

clearly establishing the overlapping and important

involvement of these proteins in pollen development

(Levitin et al. 2008; Coimbra et al. 2009). AtAGP17,

18 and 19 are known to be essential for plant growth

and development, including cell division and expan-

sion (Yang et al. 2007). AtAGP30 from Arabidopsis

expresses in cell walls of primary root and is involved

in root regeneration and seed germination (van

Hengel and Roberts, 2003). The activity of OSIAGP

Transgenic Res (2010) 19:385–397 395

123

promoter in pollen germination and pollen-tube

growth supports the fact that OSIAGP might be

important in these events.

In conclusion, we have reported isolation, charac-

terization, and promoter activity of an arabinogalac-

tan protein-encoding gene from indica rice. OSIAGP

is a pollen-preferential gene falling in the category of

late pollen genes and is speculated to play important

role in pollen-tube growth. Its promoter harbouring

regulatory elements for pollen expression and light

regulation could be of interest to the plant commu-

nity. Detailed study of its role in plant growth and

reproduction could add another function to already

growing list of functions performed by this diverse

class of arabinogalactan proteins.

Acknowledgments The research was supported by a grant

from the Department of Biotechnology, Government of India.

SA was a recipient of JRF and SRF given by University Grants

Commission.

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