Plant Cell Rep (****) **:** **

DOI **.****/s*****-009-0794-z

ORIGINAL PAPER

Isolation and promoter analysis of anther-speci c genes encoding

putative arabinogalactan proteins in Malus 3 domestica

Yeon-Ok Choi Sung-Soo Kim Sanghyeob Lee Sunggil Kim

Gi-Bo Yoon Hyojeong Kim Young-Pyo Lee Gyung-Hee Yu

Nam-In Hyung Soon-Kee Sung

Received: 21 April 2009 / Revised: 7 September 2009 / Accepted: 19 September 2009 / Published online: 5 November 2009

Springer-Verlag 2009

Abstract In this study, we searched for anther-speci c were designated MdAGP1, MdAGP2, and MdAGP3,

genes involved in male gametophyte development in apple respectively. RT (reverse transcriptase)-PCR revealed that

(Malus 9 domestica Borkh. cv. Fuji) by differential dis- the MdAGP genes were selectively expressed in the

play-PCR. Three full-length cDNAs were isolated, and the stamen. Promoter analysis con rmed that the MdAGP3

corresponding genomic sequences were determined by promoter was capable of directing anther- or pollen-

genome walking. The identi ed genes showed intronless speci c expression of the GUS reporter in tobacco and

228- to 264-bp open reading frames and shared 82 90% apple. Furthermore, expression of ribosome-inactivating

nucleotide sequence. Sequence analysis identi ed that protein under the control of the MdAGP3 promoter induced

they encoded a putative arabinogalactan protein (AGP) and complete sporophytic male sterility as we had expected.

Keywords Apple (Malus 3 domestica)

Communicated by J. R. Liu. Anther speci c gene Arabinogalactan protein

Male-sterility induction

Y.-O. Choi and S.-S. Kim contributed equally to this work.

Electronic supplementary material The online version of this

Abbreviations

article (doi:10.1007/s00299-009-0794-z) contains supplementary

AGP Arabinogalactan protein

material, which is available to authorized users.

GPI Glycosylphosphatidylinositol

Y.-O. Choi S.-S. Kim S. Lee G.-B. Yoon H. Kim ORF Open reading frame

Y.-P. Lee S.-K. Sung RIP Ribosome inactivating protein

Biotech Research Team,

UTR Untranslated region

Dongbu Advanced Research Institute,

Dongbu HiTek Co., Ltd, Daejeon 305-708, Korea

e-mail: abphta@r.postjobfree.com

S. Kim

Department of Plant Biotechnology,

Introduction

Biotechnology Research Institute,

Chonnam National University,

To produce genetically superior apples, breeders have

Gwangju 500-757, Korea

integrated traditional breeding with genetic engineering.

N.-I. Hyung

Traditional breeding usually results in the creation of new

Department of Plant Science and Technology,

varieties that replace existing ones. Genetic engineering,

Sangmyung University, Chonan 330-720, Korea

however, improves upon existing varieties by adding or

G.-H. Yu changing agriculturally important traits. Traditional meth-

Metrology and Measurement Division,

ods are quite expensive and time consuming. Extensive

Agency for Technology and Standards,

selections must be performed to choose appropriate parents

Ministry of Knowledge Economy,

and hybrids with desirable traits while minimizing inferior

Gwacheon, Gyonggi-do 427-716, Korea

123

16 Plant Cell Rep (2010) 29:15 24

AGP-encoding genes, they may participate in the intricate

ones. Furthermore, traditional breeding only produces new

process of ower development. For example, AGPNa3

varieties that resemble a parent; they are never identical to

from Nicotiana alata (Du et al. 1996) is selectively

either parent. Unlike other fruits, apple varieties have

expressed in pistils, and AtAGP18 from Arabidopsis

established markets and strong name values (Brown 1995).

(Acosta-Garcia and Vielle-Calzada 2004) is expressed in

Nevertheless, genetic engineering can improve existing

cells involved in female gametogenesis. In case of male

varieties by introducing key traits that traditional breeding

organs, several AGP genes including AtAGP23 (Becker

cannot provide. Currently, the application of genetic

et al. 2003) and BAN102 (Park et al. 2005) are preferen-

engineering is restricted to simple inherited traits such as

tially expressed in mature pollen and pollen tubes, while

disease resistance. More complex characteristics, including

PO2 from alfalfa (Qiu et al. 1997) and Sta 39-3 and -4 from

texture, color, yield, and avor, cannot be targeted by

genetic engineering. Traditional breeding is required to Brassica napus (Gerster et al. 1996) are predominantly

improve these traits and produce new varieties. expressed during the late stages of pollen development.

The transformation and regeneration needed to use In order to understand and utilize stamen develop-

genetic engineering successfully to improve existing vari- mental processes, we should have deep consideration

eties of apple has been established (Aldwinckle 1993; about both regulatory mechanism and functional roles of

stamen-speci c genes. Both 50 upstream region of genes

Hrazdina 1994). To date, 48 genetically modi ed (GM)

and roles of 50 -UTR sequences are important for under-

apples have undergone eld trials worldwide (GMO Com-

pass, http://www.gmo-compass.org/eng/database/plants/18. standing regulatory mechanisms of tissue- or organ-

apple.html). The traits targeted for improvement in these speci c genes. Therefore, identi cation of cis-acting

GM apples include pathogen and pest resistance, fruit sugar regulatory elements from promoter sequences is important

and alcohol modi cation, blossom time, rooting, and auto for this purpose. Especially, in terms of agricultural

fertility (GMO Compass). Much concern has focused on the industry, the identi cation of anther/pollen-speci c pro-

environmental impact of transgenic plants since they pro- moter is useful for further utilization, because these reg-

duce pollen that can be transported over long distances by ulatory elements can be used to induce male sterility in

wind or insects. The newly introduced transgenes in these plants. For example, tapetum-and anther-speci c promot-

plants can be transferred to related species during pollina- ers have been exploited to induce male-sterility in Sola-

tion. To prevent these genes from spreading, molecular naceous and Brassicaceae (Mariani et al. 1990; Lee et al.

approaches have focused on maternal inheritance, male 2003; Roque et al. 2007).

sterility, and seed sterility (Daniell 2002). Although each Here, we describe the isolation of three homologous

method has its advantages and disadvantages, the intro- genes encoding putative AGPs from anther cDNA library

duction of male sterility has been shown to be a very from apple and anther-speci c expression of AGPs. Fur-

promising method in producing GM apples. Male sterility thermore, we present MdAGP promoter-directed expres-

can be regulated by the nucleus or cytoplasm. To date, 12 sion in transgenic tobacco and MdAGP promoter sequence

examples of cytoplasmic male sterility (CMS) have been analysis.

identi ed in petunia, maize, sorghum, sun ower, wheat,

Brassica, and Raphanus (Hanson and Benolila 2004).

Limited application of CMS for the generation of sterile Materials and methods

male plants has forced focus on nuclear factors for the

manipulation of male sterility. Therefore, much effort has Plant materials

been placed on identifying anther- or pollen-speci c genes.

To date, many such genes have been discovered in at least The apple cultivar Fuji was provided by the National

28 species, including apple (Supplementary Table 1). Horticultural Research Institute, Suwon, Republic of

However, maternal inheritance and seed sterility via chlo- Korea. Young leaves, organs of mature owers (i.e., sepal,

roplast transformation and termination technology, respec- petal, stamen, and carpel), and young fruits approximately

tively, have yet to be demonstrated in apple plants. 7 mm in diameter were collected and immediately frozen

Arabinogalactan proteins (AGPs) constitute a family of in liquid nitrogen. All frozen samples were stored at

-70 C until further use.

extracellular glycoproteins that are widely distributed

among owering plants. AGPs are thought to be involved

in diverse aspects of plant growth, development, and Differential display-polymerase chain

organogenesis (Knox et al. 1991; van Hengel and Roberts reaction (DD-PCR)

2003; Acosta-Garcia and Vielle-Calzada 2004; Sun et al.

2004a, b; van Hengel et al. 2004). Because many studies Total RNA was extracted from leaves, sepals, petals, sta-

have demonstrated oral organ-speci c expression of mens, carpels, and young fruits and treated with DNase I to

123

Plant Cell Rep (2010) 29:15 24 17

Promoter analysis using GUS reporter gene

remove residual genomic DNA. First-strand cDNAs were

then synthesized using a commercial cDNA synthesis kit

(SUPERSCRIPTTM Preampli cation System; Life Tech- Promoter analysis was performed using a pBI vector con-

nologies, USA). Random 10-mers and 50 -T12VN-30 were struct containing the GUS reporter gene under the control

of the MdAGP3 promoter (-352 to -6 bp upstream of the

used as forward and reverse primers for each PCR reaction,

respectively. Reaction mixtures had a total volume of 20 ll start codon). This construct was transformed into tobacco

and contained 2.5 lM T12VN primer, 0.5 lM random (Nicotiana tabacum L. cv. Petit Havana SR1) through

primer, 7.5 lM dNTP, 0.5 lM [a-35S] dATP (1,250 Ci/ Agrobacterium tumefaciens LBA4404 (Hoekema et al.

1983). Agrobacterium-mediated transformation was per-

nmol), 1 U Taq DNA polymerase (TaKaRa, Japan). PCR

formed according to the method of Sung et al. (1999).

ampli cation was carried out with an initial denaturation at

94 C for 5 min that was followed by 40 cycles consisting

of 94 C for 30 s, 42 C for 1 min, and 72 C for 30 s, and Agrobacterium-mediated transformation

then by a nal 10-min extension at 72 C. PCR products of ribosome-inactivating protein (RIP)-coding gene

were separated on 6% polyacrylamide gel, transferred to a

The gene encoding RIP from Dianthus sinensis L. (Cho

hybridization membrane, and exposed to X-ray lm.

et al. 2001) were cloned into the binary vector pCAM-

Polymorphic bands were isolated, ampli ed with the same

BIA3301 under the control of the MdAGP3 promoter. The

primer pair, and cloned into pGEM-T vector (Promega,

RIP cDNA (GeneBank Accession number AF219237) was

USA). The cloned fragments were sequenced using auto-

kindly provided by Dr. Byung-Dong Kim (Seoul National

mated Big Dye DNA cycle sequencing (ABI Prism BigDye

University). The Bar gene was used as a selective marker.

Terminator Cycle Sequencing Ready Reaction Kits;

A. tumefaciens LBA4404 was used for transformation of

Applied Biosystems, USA).

tobacco. After getting the RIP transgenic plants, con r-

mation of transgene was determined by PCR reaction using

Construction of cDNA library and isolation

the primer set (bar Forward : 50 -tcgtcaaccactacatcgagaca-30

of full-length cDNA clones

and bar Reverse : 50 -ctgaagtccagctgccagaaac-30 ) designed

from bar gene sequence.

Stamens of mature owers at pre-anthesis were used for

cDNA library construction. The cDNA library was con-

Detection of GUS activity in transgenic plants

structed using a commercially available kit (Staratagene,

USA) according to the manufacturer s protocol. The cDNA

library was screened with 32P-labeled fragments isolated Histochemical and uorometric assay of GUS gene

expression in different organs of apple and transgenic

from the DD-PCR experiment. Positive cDNA clones were

tobacco plants were performed as previously described

subsequently sequenced.

(Jefferson et al. 1987). Brie y, analyzing organs of trans-

genic plant were breakdown and homogenized by vor-

Reverse transcriptase-polymerase chain reaction

texing with glass beads (710 1,180 nm, Sigma) in

(RT-PCR)

extraction buffer (50 mM sodium phosphate, pH 7.0, 0.1%

sodium lauryl Sarcosine, 10 mM EDTA, 0.1% Triton

The rst-strand cDNAs synthesized using the SUPER-

SCRIPTTM Preampli cation System were used as templates X-100, 10 mM -mercaptoethanol) and pelleting the cell

debris by centrifugation at 12,000 rpm for 15 min at 4 C.

for RT-PCR. The following primer pairs were used for

Protein concentration was measured using a Bio-Rad protein

speci c ampli cation of each of three AGP-encoding genes:

MdAGP1, 50 -GAATCTC GAAAGCTTGAAAGT-30 and assay kit(Bio-Rad) as described by the manufacture. Samples

50 -GACGCCAACAACAGCACCATT-30 ; MdAGP2, 50 -AG were normalized so that approximately equal amounts of

CTGGCCCTGTAGCTTCTA-30 and 50 -GAGTAAGTAGC protein were assayed in each uorescent GUS assay.

GTTATAGCTT-30 ; MdAGP3, 50 -TGCCACCCCTGCGGC

AGCTCC-30 and 50 -GGGAATATATTACACG TACACA

CCC-30 . The actin gene served as a positive PCR control Results

and was ampli ed using 50 -CGATGGCCAAGTCATCAC

AAT-30 and 50 -GACCCACCACTGAGCACGATG-30 . The Isolation of genes selectively expressed

in the anther of apples

PCR ampli cation was carried out with an initial denatur-

ation at 94 C for 5 min, 25 cycles of 94 C for 30 s, 58 C

for 30 s, and 72 C for 40 s, and then a nal 10-min In order to search for anther-speci c genes involved in

extension at 72 C. The PCR products were separated on a male gametophyte development, genes showing oral

organ-speci c expression were randomly ampli ed using

1.2% agarose gel.

123

18 Plant Cell Rep (2010) 29:15 24

Isolation of MdAGP promoter sequences and analysis

DD-PCR. Eleven bands detected only in owers was iso-

of organ-speci c expression of the MdAGP genes

lated, and its sequence was determined (Supplementary

Table 2). The identi ed fragment was subsequently used as

Expression pro les of the MdAGP genes in the leaves,

a probe to isolate corresponding cDNA clones from the

immature fruit, sepals, petals, carpels, and stamens were

ower cDNA library. Three positive clones were isolated

analyzed by RT-PCR. As shown in Fig. 4, MdAGP1, 2, and

from cDNA library screening. All contained short full-

3 genes were preferentially transcribed only in stamen. To

length cDNAs, with two consisting of a 228-bp open

investigate tissue speci city in more detail, we isolated the

reading frame (ORF) and the third possessing a 36-bp

promoter regions of the MdAGP genes by genome walking.

insertion in the middle of coding sequence designated as

The promoters of all three genes contained 305-bp con-

MdAGP1, 3 and 2, respectively (Fig. 1). These three genes

served sequence block (Fig. 1). In addition, the sequences

shared 82 90% nucleotide sequence identity.

of 50 UTR in MdAGP2 and MdAGP3 showed nearly 90%

AtAGP23 from Arabidopsis (Becker et al. 2003) and

identical sequences. Furthermore, two additional conserved

BAN102 from Chinese cabbage (Park et al. 2005) showed

blocks among these two sequences were identi ed in the

high homology to the apple AGP genes, particularly within

region upstream of both promoters as shown in Fig. 1.

the N- and C-terminal conserved domains (Fig. 2a). Both

To investigate the relationship between the 305-bp

genes shared 49% deduced amino acid sequence identity with

consensus promoter block and expression of MdAGPs, the

MdAGP1 (Fig. 2a) and were more closely related to MdAGP

consensus block was cloned and fused to the GUS reporter

than any other known Arabidopsis AGP gene (Fig. 3). Protein

gene (Fig. 5). Transient transfection of this reporter con-

sequences revealed that these factors possessed all charac-

struct using particle bombardment revealed that GUS was

teristics of classical AGPs. Both the N- and C-terminal

expressed only in the anther of apples, as indicated by the

regions were very hydrophobic, which is typical for most

appearance of a few tiny spots (Fig. 6). This construct was

known classical AGPs. The hydrophobic N-terminus con-

also introduced into tobacco by Agrobacterium-mediated

tained putative signal peptide for secretion (Fig. 2b). Many

transformation. As expected, GUS staining was observed

AGPs are known to be present at the outer surface of the

only in the anther and pollen sacs (Table 1, Fig. 7). Further

plasma membrane, and the N-terminal signal peptides are

analysis of promoter activity at different developmental

required for proper targeting of their protein products. The

stages of tobacco owering revealed that GUS staining was

presence of the N-terminal signal and cleavage at this site

not present in very early stage oral buds that were less

were predicted by SignalP 3.0 software (Bendtsen et al. 2004).

than 5 mm in length. Staining became visible when mi-

The C-terminus also contained a putative hydrophobic GPI

crospores were released from tetrads (Fig. 7). In contrast,

anchor attachment signal, as predicted by Big-PI (Eisenhaber

constructs lacking the consensus promoter block exhibited

et al. 2003). The most prominent feature of the classical AGPs

no GUS activity (data not shown). To investigate the level

was the presence of Pro-, Ala-, Ser-, and Thr-rich regions

and temporal pattern of GUS accumulation in transgenic

between the N- and C-terminal signal sequences. These four

plants more precisely, pollen from different developmental

residues made up approximately 50% of the deduced amino

stages was isolated. Figure 8 demonstrates that GUS

acid sequence; excluding both signal sequences, this per-

activity was not observed during early pollen development

centage increased to 86% (Fig. 2b). We have designated the

but was highest at stage 7 and also observed at stages 5 and

two identically-sized homologous genes MdAGP1 (GenBank

9 (Fig. 8). Taken together, these results indicate that

accession no. AF403122) and MdAGP3 (AF403124). The

MdAGP genes are expressed selectively in developing

larger one has been designated MdAGP2 (AF403123).

microspores and pollen grains of tobacco, and that the 305-bp

consensus promoter blocks are essential for their expression.

Taken together, these results indicate that the MdAGP genes

are selectively expressed in developing microspores and

pollen grains of tobacco, and the 305 bp consensus promoter

blocks are essential for expression of AGPs.

Expression of RIP under MdAGP promoter induced

male-sterility in tobacco

Fig. 1 Organization of genomic sequences of the three MdAGP

genes. Arrow-shaped boxes indicate coding sequences (shown 50 to To test the applicability of apple anther-speci c promoters

30 ). Filled and gray boxes represent approximately 305 bp consensus

in other species, we transformed tobacco with RIPs con-

promoter region and homologous sequence blocks, respectively.

trolled by the MdAGP3 promoter. The RIP introduced

Hatched box in the MdAGP2 coding region represents the 36-bp

transgenic plants were con rmed by the presence of bar gene

insertion. The vertical lines connect homologous sequence blocks

123

Plant Cell Rep (2010) 29:15 24 19

Fig. 2 Sequence alignment between apple, Arabidopsis, and Chinese substitutions (colon), and semi-conserved substitutions (dot) are

cabbage AGP-encoding genes and functional domain analysis of indicated. b Alignment of the primary sequences of MdAGP1,

MdAGP genes. a Alignment of deduced MdAGP amino acid MdAGP2, and MdAGP3. PAST denotes the ProAlaSerThr motif. N-

sequences with AtAGP23 from Arabidopsis and BAN102 from and C- terminal signal peptides are highlighted with rectangular

Chinese cabbage. Identical amino acids (asterisk), conserved boxes

Fig. 4 RT-PCR analysis of tissue-speci c expression of MdAGP.

Actin was included as a positive control to verify sample integrity and

to show the relative amount of synthesized cDNAs. Le leaf, Fr

immature fruit, Se sepal, Pe petal, Ca carpel, St stamen

from their reproductive organs, these transgenic tobaccos

were identical to wild type tobaccos. As we had expected,

this result indicates that the expression of 305 bp conserved

Fig. 3 Phylogenetic relationship between MdAGP and AGP-encod-

ing genes from other species. A phylogenic tree showing genetic block of MdAGP is limited in pollen.

distances between MdAGPs with known Arabidopsis AGPs. The tree

was produced by using ClustalX and TreeView softwares. The scale

bar indicates 0.1 substitutions per site. The GenBank accession

Discussion

numbers of Arabidopsis AGPs are listed in Schultz et al. (2002)

Isolation of putative AGP-encoding genes from apple

(data not shown). The RIPs are cytotoxic proteins, which

have been utilized in combination with tissue-speci c pro-

In this study, we isolated three putative AGP-encoding

moters to ablate cells in speci c tissues (Cho et al. 2001). In

genes that were speci cally expressed in the male organ of

the transgenic tobacco transformed with the RIP gene driven

apple owers. Although further biochemical analysis will

by the MdAGP3 promoter, 6 out of 9 transgenic plants

be required to con rm the functional identities of these

exhibited a male-sterile phenotype similar to those of Cho s

genes, the information obtained from their primary amino

experiment (Cho et al. 2001). The presence of the RIP

acid sequences make us reasonably sure of their identities

transgene dramatically affected ower development. The

as AGP-coding genes. The putative proteins possess a

lengths of the petal and anther lament were shorter than

relatively short backbone containing a PAST-rich region, a

those of wild type plants (Fig. 9a), and the amount of pollen

salient feature of all known AGP-coding genes. MdAGP1

grains was reduced. Examination of pollen grains from this

and MdAGP3 encode proteins of only 75 residues in

male-sterile line using scanning electron microscopy

length. Thus, these gene products can be classi ed as

revealed that all pollen grains were severely deformed

AG-peptides according to Schultz et al. (2002), who

unlikely normal plant (Fig. 9b, c). Seeds could be produced

de ne AG-peptides as being between 55 and 75 residues in

by the transformant through cross-pollination with wild type

length. All AG-peptides identi ed from Arabidopsis have

pollen, showing that only male organ was disabled. Aside

123

20 Plant Cell Rep (2010) 29:15 24

Fig. 5 Schematic diagrams of pMdAGP3-pBI101 a and pMdAGP3- Nco1 b restriction sites to facilitate directional cloning. The RIP gene

pCAMBIA3301 b constructs used for analysis of promoter from were PCR ampli ed using appropriate primer pairs containing Nco1

MdAGP3 genes, respectively. The upstream regulatory region (-352 and and SacI. The ampli ed products were cloned separately in binary

to -6 bp upstream of the start codon) were PCR ampli ed using vectors pBI101 a and pCAMBIA3301 b at the corresponding sites.

appropriate primer pairs containing Hind III and SacI a or HindII and RB and LB indicate right and left borders of T-DNA

Table 1 Effect of MdAGP3 promoter sequences on GUS reporter

expression in tobacco

Callus Leaf Root Flower

Sti Sty Ova Ant Fil Pol

No promoter - - - - - - - - -

35S promoter ? ? ? ? ? ? ? ? ?

MdAGP3 - - - - - - ? - ?

? observed GUS expression, - no visible GUS expression, Sti

stigma, Sty style, Ova ovary, Ant anther, Fil lament, Pol pollen

AGPs in Arabidopsis (Fig. 2). These two genes, which are

both selectively expressed in pollen were also contained

homologous sequence blocks upstream of the promoter

regions and in the 50 and 30 UTRs (Lalanne et al. 2004;

Park et al. 2005). These facts may imply that these two

closely related AGP-coding genes might be orthologs with

similar functions in pollen development since both Chinese

Fig. 6 Histochemical analysis of GUS activity in apple. GUS

cabbage and Arabidopsis belong to the Brassicaceae

activities in several organs of apple were determined after bombard-

ing pMdAGP3::GUS reporter constructs (a sepal, b leaf, d anther). family. Apples, which belongs to the Rosaceae family, are

The observed GUS activities were indicated by the white arrows.

distantly related to the Brassicaceae family. Thus, the

Negative control of GUS activity was shown in the anther of

structural homology of the MdAGPs with BAN102 and

bombarded with the promoterless::GUS reporter construct pBI101 (c)

AtAGP23 as well as the pollen-speci c expression of these

proteins suggests that MdAGP1, MdAGP2, and MdAGP3

been shown to contain a N-terminal signal and at least two

might have similar functional roles of BAN102 and

consecutive Ala-Pro or Ser-Pro motifs. For example, AtAGP23

AtAGP23. In addition, the conservation of sequence blocks

and BAN102, both of which showed relatively high homology

in the upstream promoter region among the three MdAGP

with the MdAGPs, are composed of only 66 residues, and they

genes indicates that these homologs may have originated

contain two consecutive Ala-Pro motifs. All known Arabid-

from triplication of the ancestral genomic sequence.

opsis AG-peptides, except for AtAGP23, contain three con-

secutive motifs. The nding that MdAGPs have two

consecutive Ala-Pro and Thr-Pro/Val-Pro motifs suggests that The anther/pollen-speci c activity of MdAGP

the two consecutive Ala-Pro or Ser-Pro motifs in the Arabid- and its promoters show known pollen-speci c

opsis AG-peptide may not be strictly conserved in other species. regulatory elements

AtAGP23 and BAN102 have 88% deduced amino acid

sequence identity, which makes then highly homologous By monitoring the localization of GUS reporter gene

considering the general low homology among known activity, the stage- or tissue-speci c regulation of genes can

123

Plant Cell Rep (2010) 29:15 24 21

Fig. 7 MdAGP3 promoter activity analysis using transgenic tobac- microspores. d. Anther carrying mature pollen grains before dehis-

cos. Tobacco was transformed with GUS reporter constructs driven by cence. an anther, ca carpel, co connective, en endothecium, ep

the MdAGP3 promoter and stained for GUS (blue). Representative epidermis, pe petal, ps pollen sac, st stomium, ta tapetum, te tetrads,

bright- eld images are shown. a Early stage ower buds 3 4 mm in vb vascular bundle

length. b Anther containing a tetrad. c Anther with developing

be identi ed. For example, the 50 anking sequence of carrying LGC1 promoter-diphtheria toxin A chain con-

Arabidopsis alpha tublin fused to GUS coding region struct showed sterile and aborted pollen (Singh et al. 2003).

showed the localization of GUS in post-miotic pollen In this study, the speci c expression of MdAGP was

grains (Carpenter et al. 1992). Transgenic tobacco plants evaluated in apple and heterologous tobacco system. To

123

22 Plant Cell Rep (2010) 29:15 24

characterize the regulatory regions of MdAGP promoter,

the conserved 305 bp region was cloned and fused to GUS

reporter gene, then resulting construct was transiently

expressed or transformed into apple and tobacco, respec-

tively. Both plants showed anther- or pollen-speci c GUS

activity. However, the other constructs exclude 305 bp

consensus sequences showed no GUS activity. The dif-

ferent GUS expression patterns between apple and tobacco

may explained by two ways. Apple and tobacco may have

different regulatory mechanisms for expression of MdAGP

promoter region. To investigate the level and temporal

pattern of GUS accumulation in transgenic tobacco plants

more precisely, a series of pollen development stage was

isolated. GUS activity was not observed at early stage of

pollen development, however, late stage of pollen devel-

Fig. 8 The speci city of 305 bp conserved upstream sequences of opment showed GUS activity.

MdAGP3 promoter driven GUS activity in different pollen develop- Promoters and cis-acting regulatory elements of genes

ment stage of tobacco ower buds. For GUS uorometric assay,

that express in anther- or pollen-speci c manner give

20-lg proteins from each development stage of pollens were used

opportunity for target expression of desirable genes in

for enzyme assay. Mean of the speci c Gus activity (in nmol of

4 MU/mg protein/h) for each dissected anther of three transgenic speci c stage of male gametophyte development. For this

plants of four independent transgenic lines is shown. Standard error purpose, several anther/pollen-speci c promoters have

bars are also shown. The stage 1, 3, 5, 7 and 9 represent the length of

been identi ed in various species (Guerrero et al. 1990;

ower bud as 8, 14, 20, 28, 43 mm, respectively [see detail

van Tunen et al. 1990; Twell et al. 1991; John and

information in Koltunow et al. (1990)]

Fig. 9 Effect of MdAGP3

promoter-driven expression of

RIP on tobacco phenotypes.

a Open owers. Left wild

type, Right transgenic tobacco.

b, c Scanning electron

micrographs of pollen grains

from wild type (b) and

transgenic tobacco (c)

123

Plant Cell Rep (2010) 29:15 24 23

Du H, Simpson R, Clarke A, Bacic A (1996) Molecular character-

Petersen 1994; Tsuchiya et al. 1994; Eyal et al. 1995;

ization of a stigma-speci c gene encoding an Arabinogalactan-

Hamilton et al. 1998; Rogers et al. 2001; Gomez et al.

protein (AGP) from Nicotiana alata. Plant J 9:313 323

2004). In this study, we found one pollen-speci c cis- Eisenhaber F, Eisenhaber B, Kubina W, Maurer-Stroh S, Neuberger

acting regulatory element AGAAA (-41 bp position) G, Schneider G, Widpaner M (2003) Prediction of lipid

posttranslational modi cations and localization signals from

from searching of sequences upstream to the transcription

protein sequences: big-Pi NMT and PTS1. Nucleic Acids Res

start site (?1) of MdAGP. Same cis-acting regulatory

31:3631 3634

element also observed in other plant species. For example, Eyal Y, Curie C, McCormick S (1995) Pollen speci city elements

the activity of pollen-speci c LAT52 was dependent on reside in 30 bp of the proximal promoters of two pollen-

expressed genes. Plant Cell 7:373 384

two regulatory elements, AGAAA and TCCACCATA

Gerster J, Allard S, Robert L (1996) Molecular characterization of

(Bate and Twell 1998). In promoter of OSIPA, the

two Brassica napus pollen-expressed genes encoding putative

AGAAA sequence were observed at -424 and -1,425 bp Arabinogalactan proteins. Plant Physiol 110:1231 1237

position promoter and these may have an important role in

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

drives anther-speci c gene expression in different plant species.

imparting the pollen-speci c expression (Gupta et al.

Planta 219:967 981

2007). Above results indicate that the AGAAA element in

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

MdAGP promoter may have important role for anther/ JP (1990) Promoter sequences from a maize pollen-speci c gene

pollen-speci c expression. direct tissue-speci c transcription in tobacco. Mol Gen Genet

224:161 168

Furthermore expression of RIP gene under the control of

Gupta V, Khurana R, Tyagi AK (2007) Promoters of two anther-

the consensus promoter region of the MdAGP genes pro-

speci c genes confer organ-speci c gene expression in a

duced male-sterile tobacco. This result indicate that the stage-speci c manner in transgenic systems. Plant Cell Rep

305 bp consensus promoter block of the MdAGP genes 26:1919 1931

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

retains the ability to direct anther-speci c expression in a

pollen-speci c promoter contains separable pollen-speci c and

distantly related heterologous system such as tobacco.

quantitative elements. Plant Mol Biol 38:663 669

Further detail analysis of availability of this regulatory Hanson MR, Benolila S (2004) Interactions of mitochondrial and

element into various plants will be tried. nuclear genes that affect male gametophyte development. Plant

Cell 16(Suppl):S154 S169

Hoekema A, Hirsch P, Hooykaas P, Schilperoort R (1983) A

Acknowledgments This work was supported by a grant (Code

binary vector strategy based on separation of vir- and T-region

200***********) from BioGreen21 program, Rural Development

of the Agrobacterium tumefaciences Ti-plasmid. Nature 303:

Administration, Republic of Korea.

179 181

Hrazdina G (1994) Genetic engineering of Mclntosh apple to

prevent softening during storage. Nk Fruit Q Winter:9

Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-

References glucuronidase as a sensitive and versatile gene fusion marker in

higher plants. EMBO J 6:3901 3907

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

Acosta-Garcia G, Vielle-Calzada J (2004) A classical arabinogalactan

pollen-speci c polygalacturonase mRNA: tissue and temporal

protein is essential for the initiation of female gametogenesis in

speci city of its promoter in transgenic tobacco. Plant Mol Biol

Arabidopsis. Plant Cell 16:2614 2628

26:1989 1993

Aldwinckle H (1993) Improvement of New York apple varieties and

Knox P, Linstead PJ, Peart J, Cooper C, Roberts K (1991)

root stocks by genetic engineering. NY Fruit Q Winter:3

Developmentally regulated epitopes of cell surface arabinoga-

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

lactan proteins and their relation to root tissue pattern formation.

promoter: pollen-speci c transcription is developmentally regu-

Plant J 1:317 326

lated by multiple stage-speci c and co-dependent activator

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

elements. Plant Mol Biol 37:859 869

(1990) Different temporal and spatial gene expression patterns

Becker JD, Boavida LC, Carneiro J, Haury M, Feijo JA (2003)

occur during anther development. Plant Cell 2:1201 1224

Transcriptional pro ling of Arabidopsis tissue reveals the unique

Lalanne E, Honys D, Johnson A, Borner G, Lilley K, Dupree P,

characteristics of the pollen transcriptom. Plant Physiol 133:

Grossniklaus U, Twell D (2004) SETH1 and SETH2, two

713 725

components of the glycosylphosphatidylinositol anchor biosyn-

Bendtsen J, Nielsen H, Heijne G, Brunak S (2004) Improved

thetic pathway, are required for pollen germination and tube

prediction of signal peptides: SignalP 3.0. J Mol Biol 340:

growth in Arabidopsis. Plant Cell 16:229 240

783 795

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

Brown SK (1995) Genetic improvement of apple: the roles of plant

Induction of male sterile cabbage using a tapetum-speci c

breeding and biotechnology. NY Fruit Q 3:2 5

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

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

Rep 22:268 273

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

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

Plant Cell 4:557 571

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

Cho H, Kim S, Kim M, Kim B (2001) Production of transgenic male

ribonuclease gene. Nature 347:737 741

sterile tobacco plants with the cDNA encoding a ribosome

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

inactivating protein in Dianthus sinensis L. Mol Cell 11:326 333

pollen-preferential gene, BAN102 \ from Chinese cabbage.

Daniell H (2002) Molecular strategies for gene containment in

Plant Cell Rep 24:663 670

transgenic crops. Nature Biotechnol 20:581 586

123

24 Plant Cell Rep (2010) 29:15 24

Qiu X, Wu Y, Du S, Erickson L (1997) A new Arabinogalactan glycosylphosphatidylinositol-anchored arabinogalactan-protein.

protein-like gene expressed in the pollen of alfalfa. Plant Sci Physiol Plant 120:319 327

124:41 47 Sung S, Yu G, An G (1999) Characterization of MdMADS2, a

Rogers HJ, Bate N, Combe J, Sullivan J, Sweetman J, Swan C, member of the SQUAMOSA subfamily of genes, in apple. Plant

Lonsdale DM, Twell D (2001) Functional analysis of cis- Physiol 120:969 978

regulatory elements within the promoter of the tobacco late Tsuchiya T, Toriyama K, Ejiri S, Hinata K (1994) Molecular

pollen gene g10. Plant Mol Biol 45:577 585 characterization of rice genes speci cally expressed in the anther

Roque E, Gomez MD, Ellul P, Wallbraun M, Madueno F, Beltran J-P, tapetum. Plant Mol Biol 26:1737 1746

Canas LA (2007) The PsEND1 promoter: a novel tool to produce Twell D, Yamaguchi J, Wing RA, Ushiba J, McCormick S (1991)

genetically engineered male-sterile plants by early anther Promoter analysis of genes that are coordinately expressed

ablation. Plant Cell Rep 26:313 325 during pollen development reveals pollen-speci c enhancer

Schultz CJ, Rumsewicz MP, Johnson KL, Jones BJ, Gaspar YM, sequences and shared regulatory elements. Genes Dev 5:496

Bacic A (2002) Using genomic resources to guide research 507

directions. The arabinogalactan protein gene family as a test van Hengel A, Roberts K (2003) AtAGP30, an arabinogalactan-

case. Plant Physiol 129:1448 1463 protein in the cell walls of the primary root, plays a role in root

Singh M, Bhalla PL, Xu H, Singh MB (2003) Isolation and regeneration and seed germination. Plant J 36:256 270

characterization of a owering plant male gametic cell-speci c van Hengel A, Barber C, Roberts K (2004) The expression patterns of

promoter. FEBS Lett 542:47 52 arabinogalactan-protein AtAGP30 and GLABRA2 reveal a role

Sun W, Kieliszewski MJ, Showalter AM (2004a) Overexpression of for abscisic acid in the early stages of root epidermal patterning.

tomato LeAGP-1 arabinogalactan-protein promotes lateral Plant J 39:70 83

branching and hampers reproductive development. Plant J van Tunen AJ, Mur LA, Brouns GS, Rienstra J-D, Koes RE, Mol

40:870 881 JNM (1990) Pollen- and anther-speci c chi promoters from

Sun W, Zhao Z, Hare MC, Kieliszewski MJ, Showalter AM petunia: tandem promoter regulation of the chiA gene. Plant Cell

(2004b) Tomato LeAGP-1 is a plasma membrane-bound, 2:393 401

123

Contact this candidate