The transcript composition of egg cells changes significantlyfollowing fertilization in wheat (Triticum aestivum L.)

Stefanie Sprunck1, Ute Baumann2, Keith Edwards3, Peter Langridge2 and Thomas Dresselhaus1,*

1Developmental Biology and Biotechnology, Biocenter Klein Flottbek, University of Hamburg, Ohnhorststrasse 18, D-22609

Hamburg, Germany,2Australian Center for Plant Functional Genomics, University of Adelaide,Waite Campus, Hartley Grove, SA 5064, Australia, and3Department of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK

Received 1 November 2004; accepted 23 November 2004.*For correspondence (fax þ49 40 42816 229; e-mail dresselh@botanik.uni-hamburg.de).

Summary

Here, we report the transcript profile of wheat egg cells and proembryos, just after the first cell division.

Microdissected female gametophytes of wheat were used to isolate eggs and two-celled proembryos to

construct cell type-specific cDNA libraries. In total, 1197 expressed sequence tags (ESTs) were generated.

Analysis of these ESTs revealed numerous novel transcripts. In egg cells, 17.6% of the clustered ESTs

represented novel transcripts, while 11.4% novel clusters were identified in the two-celled proembryo.

Functional classification of sequences with similarity to previously characterized proteins indicates that the

unfertilized egg cell has a higher metabolic activity and protein turnover than previously thought. Transcript

composition of two-celled proembryos was significantly distinct from egg cells, reflecting DNA replication as

well as high transcriptional and translational activity. Several novel transcripts of the egg cell are specific for

this cell. In contrast, some fertilization induced novel mRNAs are abundant also in sporophytic tissues

indicating a more general role in plant growth and development. The potential functions of genes based on

similarity to known genes involved in developmental processes are discussed. Our analysis has identified

numerous genes with potential roles in embryo sac function such as signaling, fertilization or induction of

embryogenesis.

Keywords: egg cell, fertilization, embryogenesis, genomics, wheat.

Introduction

Fertilization leads to a remarkable biologic phenomenon:

the process of embryogenesis. Once activated, the ferti-

lized egg cell starts to divide and progeny cells undergo a

series of specification and differentiation steps, finally

generating a new organism consisting of different cell

types, tissues and organs. The molecular mechanisms of

fertilization and early embryogenesis have been most

intensively studied in animals. In most of the species

studied, maternally stored mRNAs of the egg were iden-

tified as involved in the establishment of embryonic axes,

diversification of cell types and morphological changes

during early embryogenesis (Angerer and Angerer, 2000;

Mohr et al., 2001; Nishida, 1997; Pellettieri and Seydoux,

2002; St Johnston, 1995; Wylie et al., 1996). The transition

from maternal to zygotic/embryonic control of gene

expression (ZGA/EGA) is the first major transition that

occurs following fertilization. Thereafter, transcript profiles

dynamically change throughout embryogenesis (Baugh

et al., 2003; Makabe et al., 2001). Nevertheless, the time

point of transition appears to be species-dependent: ZGA/

EGA occurs at the two-celled stage in mice (Adenot et al.,

1997), the four- to eight-celled stage in cows and humans

and the eight- to 16-celled stage in sheep and rabbits

(Kanka et al., 2003; Nothias et al., 1995). In Caenorhabditis,

the first embryonic transcription is detected in blastomeres

at the four-celled stage (Edgar et al., 1994), while major

embryonic transcription in zebra fish and Xenopus occurs

after 10–12 cell cycles at the beginning of the midblastula

transition (Kane and Kimmel, 1993; Nakakura et al., 1987;

Newport and Kirschner, 1982).

Compared with animals, little is known about transcripts

stored in egg cells of flowering seed plants (angiosperms)

660 ª 2005 Blackwell Publishing Ltd

The Plant Journal (2005) 41, 660–672 doi: 10.1111/j.1365-313X.2005.02332.x

and activation of the zygotic genome after fertilization.

Here, the egg cell is part of the female gametophyte

(embryo sac) which consists of four different cell types: the

egg cell, two synergids, the large central cell and some

antipodal cells (Yadegari and Drews, 2004). During double

fertilization, a phenomenon unique to angiosperms, the

two female gametes (egg and central cell) each fuse with

one sperm cell delivered by the pollen tube. They then

develop into the embryo and endosperm, respectively. As

the embryo sac is typically surrounded by sporophytic

tissues of the ovule and ovary, access to female gametes

and very early embryo and endosperm stages is ham-

pered. Gametic and zygotic transcripts of seed plants are

thus poorly represented in current databases of expressed

sequence tags (ESTs) although some have been generated

through sequencing from cDNA libraries produced from

whole floral tissues. However, the use of these complex

tissues results in under representation of genes expressed

at low levels and in only one or a few cell types. Methods

for the isolation of single cells from the embryo sac of

maize, barley, wheat and rice have been described (Holm

et al., 1994; Kranz et al., 1991; Kumlehn et al., 1998; Zhao

et al., 2000) and the generation of cDNA libraries

from maize and wheat egg cells have been reported

(Dresselhaus et al., 1994; Kumlehn et al., 2001), but our

knowledge of transcript compositions of eggs, zygotes and

proembryos is still limited. Only a few genes have been

reported to be expressed in egg cells of maize and Arabid-

opsis, some of which alter their expression pattern after

fertilization (e.g. Cordts et al., 2001; Dresselhaus et al.,

1999a; Haecker et al., 2004; Heuer et al., 2001; Perry et al.,

1996). Analysis of cell cycle regulatory genes and ribosomal

genes in maize indicated that plant zygotic gene activation

occurs earlier compared with the animal systems investi-

gated so far (Dresselhaus et al., 1999b1 ; Sauter et al., 1998).

To develop a broad picture of the genes involved in the

development and function of female gametophyte cells,

we used a genomics-based approach involving the gen-

eration of ESTs from cell type-specific cDNA libraries.

Applying an experimental technique to microdissect

wheat embryo sac cells (Kumlehn et al., 1998), we isola-

ted single cells of the unfertilized embryo sac as well as

defined stages of zygotes and early embryos. We gener-

ated stage-specific cDNA libraries for the egg cells and

the proembryos, which have completed the first zygotic

cell division. Randomly selected clones from these librar-

ies were sequenced and analyzed. Results obtained from

EST analyses, including functional classification and

comparative studies on the transcript composition of the

egg cell and two-celled proembryo, as well as expression

studies of selected genes are shown. The function of

putative regulators of various processes related to

double fertilization and embryonic genome activation are

discussed.

Results

Isolation and cultivation of single cells from the female

gametophyte of wheat

In order to obtain single gametes and zygotes of defined

developmental stages, an experimental system based on a

microdissectionmethod to isolate embryo sac cells of wheat

(Kumlehn et al., 1998) was established. The isolation pro-

cedure resulted in intact and viable cells free of maternal

tissues (Figure 1c–h). Egg cells and early zygotes were iso-

lated mechanically from ovaries without using cell wall-

degrading enzymes. The isolation procedure is rapid and

helps in minimizing cellular responses triggered by enzy-

matic cell wall degradation of the embryo sac and the sur-

rounding nucellus cells. Wheat ovary tips were dissected at

the micropylar region, either at the time of flowering using

previously emasculated spikelets (Figure 1a), or 4–6 h after

hand-pollination (hap). Ovular tissue containing the egg

apparatus (Figure 1b) was excised in 0.55 M mannitol solu-

tion and egg cells (Figure 1c) or zygotes (Figure 1d) were

released from the ovule tips using fine glass needles. Col-

lected cells were either directly frozen for later mRNA

isolation or kept in mannitol to monitor zygotic embryo

development in vitro. The nucleus of the mature egg cell is

surrounded by dense cytoplasm and typically contains one

large nucleolus (Figure 1c; arrow). Around 13 hap zygotes

were clearly distinguished from unfertilized egg cells by the

occurrence of a second nucleolus (Figure 1d). Around

22 hap the first zygotic cell division was observed (Fig-

ure 1e) and two-celled proembryos were visible 23–24 hap

(Figure 1f). Isolated zygotes autonomously undergo the first

cell divisions, without adding supplements such as plant

hormones, conditionedmedium or feeder cells (Figure 1f,g).

The microdissection technique described here was also

successful for isolating in vivo embryos as early as

2–3 days after fertilization (Figure 1h), giving the possibility

of analyzing gene expression in very young wheat

embryos.

Bioinformatic analysis of ESTs

Unfertilized egg cells and two-celled proembryos were used

to generate cDNA libraries. Subsequently, single-run partial

sequencing of randomly selected cDNA clones (ESTs) was

performed. As cDNAs were cloned in a non-directional

manner, both 5¢ and 3¢ read sequenceswere generated. After

DNA sequencer trace data passed an automated cleanup

pipeline, a total of 1197 ESTs were used for analysis. The

sequences of each library were clustered and assembled

into contigs. Thus, 735 ESTs from the egg cell form 404

independent clusters including 310 singletons (77%), while

462 sequences from the two-celled proembryo generated

298 clusters, containing 236 singletons (79%).

Transcriptome of wheat eggs and proembryos 661

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 41, 660–672

The consensus sequences of the clusters were used for

BLASTN and BLASTX searches at the NCBI non-redundant

database (nr), dbEST and SwissProt database. In addition,

we performed BLASTN searches against the TIGR-assembled

wheat genes (Quackenbush et al., 20012 ). BLASTN searches

against the NCBI database category of non-mouse and non-

human ESTs resulted in 629 egg cell ESTs (333 clusters) and

418 two-celled proembryo ESTs (264 clusters) matching

significantly to annotated ESTs mainly generated from

different tissues of wheat, barley or rice (Figure 2, NCBI

dbEST Poaceae). Among these, only 276 ESTs represent

genes present in both, the egg cell and the two-celled

proembryo. Interestingly, 106 egg cell ESTs (71 clusters) and

44 proembryo ESTs (34 clusters) did not match annotated

ESTs and were thus considered as ‘novel’ transcripts. Seven

of these ‘novel’ transcripts were common to the egg cell and

early embryo (Figure 2). In total, a surprisingly small number

of 23.6% of all ESTs (representing 5.4% of all clusters) were

present in both egg cell and two-celled proembryo.

Clusters were further categorized according to their

BLASTX and BLASTN search results into (i) transcripts

encodingproteinsof knownfunction (similar to characterized

proteins), (ii) transcripts of unknown function, present in

dbEST only, (iii) transcripts encoding hypothetical proteins

[similar to uncharacterized genes, such as predicted open-

reading frames (ORFs), unknown proteins, ‘similar to’ and

‘-like’ proteins] showing additional similarity to published

ESTs or cDNAs, (iv) transcripts encoding hypothetical pro-

teins (similar to computer-predictedORFs) without similarity

to any EST or cDNA and (v) novel transcripts of unknown

function (Figure 3). Classification of sequences derived from

the egg cell revealed that 177 of the 404 clusters (43.8%)

represent characterized proteins, while the remaining 56.2%

represent genes of unknown function. Among these, 69

clusters (17.1%) showed a match to annotated ESTs only

(Figure 3a). Significant similarities to hypothetical proteins

predicted from the Arabidopsis or rice genome sequences

were identified for 98 egg cell clusters (24.2%). Among these,

stigma

micropyle

(a) (b) (c) (d)

(e) (f) (g) (h)

Figure 1. Isolation of egg cells and zygotes from the female gametophyte of wheat and the development of zygotes into proembryos in culture.

(a) Mature unpollinated ovary, the arrow indicates direction of section.

(b) Isolated ovule tip showing the egg apparatus consisting of egg cell (arrow) and two flanking synergids (open triangles).

(c) Egg cell containing one nucleolus (arrow).

(d) Two zygotes, 13 hap, each containing two nucleoli (arrows).

(e) Cytokinesis of first zygotic cell division, appearing 20 hap.

(f) Two-celled proembryo, 22 hap.

(g) Four-celled proembryo, 30 hap.

(h) Isolated embryo, 3 days after pollination (bars: 25 lm; hap, hours after pollination).

2-celled proembryoEgg cell

NCBI dbEST Poaceae(>1.5 × 106)

284487

102 41

276

7

Figure 2. Distribution of 735 ESTs derived from egg cells (blue) and 462 ESTs

from the two-celled proembryos (red) among each other and the Poaceae EST

database (yellow), containing more than 1.5 Mio. ESTs (as of March 2004).

662 Stefanie Sprunck et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 41, 660–672

11 hypothetical proteins (2.7%) are novel as they represent

computer-predicted ORFs with no match to annotated ESTs

ormRNAsequences.Moreover, 60 clusters (14.9%) represent

completely novel transcripts lacking any similarity to anno-

tated genes, ESTs or proteins. Taken together, 17.6% of all

clusters from the egg cell can be considered as ‘novel.’

The same classificationwas conducted for the 298 clusters

derived from the two-celled proembryo. Here, 163 clusters

(54.7%) significantly matched characterized proteins (Fig-

ure 3b). Moreover, 47 clusters (15.8%)matched to annotated

ESTs only, while 54 clusters (18.1%) of the two-celled

proembryo ESTs showed significant similarities to hypo-

thetical proteins of Arabidopsis or rice. All clusters with

similarity to predicted proteins revealed additional matches

in the Poaceae dbEST. Finally, 34 clusters (11.4%) represent

‘novel’ sequences.

Composition and dynamics of the egg cell and proembryo

transcriptome

Independent clusters derived from egg cells and pro-

embryos were sorted according to decreasing cluster size.

The 20 largest clusters of egg cell and two-celled proembryo

sequences are shown in Table 1a,b, respectively. Largest

egg cell clusters are quite distinct compared with those from

the two-celled proembryo.

Egg cell. The largest egg cell cluster is formed by ESTs

showing sequence similarity to ECA1, a cDNA isolated from

barley microspore cultures (Vrinten et al., 1999). Although

this is the largest cluster in egg cells (6.67% of ESTs;

Table 1a), TaECA1-like ESTs were not identified among the

two-celled proembryo ESTs. The second largest egg cell

cluster shows similarity to cyclophilin A1 (CyP1; Cluster EC-

2). In general, ESTs encoding CyP1 are highly represented in

several wheat cDNA libraries generated from different tissue

types (UniGene Cluster Ta.1078; NCBI). In the egg cell, 4.6%

of ESTs encode CyP1. This number decreases to 1% in the

two-celled proembryo (Table 1b; Cluster 2C-1524). The third

largest cluster of the egg cell is formed by ESTs encoding a

cytosolic ascorbate peroxidase (Cluster EC-3). However,

similar ESTs were not identified among the largest cluster of

the two-celled proembryo. Transcripts encoding histones

and ribosomal proteins were less strongly represented

among the most abundant classes of egg cell ESTs, com-

paredwith the two-celled proembryo. Nevertheless, the core

histones H4 (Cluster EC-6) and H2A (Cluster EC-22) as well as

the histone variant H3.3 (Cluster EC-9) are present (Table 1a).

Several ESTs with similarity to ubiquitin and ubiquitin-like

proteins are found among the most prevalent transcripts

(Clusters EC-5, EC-7, EC-16, EC-17, EC-57). In particular, ESTs

with similarity to rice ubiquitin-like smt3 protein (Cluster EC-

17) and barley ubiquitin-tail fusion protein 1 (Cluster EC-7)

are abundant in egg cells, but missing entirely in ESTs

derived from the two-celled proembryos. The same was

found for ESTs with similarity to small heat shock proteins

(sHSPs), sHSP16.9 and sHSP17.9: transcripts encoding these

sHSPs are abundant in the egg cell (Clusters EC-10, EC-430)

but not in the two-celled proembryo. We also found novel

sequences among the largest clusters of the egg cell: four

clusters show similarity to predicted hypothetical proteins

from rice and Arabidopsis (Clusters EC-4, EC-16, EC-18,

EC-57), while Cluster EC-12 represents a novel gene.

Two-celled proembryo. The largest clusters of the two-cel-

led proembryo are generated by ESTs encoding the histones

H4, H3 and H2B-6 (Table 1b; Clusters 2C-1520, 2C-1521,

2C-1522). Another seven clusters represent various ribo-

somal proteins (Clusters 2C-1523, 2C-1528, 2C-1529,

2C-1531, 2C-1532, 2C-1537, 2C-1539). In particular, tran-

scripts of the ribosomal protein L39 (RPL39; Cluster 2C-1523)

are abundant in the two-celled proembryo. Other transcripts

encode a 12-oxophytodienoic acid reductase involved in

jasmonic acid synthesis (Cluster 2C-1527), and a putative

calmodulin-binding protein (Cluster 2C-1527). Among the

most prevalent transcripts are also four clusters where no

BLASTX hit was found (Clusters 2C-1525, 2C-1526, 2C-1533,

2C-1566).

(a) Egg cell (404 clusters) (b) 2-celled proembryo (298 clusters)

Similar to characterized proteins

Similar to sequences present in dbEST only

Novel transcripts

Similar to hypothetical proteins and ESTs

Similar to hypothetical proteins only

15.8%

54.7%

18.1%

11.4%17.1%

43.8%

21.5%

2.7%

14.9%

Figure 3. Categorization of contigs derived

from the egg cell (a) and two-celled proembryo

(b) cDNA libraries. In the egg cell, 60 contigs

(14.9%) represent novel transcripts. In addition,

11 contigs (2.7%) show similarity to predicted

proteins but no similarity to any expressed

sequence. In the two-celled proembryo, 34 con-

tigs (11.4%) represent novel transcripts, showing

nomatch to published sequences. The portion of

sequences with similarity to characterized pro-

teins is significantly higher in the two-celled

proembryo (54.7%) compared with the egg cell

(43.8%).

Transcriptome of wheat eggs and proembryos 663

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 41, 660–672

Comparative functional classification of ESTs encoding

characterized proteins

Sequences encoding characterized proteins were further

classified into functional groups to compare the status of the

unfertilized egg with that of the two-celled proembryo. In

total, 359 clusters (51%) of both cell types containing 753

ESTs revealed significant matches to characterized proteins,

to proteins with putative functions or to proteins with known

functional domains. We defined 11 major functional categ-

ories as shown in Figure 4. However, several proteins can-

not be classified strictly into one of these categories because

their biologic role is also relevant to another functional

group.

Table 1 The 20 largest independent contigs derived from egg cells (a) and two-celled proembryos (b), sorted according to cluster size.Functional annotations of BLASTX results are given where significant similarity to characterized genes or proteins were found

Cluster ID Blast · resulta Accession Organism Number of ESTs

(a) Egg cellEC-1 ECA1-like gene familyb AAF23356 H. vulgare 49EC-2 Cyclophilin A-1 AAK49426 T. aestivum 34EC-3 Ascorbate peroxidase CAA06996 H. vulgare 19EC-6 Histone H4 P59258 T. aestivum 15EC-4 Hypothetical protein BAB44057 O. sativa 14EC-5 Polyubiquitin Q9VZL4 D. melanogaster 13EC-7 Ubiquitin/ribosomal protein CEP52 S33633 O. sativa 13EC-57 Hypothetical proteinc BAC83160 O. sativa 12EC-9 Histone H3.3 P59169 A. thaliana 10EC-10 16.9 kDa class I HSP P12810 T. aestivum 10EC-12 No hit – – 10EC-14 Ribosomal protein L39 CAA64728 Z. mays 8EC-15 Putative ribosomal protein S17 AAN61484 O. sativa 6EC-16 Hypothetical proteinc BAC83160 O. sativa 6EC-17 Ubiquitin-like smt3 protein P55857 O. sativa 6EC-18 Hypothetical proteind BAB07973 O. sativa 6EC-22 Histone H2A CAA64356 T. aestivum 5EC-24 Putative lipid transfer protein AAN05565 O. sativa 5EC-430 17.9 kDa class I HSP AAK54445 O. sativa 5EC-51 Pathogenesis-related protein CAA38223 Zea mays 4

(b) Two-celled proembryo2C-1520 Histone H4 P59258 T. aestivum 392C-1521 Histone H3 CAA25451 T. aestivum 182C-1522 Histone H2B-6 BAA07156 T. aestivum 122C-1523 Ribosomal protein L39 CAA64728 Z. mays 82C-1524 Cyclophilin A-1 AAK49426 T. aestivum 52C-1525 No hite – – 42C-1526 No hit – – 42C-1527 Putative calmodulin-binding proteinf CAE02429 O. sativa 42C-1529 Putative ribosomal protein S12 BAC20920 O. sativa 42C-1537 Ribosomal protein S19 P40978 O. sativa 42C-1528 Ribosomal protein L18a Q943F3 O. sativa 32C-1531 Putative acidic ribosomal protein P2A BAA99431 O. sativa 32C-1532 Putative ribosomal protein S5 BAB64234 O. sativa 32C-1533 No hit – – 32C-1534 Histone H2A AAL40108 T. aestivum 32C-1535 Polyubiquitin S20925 Z. mays 32C-1536 12-oxophytodienoic acid reductase BAC20139 O. sativa 32C-1538 Putative snRNP polypeptide G O82221 A. thaliana 32C-1539 Putative ribosomal protein L37a BAB90039 O. sativa 32C-1566 No hit – – 3

aNCBI database nr.bContig comprises transcripts of at least five different members of a gene family with similarity to ECA1 from barley.cHypothetical protein contains conserved domain COG5227 (NCBI Conserved Domain Summary) of smt3 ubiquitin-like proteins.dHypothetical protein contains conserved domain pfam 00026 of aspartyl protease and is predicted to be extracellular located (SignalP).eCluster shows similarity to members of UniGene Cluster Ta.896 transcribed sequences which have moderate similarity to protein NP_565058(Arabidopsis) containing the domain KOG2315, a predicted translation initiation factor related to eIF-3a.f2C-1527 matches to UniGene Cluster Ta.9469 which has moderate similarity to a calmodulin-binding protein from Arabidopsis (NP_565441).

664 Stefanie Sprunck et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 41, 660–672

Transcripts encoding proteins involved in primary and

secondary metabolism form the largest functional group of

the unfertilized egg cell (category 1; Figure 4, blue bar). In

contrast, genes encoding proteins involved in translation

represent the largest functional group in two-celled pro-

embryos (category 4; Figure 4, yellow bar). The high abun-

dance of many different ribosomal proteins (seven large

clusters, listed in Table 1b, and additional 47 clusters with

fewer EST members) reflects high activity in ribosome

biosynthesis and mRNA translation. Nevertheless, many

transcripts for ribosomal proteins are also present in the egg

(category 4; Figure 4, blue bar). As already indicated by the

largest clusters (Table 1b), a much higher number of ESTs in

the two-celled proembryo encode proteins involved in

replication or establishment of DNA and chromatin struc-

ture, reflecting the onset of DNA replication after fertilization

(Figure 4b; category 2). Several singletons from the two-

celled wheat proembryo are present in this category,

representing S-phase-specific genes such as a wheat homo-

log of the rice replication protein A1 (van der Knaap et al.,

1997) involved in DNA replication, recombination and repair,

a transcript similar to the rice S-phase-specific ribosomal

protein RSPSP94 (accession AF052503), a homolog of the

rice replication factor C 36 kDa subunit (Furukawa et al.,

20033 ) and a homolog of the retinoblastoma-associated

protein ZmRbAp1 from maize (Rossi et al., 2001).

It is striking that the egg cell contains many transcripts

encoding proteins involved in protein folding and stability

(Figure 4b; category 5) as well as protein modification and

degradation (Figure 4b; category 6), whereas fewer such

transcripts are represented in the two-celled proembryo. In

the egg cell, category 5 ismainly represented by cyclophilins

and numerous different sHSPs, while category 6 is

predominantly composed of transcripts encoding ubiquitin,

ubiquitin-related proteins, 26S proteasome subunits, but

also novel or hypothetical F-box proteins.

A heterogeneous group of ESTs with similarity to proteins

involved in defense and/or stress response forms category

10, which is larger in egg cells relative to two-celled

proembryos. Among the egg cell ESTs are transcripts similar

to barley Rar1 and SGT1, two important signaling compo-

nents of resistance (R) gene-mediated plant innate immune

responses (Azevedo et al., 2002; Shirasu et al., 1999). The

high percentage of egg cell ESTs in category 11 (miscella-

neous proteins) is almost exclusively formed by one large

cluster containing the members of the TaECA1-like gene

family of unknown function.

Expression analysis of candidate genes from egg cells and

proembryos

We focused expression studies on novel transcripts identi-

fied in the egg cell and the two-celled proembryo, as well as

on candidates encoding putative developmental regulators

(Figures 5 and 6). Expression patterns were analyzed by RT-

PCR in order to give the required sensitivity. As controls,

cDNA from egg cells, two-celled proembryos, and central

cells were used.

Egg cell transcripts. Expression of TaECA1-like genes, the

largest cluster in the egg cell, was not detected in any

vegetative tissue, in anthers or 12-day-old developing cary-

opsis of wheat (Figure 5). Expression of TaECA1-like genes

was only found in the tissue containing the unfertilized egg

cell (pistil), and isolated egg cells. After fertilization, TaECA1-

like transcripts appear to be downregulated. A similar

expression pattern was observed for Cluster EC-12, repre-

senting a novel transcript (Table 1a). Two other transcripts

(Clusters EC-4, EC-70) were detected both in the egg cell and

the two-celled proembryo, but not in vegetative tissue (Fig-

ure 5). Cluster EC-70 encodes a novel zinc-finger-containing

protein, while Cluster EC-4 represents a cDNAwith similarity

to a hypothetical protein of rice (Table 1a). Unlike EC-70, EC-

4 appears to be downregulated in older embryos, as it is not

detectable in kernels 12 dap. A novel gene represented by

Cluster EC-57 encodes a small polypeptide with a conserved

Categories

(a) Functional categorization of cluster

0

5

10

15

20

25

30

35

40

1 2 3 4 5 6 7 8 9 10 11

% C

lust

er

Categories

(b) Functional categorization of ESTs

0

5

10

15

20

25

30

35

1 2 3 4 5 6 7 8 9 10 11

% E

ST

s

Figure 4. Functional classification of 360 contigs (a) containing 756 ESTs (b)

with similarity to characterized proteins. Functional categories are (1) primary

and secondary metabolism, (2) DNA and chromatin structure (including

histones), (3) transcription and RNA processing, (4) translation (including

ribosomal proteins), (5) protein-folding, -stability and -transport, (6) protein

modification and degradation, (7) cytoskeleton, (8) signal transduction, (9) cell

cycle regulation, (10) defense and stress response, (11) miscellaneous.

Percentages of egg cell contigs (193) and ESTs (454) are represented by blue

bars, red bars represent the percentages of contigs (167) and ESTs (302) from

the two-celled proembryo.

Transcriptome of wheat eggs and proembryos 665

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 41, 660–672

domain similar to Smt3 ubiquitin-like proteins (Table 1a).

Expression of EC-57 was detected in egg cells, central cells,

proembryos and unfertilized pistils but not in vegetative

tissues, except for a very weak signal in root tissue (Fig-

ure 5). Another group of novel transcripts appeared to be

expressed in male and female reproductive tissues (Fig-

ure 5, Clusters EC-123, EC-50, EC-217, EC-52). With the

exception of EC-52, this group of transcripts is downregu-

lated later in kernel development. While mRNAs of Clusters

EC-217 and EC-52 were detected in the female gametes, the

proembryo and anthers, Cluster EC-123, encoding an

Armadillo repeat (ARM) domain-containing protein, was not

detected in central cells and proembryos. ARM-repeat pro-

teins are characterized by degenerated sequence motifs of

about 42 amino acids originally found in the Drosophila

segment polarity protein armadillo (Nuesslein-Volhard and

Wieschaus, 1980; Riggleman et al., 1989) and regulate a

variety of cellular processes including cell adhesion, intra-

cellular signaling and gene expression (Hatzfeld, 1999).

Candidate genes encoding putative developmental regu-

lators, such as proteins with similarity to an SKP1-related

Fimbriata-associated protein (FAP) from Antirrhinum

(Ingram et al., 1997), a Brassinolide enhanced gene of

unknown function from rice (Yang and Komatsu, 2004), a

putative heterogeneous RNA-binding protein from rice

(accession BAC56813), and a putative lipid transfer protein

(LTP) from rice (accessionAAN05565; Table 1a)were present

in almost all tissues and cells tested (Figure 5; EC-24; EC-75,

EC-91, EC-386). Interestingly, two egg cell ESTs (Cluster

EC-95) encode a protein containing the conserved motif of

the LOB (LATERAL ORGAN BOUNDARIES) domain gene

family. Members of this gene family are thought to play a

role in boundary establishment or communication between

the meristem and initiating lateral organs as well as adaxial–

abaxial polarity establishment (Lin et al., 2003). In addition

to a presence in egg cells, the LOB-like gene transcript was

detected in anthers, pistils, kernels 12 dap, as well as in

coleoptiles, primary leaves and stems (EC-95; Figure 5).

Transcripts of the two-celled proembryo. Several candi-

dates of the two-celled proembryo which potentially encode

proteins involved in development or which match to pub-

lished ESTs of yet unknown function are expressed in almost

all vegetative tissues tested, but their transcripts were not

detected among cDNA from the female gametes before

fertilization (Figure 6). These include Cluster 2C-1570, which

encodes a protein similar to a SET domain protein from rice

(accession BAD05333) and Cluster 2C-1645, which encodes a

protein similar to the WD-repeat protein ZmRbAp1 from

maize (accession AF250047). Corresponding genes appear

to be newly expressed after fertilization. Two other genes

which appear to be constitutively expressed after fertiliza-

tion are represented by Clusters 2C-1696 and 2C-1726.

Cluster 2C-1696 encodes a protein similar to a translationally

controlled tumor protein (TCTP) from wheat (accession

AAM34280). TCTPs are highly conserved cytoplasmic

calcium-binding proteins, present in all eukaryotics and

put. RNA-binding protein

ECA1-like

Novel F-box protein

Hypothetical protein

Novel Zn-finger protein

Novel

Novel, SMT3-like

Hypoth. ARM protein

Novel

LOB domain protein

Lipid transfer protein

BLE-like

Fimbriata assoc. protein

GAP-DH

Novel F-box protein

EC-1

EC-12

EC-4

EC-70

EC-123

EC-50

EC-217

EC-57

EC-52

EC-95

EC-24

EC-386

EC-75

EC-91

Control

Co

leo

pti

le

Pri

mar

y le

af

Ste

m

Ro

ot

w.o

. tip

Eg

g c

ell

Pis

til

An

ther

s

Co

ntr

ol

Ro

ot

tip

Mat

ure

leaf

Ker

nel

, 12d

ap

2-ce

lled

pro

emb

ryo

Cen

tral

cel

l

Figure 5. Expression profiles for transcripts derived from the egg cell cDNA

library (compare also with Table 1a). Expression was examined by RT-PCR

using DNAse-treated total RNA from different tissues of wheat. As controls,

cDNA fromegg cells, central cells and two-celled proembryos have been used.

Unknown protein

Novel

GASA-domain protein

SET-domain protein

RbAP1-like

TCTP

BI-1 like

GAP-DH

Novel

Novel

2C-1566

2C-1563

2C-1533

2C-1526

2C-1807

2C-1570

2C-1645

2C-1696

2C-1726

Control

Co

leo

pti

le

Pri

mar

y le

af

Ste

m

Ro

ot

w.o

. tip

Eg

g c

ell

Pis

til

An

ther

s

Co

ntr

ol

Ro

ot

tip

Mat

ure

leaf

Ker

nel

, 12d

ap

2-ce

lled

pro

emb

ryo

Cen

tral

cel

l

Figure 6. Expression profiles for transcripts derived from the two-celled

proembryo cDNA library (compare also with Table 1b). Expression was

examined by RT-PCR using DNAse-treated total RNA from different tissues of

wheat. As controls, cDNA from egg cells, central cells and two-celled

proembryos have been used.

666 Stefanie Sprunck et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 41, 660–672

implicated in cellular processes such as cell cycle progres-

sion, malignant transformation and the protection against

apoptosis (Bommer and Thiele, 2004). Cluster 2C-1726 rep-

resents the transcript of a protein similar to the barley BAX

Inhibitor-1 (BI-1), an intracellular multi-membrane spanning

protein and cell death inhibitor (Huckelhoven et al., 2001).

A small protein of 105 amino acids containing a GASA

domain (pfam02704; present in a gibberellin-regulated pro-

tein family of Arabidopsis) is encoded by Cluster 2C-1807. As

with other candidate genes analyzed by RT-PCR, expression

of 2C-1807 was not detected in the gametes before fertiliza-

tion. After fertilization, 2C-1807 was expressed in all the

tissues tested, except mature leaves (Figure 6). A gene of

unknown function with similarity to barley ESTs derived

from embryo sacs, embryos as well as callus is represented

by Cluster 2C-1563. Expression of 2C-1563 was detected in

unfertilized pistils, developing kernels and very weakly in

stems and root tips, indicating a potential role in cell division

(Figure 6). Two novel clusters of the two-celled proembryo

(2C-1526 and 2C-1533) represent genes expressed weakly in

some, but not all vegetative tissues (Figure 6), while the

novel gene represented by Cluster 2C-1566 is not detectable

in vegetative tissues.

Discussion

Transcriptional activity and protein metabolism seems high

in the unfertilized egg cell

Based on our experiments we conclude that mature egg

cells of plants are not as quiescent as previously thought

(Diboll, 1968; Mogensen, 1982; Taylor and Vasil, 1995). The

high transcriptional activity of wheat egg cells4 was already

indicated by the presence of a large nucleolus (Kumlehn

et al., 1999), the nuclear sub-compartment of rRNA gene

transcription, rRNA processing and ribosome assembly.

This coincides with our identification of numerous egg cell

transcripts encoding ribosomal proteins. Strong transcrip-

tional activity is also indicated by the presence of the histone

variant H3.3 among the largest clusters of the wheat egg cell,

as H3.3 is incorporated into the chromatin of non-dividing

cells and accumulates in regions of actively transcribed

genes in Drosophila (Ahmad and Henikhoff, 2002; McKittrick

et al., 2004), probably by replacing the core histone H3. This

is further supported by the observation that H3.3 was not

present among the ESTs from the two-celled proembryo,

where 3.9% of ESTs encode H3, which is also a marker for

DNA replication (van der Knaap et al., 1997).

We also identified many egg cell transcripts that encode

proteins involved in primary and secondary metabolism,

leading us to the assumption that the unfertilized wheat egg

cell is in a state of high metabolic activity, at least shortly

before anthesis. Furthermore, the egg cell seems to be very

active in mRNA translation and protein turnover, as it

comprises not only a high number of transcripts encoding

ribosomal proteins, but also CyP1, sHSPs, and proteins of

ubiquitin-related protein-conjugation systems. CyP1 is a

peptidyl-prolyl cis-trans isomerase which catalyzes protein

folding by accelerating the slow step of cis-trans isomeriza-

tion of peptidyl-prolyl bonds (Boston et al., 1996). As a

member of the hsp90-based chaperone system, CyP1 is

involved in post-translational modification and protein

turnover. Interestingly, many signaling proteins in animals

(including steroid hormone receptors and kinases) are

regulated by this hsp90-based chaperone system (Buchner,

1999), so this may also be likely for wheat eggs.

Another remarkable finding was the discovery of several

known and novel transcripts encoding ubiquitin, ubiquitin-

related and F-box proteins, indicating that ubiquitination

may be an important post-translational regulatory pathway

for the unfertilized egg cell, both to establish and tomaintain

its functional status. Selection of ubiquitinated target pro-

teins to proteolysis by the 26S proteasome complex is

accomplished by ubiquitin-conjugating enzymes (E2s) and

ubiquitin-protein ligases (E3s). In particular, F-box proteins

linked to the E3 complex are responsible for specific

substrate recognition and binding (Kuroda et al., 2002) and

thus may target individual proteins for proteolysis. Besides

removing abnormal polypeptides, selective degradation of

short-lived regulatory proteins using the ubiquitin/26S pro-

teasome pathway is a powerful regulatory mechanism in a

wide variety of cellular processes in plants including the

regulation of cell cycle, flower development, hormonal

signal transduction, self-incompatibility and pathogen

response (Smalle and Vierstra, 2004; Sullivan et al., 2003).

Moreover, data from yeast and animals indicate other

functions of ubiquitin that do not necessarily involve the

proteasome. Several ubiquitin-like proteins are involved in

ubiquitin-like protein conjugation systems regulating his-

tone modification, protein stabilization, protein localization

and internalization of plasma membrane proteins (Hicke,

2001). It is thus tempting to speculate that specifically

expressed F-box proteins and ubiquitin-related proteins

may be used to prevent the onset of DNA replication and

other cell cycle-related processes in the unfertilized egg cell,

until the sperm fusion triggers zygotic development.

Zygotic gene activation occurs shortly after fertilization in

wheat

Zygotic gene activation in plants has been proposed based

on expression analyses of single genes (Dresselhaus et al.,

1999a; Sauter et al., 1998; Weijers et al., 2001). The appear-

ance of a second nucleolus in zygotes of maize and wheat

has been discussed as a suitable indication for successful

fertilization (Faure et al., 1994; Kumlehn et al., 1998). In

maize, the second nucleolus is derived from the sperm cell

genome (Mol et al., 1994), indicating the activation of

Transcriptome of wheat eggs and proembryos 667

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 41, 660–672

paternal rDNA shortly after karyogamy of egg and sperm

nuclei. The present study substantiates the assumption that

ZGA in higher plants occurs earlier than in animals: the

significant changes in transcript composition observed in

proembryos just after the first cell division mirrors the

completed transition towards zygotic gene expression.

Several transcripts derived from the two-celledproembryo

were found to be fertilization-induced, ofwhichmany remain

constantly expressed throughout plant development. Such

transcripts encode, for example, a homolog of the maize

retinoblastoma-associated protein ZmRbAp1, as well as a

SET domain protein. RbAp-like proteins are components of

multiprotein complexes related to chromatin assembly dur-

ing DNA replication, chromatin remodeling and histone

acetylation activities (Ach et al., 1997). Transcripts of ZmR-

bAp1 are abundant during the initial stages of endosperm

formation, and mRNAs are localized in shoot apical meri-

stems and leaf primordia of developing embryos (Rossi

et al., 2001). Our results show that the ZmRbAp1 homolog of

wheat is not expressed in gametes, but in embryos very early

after fertilization aswell as in vegetative tissues, suggesting a

general role in chromatin assembly or modification during

sporophytic development. SET domain proteins are very

important for developmental and epigenetic regulation of

gene expression. Several plant SET domain proteins appear

to control chromatin states via histonemethylation (Springer

et al., 2003). However, transcripts of the fertilization-induced

SET domain protein of wheat proembryos are detected in all

sporophytic tissues and thus appear to have a general

function in plant growth and development.

Genes specifically expressed in egg cells and two-celled

proembryos encode not only novel proteins but also

putative secreted peptides of unknown function

Thesequencingofonly 1197ESTshas led to the identification

of many clusters representing novel transcripts, although

more than 1.5 million Poaceae ESTs are present in the public

EST database. This may reflect the under representation of

gametic transcripts in current databases. Interestingly, the

egg cell containsmorenovel clusters (71), comparedwith the

34 novel clusters of the proembryo. As 11 egg cell clusters

were not similar to any published EST but to annotated hypo-

thetical genesdetected in genomesofArabidopsis and/or rice,

weconclude that thesegenesmight beexclusively expressed

in the gametes of Arabidopsis and/or rice. It seems likely that

some of the 105 novel transcripts encode proteins required

for specific functions of the gamete and the early embryo,

respectively. Expression analyses verified that several novel

transcripts and transcripts with similarity to hypothetical

proteins are abundant in the egg cell prior to fertilization, but

either not or only barely detectable in the two-celled proem-

bryo. The majority of these genes were not expressed in

vegetative tissues, indicating that they might either specify

egg cell identity, or function in processes such as micropylar

pollen tube guidance, fertilization or the onset of embryo-

genesis. Such are transcripts for novel F-box proteins, a zinc-

finger protein, an SMT3-like and an ARM-repeat-containing

protein as well as some completely unknown proteins.

Strikingly, a number of transcripts encode small and

putative secreted proteins. These include transcripts form-

ing the largest cluster of the egg cell with similarity to ECA1

(early culture abundant1), a cDNA of unknown function

previously isolated from androgenic barley microspores

(Vrinten et al., 1999). We compared the 5¢ and 3¢ UTRs of all

wheat ECA1-like (TaECA1-like) ESTs and identified at least

five distinct transcript groups, representing members of a

gene family. All TaECA1-like transcripts encode small pro-

teins of around 151 amino acids, containing six conserved

cysteine residues and a putative signal peptide for extra-

cellular localization. Although the largest cluster in egg cells,

TaECA1-like ESTs were not identified among the two-celled

proembryo ESTs. Interestingly, Cordts et al. (2001) previ-

ously reported about genes encoding cysteine-rich secreted

peptides (ZmES1-4) that are highly and specifically ex-

pressed in cells of the maize female gametophyte and are

downregulated after fertilization. An important role for

ZmES1-4 in the fertilization process can be assumed, as

maize knockout plants are female-sterile5 (S. Amien and

T. Dresselhaus, unpublished data). In general, small extra-

cellular proteins are attractive candidates for signaling

molecules, and may serve as ligands for receptor-like

kinases (Dresselhaus and Sprunck, 2003; Ryan et al., 2002).

Such peptides might mediate interactions between male

and female gametes, communication among the cells of the

female gametophyte, or interactions with maternal tissues.

Physiological and genetic data support the assumption

that cells of the female gametophyte produce diffusible

signals involved in pollen tube attraction and reception

(Higashiyama et al., 2001; Huck et al., 2003; Rotman et al.,

2003; Shimizu and Okada, 2000). Another gene which is

expressed in both the egg cell and the proembryo encodes a

putative LTP. LTPs are secreted hydrophilic peptides which

are characterized by a conserved signature motif of eight

cysteines (Kader, 1997). They are abundant and widespread

in plants, forming a broad family of proteins whose biologic

role is still under discussion, but there is evidence that some

LTPs might act as signaling molecules (Buhot et al., 2001).

Other transcripts found in the egg cell and the proembryo

encode a peptide similar to the pathogenesis-related small

protein PR1 from maize (Casacuberta et al., 1991). This

peptide is secreted and contains six cysteines. A fertilization-

induced transcript encodes a predicted secreted small

protein of 105 amino acids containing a 25-amino acid

signal peptide and a motif of 12 cysteines. Interestingly, this

small protein contains a GASA domain (pfam 02704), found

in a gibberellin-regulated cysteine-rich protein family from

Arabidopsis. The expression of such GASA proteins is

668 Stefanie Sprunck et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 41, 660–672

upregulated by the plant hormone gibberellin and most of

these members appear to have a role in developmental

control (Aubert et al., 1998; Herzog et al., 1995).

Conclusions

By generating and analyzing a limited number of ESTs from

cell type-specific cDNA libraries we were able to greatly in-

crease our knowledge about changes in gamete expression

profiles and thus, the transcriptional consequence of fertil-

ization. We have shown that transcriptional changes take

place considerably and soon after fertilization in wheat,

providing now a clear proof that ZGA occurs early in wheat,

and possibly in other cereals. It is also clear now that mature

egg cells of wheat are not quiescent. Especially protein

biosynthesis, protein modification and protein degradation

as well as signaling seem to be crucial biologic functions for

the egg cell before fertilization, while translation, DNA rep-

lication, chromatin remodeling, and other cell cycle-related

processes are characteristic of early zygote/embryo devel-

opment. Especially the identified novel genes which are

specifically expressed in each, the egg cell and the pro-

embryo will be interesting for functional analysis, as they

may be involved in regulating egg cell identity, fertilization

as well as the initiation of embryogenesis.

Experimental procedures

Isolation of wheat embryo sac cells before and after

fertilization

Spikes of Triticum aestivum cv. ‘Florida’ were emasculated

2–4 days before anthesis and covered with bags to prevent

fertilization. Egg cells were isolated mechanically from

microdissected ovules in 0.55 M sterile mannitol using fine-

tipped glass needles and an inverted microscope, as des-

cribed by Kumlehn et al. (1999). Single cells were transferred

into 0.5 ml reaction tubes by using a glass capillary inter-

faced with a hydraulic system to a micropump. Collected

cells were immediately frozen in liquid nitrogen. Wheat

zygotes were isolated 4–6 h after hand-pollination of recep-

tive stigmata by using the same procedure, but transferring

the zygotes into fresh 0.55 M mannitol solution. Defined

stages of zygotes and proembryos were selected using an

inverted microscope. In vivo embryos were isolated using

the similar procedure to that described above.

mRNA isolation and cDNA synthesis

mRNA was isolated from 12 egg cells and 20 proembryos

at the two-celled stage, using the Dynabeads� mRNA

DIRECTTM Micro kit (Dynal, Hamburg, Germany)6 following

themanufacturer’s guidelines with but scaled down to 50 ll.Annealed mRNA was isolated using a magnetic particle

transfer device (PickPenTM; Bio-Nobile, Turku, Finland).7

Subsequently, the SMARTTM PCR cDNA synthesis kit (BD

Biosciences, Heidelberg, Germany)8 was used for cDNA

synthesis. First-strand cDNA, LD-PCR, and determination

of optimal cycle numbers for generating a population

of representative cDNAs was performed according the

manufacturer’s guidelines but using a digoxigenin-11-dUTP

(Roche Applied Science, Mannheim, Germany)9 labeled

fragment of wheat GAPDH as a probe.

Library construction and sequencing

cDNA (150 ll) was used for polishing, according to the

SMARTTM PCR cDNA synthesis kit (BD Biosciences).

Subsequently, 3 lg of EcoRI (NotI) adapters (Invitrogen,

Karlstuhe, Germany)10 were ligated to blunt-end cDNA, using

T4 ligase (New England Biolabs, Frankfurt, Germany).11

Remaining adapters and fragments below 0.3 kb were

removed by electrophoresis in 0.8% low-melting point

agarose (Seaplaque GTG). Afterwards, cDNA was extracted

using b-agarase I (New England Biolabs). After phosphory-

lation of EcoRI cohesive ends (10 U/ll T4 polynucleotide

kinase; New England Biolabs), a second purification step

using ChromaspinTM columns (BD Biosciences) was per-

formed. The cDNAwas then ligated into predigested lambda

ZAP� II/EcoRI/CIAP vector (Stratagene, Amsterdam, the

Netherlands).12 The titer of the unamplified libraries was

1.43 · 106 pfu ml)1 for egg cells and 3.2 · 105 pfu ml)1 for

the two-celled proembryo, respectively. After amplification

and in vivo excision, cloneswere randomly picked from each

library and used to generate ESTs. Insert sizes ranged from

300 to 3000 bp, with an average of 900 bp. The average

readable sequence length of ESTs was about 500 bp. DNA

sequencer trace data subsequently passed an automated

cleanup pipeline including PHRED to call bases and assign

quality values, followed by CROSS_MATCH to align

sequences and to eliminate vector sequences.

Bioinformatics

The sequences of each library were clustered using blast-

clust (NCBI) and assembled into contigs using Vector NTI 8

(Invitrogen). The contig’s consensus sequence or the lon-

gest representative was used for BLASTN searches against

NCBI’s non-redundant (nr) database and the EST database,

and for BLASTX searches against NCBI’s nr database and

SWISSPROT (March 2004). A number of cDNAs resulted in

limited sequence information (100–250 bp) from non-coding

regions. Therefore, BLASTN searches against the TIGRWheat

Gene Index Release 8.0 (Quackenbush et al., 2001) were

performed, using the BLASTN algorithm. If a match with

>95% sequence identity over the total length of the query

sequence was found, the matching sequence was retrieved

and used in subsequent BLASTX searches in place of the

Transcriptome of wheat eggs and proembryos 669

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 41, 660–672

original EST. A sequence was considered novel if it did not

show a significant match with a sequence of the NCBI

databases (nr, EST) or to the TIGR-assembled wheat

consensus sequences using the BLASTN algorithm (Altschul

et al., 1997). The significance threshold used for BLASTN

searches were: score >115, expected value <e)25.

For BLASTX searches, the cutoff for a significant match for

all but the short sequences was an e-value of <e)15, score

‡80. Matches to short query sequences (below 260 bp) were

inspected and categorized manually. Clusters encoding

proteins of known function were manually categorized into

broad functional groups using the Munich Information

Centre for Protein Sequences classification as guidance.

Expression analysis by RT-PCR

RNA was isolated from vegetative and generative tissues

using TRIzol� reagent (Invitrogen), essentially following the

manufacturer’s protocol. Starch containing tissues such as

caryopsis were extracted twice, using 3 ml of TRIzol�

reagent per 100 mg of tissue. The quality of the total RNA

preparation was analyzed by denaturating agarose gel elec-

trophoresis. Before RT-PCR, 1 lg of total RNA was digested

with DNAse (RNAse free; Invitrogen) and subsequently used

for first-strand cDNA synthesis using Oligo(dT)23 (Sigma,

Taufkirchen, Germany)13 and Superscript II reverse transcrip-

tase (Invitrogen), following the manufacturer’s protocol but

adding RNAseOUTTM (Invitrogen). Quality and amount of

generated cDNAswas analyzed by PCRwith intron-spanning

primers directed against wheat GAPDH (TaGAP1, 5¢-AGG-

GTGGTGCCAAGAAGGTCA-3¢; TaGAP2, 5¢-TATCCCCACTC-GTTGTCGTA-3¢). Primer pairs directed against selected

sequence clusters were designed according to PrimerSelect

(Lasergene, GATC Biotech AG, Konstanz, Germany),14 result-

ing in amplified products of 120–600 bp length. Sequences

of gene-specific primers are available on request. GAPDH

PCR reactions were carried out for 30 cycles, using 2 ll ofcDNA. PCR reactions for transcript-specific primers of

selected candidates were carried out for 38 cycles, using 2.5

of cDNA as template.

Acknowledgements

This work was funded by the Grains Research DevelopmentCorporation, Australia. We thank Gary Barker and Ian Wilson(University of Bristol, UK) for sequencing and editing the ESTs aspart of the BBSRC Investigating Gene Function program. We aregrateful to Jochen Kumlehn for his excellent introduction to thetechnique of wheat microdissection.

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Accession numbers: The GenBank numbers for the sequences mentioned in this article are AL830671–AL831324, CV 973579–CV 973658 (eggcell) and CV 973119–CV 973578 (proembryo).

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