Plant Biotechnology Journal
(2008)
6
, pp. 465–476 doi: 10.1111/j.1467-7652.2008.00333.x
© 2008 ACPFGJournal compilation © 2008 Blackwell Publishing Ltd
465
Blackwell Publishing LtdOxford, UKPBIPlant Biotechnology Journal1467-76441467-7652© 2008 Blackwell Publishing LtdXXXOriginal Articles
Endosperm-specific promoters from rice
Ming Li
et al.
Spatial and temporal expression of endosperm transfer cell-specific promoters in transgenic rice and barley
Ming Li
1
, Rohan Singh
1
, Natalia Bazanova
2
, Andrew S. Milligan
2
, Neil Shirley
2
, Peter Langridge
2
and Sergiy Lopato
2,
*
1
Plant and Pest Science, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia
2
Australian Centre for Plant Functional Genomics, Hartley Grove, Urrbrae, SA 5064, Australia
Summary
Two putative endosperm-specific rice genes,
OsPR602
and
OsPR9a
, were identified from
database searches. The promoter regions of these genes were isolated, and transcriptional
promoter:
β
-glucuronidase (GUS) fusion constructs were stably transformed into rice and
barley. The GUS expression patterns revealed that these promoters were active in early grain
development in both rice and barley, and showed strongest expression in endosperm
transfer cells during the early stages of grain filling. The GUS expression was similar in both
rice and barley, but, in barley, expression was exclusively in the endosperm transfer cells and
differed in timing of activation relative to rice. In rice, both promoters showed activity not
only in the endosperm transfer cells, but also in the transfer cells of maternal tissue and in
several floral tissues shortly before pollination. The expression patterns of
OsPR602
and
OsPR9a
in flowers differed. The similarity of expression in both rice and barley suggests that
these promoters may be useful to control transgene expression in the transfer cells of cereal
grains with the aim of altering nutrient uptake or enhancing the barrier against pathogens
at the boundary between maternal tissue and the developing endosperm. However, the
expression during floral development should be considered if the promoters are used in rice.
Received 9 February 2008;
accepted 26 February 2008
.
*
Correspondence
(fax +63 8 830 37 102,
e-mail [email protected])
Keywords:
barley, endosperm, grain,
promoter, rice, transfer cells.
Introduction
The cereals rice, wheat, maize and barley are major sources
of human food and animal feed, and also provide the raw
material for many industries. Some of the limitations of
conventional breeding of cereals may be overcome by applying
the techniques of genetic transformation or engineering
(Vonwettstein, 1993; Sinclair
et al
., 2004; Shrawat and Lörz,
2006). An important application of genetic engineering is
expected to be in the improvement of grain size, quality
and yield by the modulation of the levels of expression of
particular gene(s), whilst retaining the desirable qualities
in the selected cultivar. Negative effects of constitutive
transgene expression on plant development may be prevented
by the use of tissue- or development-specific promoters
(Potenza
et al
., 2004). Endosperm-specific promoters are of
great importance for the development of modified grains
with altered grain size, shape and composition, as well as
biotic and abiotic stress tolerance.
After cellularization, the endosperm of cereals comprises
at least four different tissues which have specialized roles: the
endosperm transfer cells (ETCs), embryo surrounding region
(ESR), starchy endosperm and aleurone. ETCs are responsible
for the uptake of nutrients and act as a barrier against
pathogens. The ESR protects the embryo, transfers nutrients
to the embryo and acts as a channel for regulatory cross-talk
between the endosperm and embryo. Starchy endosperm
cells make up the bulk of the endosperm and accumulate
storage proteins and starch. The aleurone layer is important
as the site of synthesis of enzymes which hydrolyse the
storage reserves in the endosperm cells at the time of seed
germination (Becraft, 2001; Olsen, 2001, 2004).
Most of the endosperm-specific promoters from monocots
described in the literature have been shown to be active in
466
Ming Li
et al.
© 2008 ACPFGJournal compilation © 2008 Blackwell Publishing Ltd,
Plant Biotechnology Journal
,
6
, 465–476
the late phases of grain development, and are expressed
mainly in starchy endosperm (Russell and Fromm, 1997;
Lamacchia
et al
., 2001; Qu and Takaiwa, 2004; Su
et al
.,
2004). They show maximal activity at the middle of grain
maturation, when cell proliferation and differentiation are
mostly complete. These promoters are useful for biotechno-
logical projects attempting to increase starch and protein
content or to enrich grain with particular amino acids
(Vonwettstein, 1993). Strong endosperm-specific promoters
of storage proteins are also widely used for the high-level
expression of proteins of non-grain origin, with the aim of
large-scale protein production for applications such as the
production of pharmaceuticals and in industry (Wang
et al
.,
1994b; Hood and Jilka, 1999; Hood
et al
., 2002). However,
for the manipulation of grain size and shape, it is probable
that the expression of transgenes will be needed before or
during cellularization and in tissues involved in nutrient
transfer to the developing endosperm, such as the ETC layers.
ETCs are in contact with cells of the maternal tissue. These
cells transfer efficiently sugars and amino acids from
maternal tissue to the endosperm (Wang
et al
., 1994b; Becraft,
2001; Olsen, 2004). To increase the efficiency of transfer,
ETCs develop cell wall ingrowths, which increase the surface
area by up to 22-fold (Wang
et al
., 1994b).
In maize, transfer cells are restricted to the aleurone layer
and three to four adjacent layers of starchy endosperm
(Thompson
et al
., 2001; Olsen, 2004). In barley and wheat,
transfer cells are formed from maternal nucellar cell pro-
jections adjacent to the endosperm cavity and at least one layer
of ETCs on the other side of the endosperm cavity (Wang and
Fisher, 1994; Wang
et al
., 1994a,b; Broekaert
et al
., 1997;
Olsen, 2004). In rice, the pericarp has four vascular bundles,
three of which are situated on the ventral part and side faces
of the grain and are responsible for the supply of nutrients
and water for the development of the pericarp. The fourth
vascular bundle is situated on the dorsal side of the grain and
transports organic solutes and minerals to the endosperm.
In the portion of aleurone adjacent to the vascular bundles,
there are three to five layers of cells responsible for the transfer
of nutrients to the starchy endosperm (Hoshikawa, 1989).
Several types of gene have been found to be expressed
only or mainly in ETCs (Thompson
et al
., 2001; Olsen, 2004).
Most encode low-molecular-weight, cysteine-rich proteins
with hydrophobic signal peptides. Four types of these
proteins have been found in maize basal endosperm transfer
layers (BETLs). BETL-1 and BETL-3 show sequence homology
to defensin-like proteins; BETL-2 has no homologous
sequences; BETL-4 has some homology to the Bowman–Birk
family of
α
-amylase/trypsin inhibitors (Hueros
et al
., 1995,
1999b). As defensins and low-molecular-weight trypsin
inhibitors have been shown to inhibit the growth of fungi and
bacteria (Broekaert
et al
., 1997), BETL proteins may help
protect the grain from infection. Defensins can also alter the
permeability of fungal plasma membranes, and hence may
act as regulators of transport through the plasmalemma
(Thompson
et al
., 2001).
The
BETL-1
promoter was isolated and used to direct
β
-glucuronidase (GUS) gene expression in transgenic maize.
GUS activity was found exclusively in BETL cells (Hueros
et al
.,
1999a,b). Expression of BETL proteins is greatly reduced in
the maize
rgf1
(
reducing grain filling1
) mutant, which also
shows decreased uptake of sugars in endosperm cells at
5–10 days after pollination (DAP) (Maitz
et al
., 2000). The
identification and characterization of homologues of
BETL
genes in other grasses have not been reported.
One further class of ETC genes, encoding low-molecular-
weight, cysteine-rich proteins, was identified in the barley
transfer cell domain of the endosperm coenocyte. This cell
type gives rise to the cells that differentiate into transfer cells
(Doan
et al
., 1996). The gene was designated
Endosperm 1
(
END1
). The expression activities of the barley gene
HvEND1
and its orthologue from wheat were studied using
in situ
hybridization (Doan
et al
., 1996; Drea
et al
., 2005) to show
that
END1
is expressed in the coenocyte above the nucellar
projection during the free-nuclear division stage. After cellu-
larization,
END1
transcripts accumulated mainly in the ventral
endosperm over the nucellar projection, but, from 8 DAP, a
low level of expression was also detected in the modified
aleurone and the neighbouring starchy endosperm (Doan
et al
., 1996).
Several other ETC genes have also been identified and
partially characterized. These include
ZmTCRR-1
(Muniz
et al
.,
2006), the transcription factor
ZmMRP-1
(Gomez
et al
., 2002)
and
meg1
(
maternally expressed gene1
) (Gutierrez-Marcos
et al
., 2004) from maize.
In this study, the promoters of two rice genes, designated
OsPR602
and
OsPR9a
, were isolated and characterized. They
were cloned into the pMDC164 plant transformation vector
upstream of the GUS reporter gene, and the activity of the
promoters was analysed using stable
Agrobacterium
-
mediated transformation of rice and barley. The spatial and
temporal activities of the promoters were studied using
whole-mount and histological analysis. It was shown that
OsPR602
and
OsPR9a
promoters lead to GUS expression
in ETCs of developing caryopses in both rice and barley.
However, the promoters exhibit less specific expression in
rice than in barley, as rice shows expression in certain flower
tissues shortly before pollination.
Endosperm-specific promoters from rice
467
© 2008 ACPFGJournal compilation © 2008 Blackwell Publishing Ltd,
Plant Biotechnology Journal
,
6
, 465–476
Results
Identification and isolation of promoter regions of
OsPR602
and
OsPR9a
A cDNA library was prepared from the liquid endosperm of
wheat at 3–6 DAP (Lopato
et al
., 2006). Inserts from 100
randomly selected clones were sequenced, and several cDNAs
encoding full-length, low-molecular-weight, cysteine-rich
proteins were identified. One such clone, designated
TaPR60
(Accession Number EU264062), showed protein sequence
identity with
HvEND1
(Doan
et al
., 1996) (Figure 1a). Another
cDNA, designated
TaPR9
(Accession Number EU264058),
encoded a short protein with no close homologues in the
databases, although the position of some cysteines was
similar to the position of cysteines in BETL-3, a defensin-like
protein from maize (Hueros
et al
., 1995) (Figure 1b).
Northern blot hybridization suggested that both
TaPR9
and
TaPR60
mRNAs were transcribed and accumulated at
high levels in the developing grain between 6 and 10 DAP,
but were not detectable in any other tissues tested (Figure 2a).
Quantitative real-time polymerase chain reaction (PCR)
analysis showed that the amount of
TaPR9
mRNA started to
increase in the grains at 6–7 DAP, reached a maximum at
8–10 DAP and remained present until 16–20 DAP (Figure 2b).
The level of
TaPR60
transcripts was up to 100-fold higher
than that of
TaPR9
transcripts. These were first seen at 3 DAP,
reached a maximum expression level at 7–8 DAP and were
not detected at 18–20 DAP. At 5 DAP, the expression of both
TaPR9
and
TaPR60
was found mainly in the liquid fraction of
the endosperm (Figure 2b). As the objective of the research
was to isolate cereal promoters that showed specific expression
during early endosperm development, the rice genome
sequence was used to identify the promoter regions. The
amino acid sequences of TaPR60 and TaPR9 allowed the
identification of rice homologues, designated as
OsPR602
and
OsPR9a
, respectively, in expressed sequence tag (EST)
databases. ESTs for
OsPR602
originated from cDNA libraries
prepared from the panicle or pistil a short time after flowering
[Institute for Genomic Research (TIGR) libraries #ILF, #68F,
#IL6, #IL0, #IKS, #IJM]. The EST for
OsPR9a
was represented
by a singleton in a library prepared from rice immature seed
(TIGR library #OS36). Contigs containing full-length coding
sequences were found for both
OsPR602
and
OsPR9a
[Accession
Numbers CA767165 (EST), EU264061 (gene) and CA760707
(EST), EU264060 (gene), respectively]. Quantitative real-time
PCR analysis showed that the amount of
OsPR602
mRNA
started to increase in the grains at 2–5 DAP, with a very small
amount remaining detectable after 11 DAP. A very low copy
number of
OsPR602
mRNA was also found in pre-anthesis
panicles (Figure 2b).
OsPR9a
mRNA was detected in pre-
anthesis panicles and in panicles between 2 and 11 DAP, but
was not detectable at 12–18 DAP. No expression of either
gene was detected in the other tissues tested (Figure 3). The
level of
OsPR602
transcripts in panicles was up to several
100-fold higher than that of
OsPR9a
transcripts.
The nucleotide sequences of the two EST contigs were used
to identify the translational start site of the genes in the rice
genomic database. DNA fragments of 2808 and 1730 bp,
upstream of the translational start site of
OsPR602
and
OsPR9a
,
respectively, were isolated by PCR using rice (
Oryza sativa
ssp.
japonica cv. Nipponbare) genomic DNA as template. These
promoter sequences were then cloned into the plant trans-
formation vector pMDC164 (Curtis and Grossniklaus, 2003) to
provide the transcriptional GUS fusion promoter constructs
designated as pMDC164-OsPR602 and pMDC164-OsPR9a.
Computer analysis for putative
cis
-elements in
the promoters of
OsPR602
and
OsPR9a
Computer analysis of the
OsPR602
and
OsPR9a
promoters
using
PLACE
software (http://www.dna.affrc.go.jp/PLACE/
Figure 1 Multiple alignments of protein sequences of OsPR9a to TaPR9 (a) and OsPR602 to TaPR60 and HvEND1 (b). Identical amino acids are shown in black boxes; similar amino acids are shown in grey boxes.
468
Ming Li
et al.
© 2008 ACPFGJournal compilation © 2008 Blackwell Publishing Ltd,
Plant Biotechnology Journal
,
6
, 465–476
signalscan.html) revealed several putative
cis
-elements which
could be involved in endosperm-specific activation. These
included: the GCN4-like motif (GLM; 5
′
-ATGAG/CTCAT-3
′
),
recognized by bZIP proteins of the Opaque2 subfamily
(Albani
et al
., 1997; Wu
et al
., 1998; Conlan et al., 1999;
Onate et al., 1999; Vicente-Carbajosa and Carbonero, 2005);
the prolamin box (PB; 5′-TGTAAAG-3′) recognized by one
zinc finger (DOF) class of transcription factors (Mena et al.,
1998; Lijavetzky et al., 2003; Yanagisawa, 2004); and a motif
for the R2R3 subclass of MYB transcription factors (5′-AAC/
TA-3′) (Suzuki et al., 1998; Diaz et al., 2002). It has been
demonstrated previously that DOF transcriptional factor(s)
interact with R2R3MYB (GAMYB) in vivo to activate
endosperm-specific promoters (Diaz et al., 2005).
The recently discovered cis-element from the BETL-1
promoter (Barrero et al., 2006), which comprises two -TATCTC-
repeats and specifically interacts with ZmMRP-1, was not
identified in either the OsPR602 or OsPR9a promoter. Two
single -TATCTC- sequences were found at 1189 and 1677 bp
upstream of the translational start site in the OsPR602
promoter. However, no confirmation of the role of predicted
cis-elements in the activation of the OsPR602 or OsPR9a
promoter was undertaken.
Generation of transgenic plants
pMDC164-OsPR602 and pMDC164-OsPR9a constructs were
transformed into Agrobacterium tumefaciens strain AGL1,
and the presence of plasmid in selected colonies was
confirmed by PCR using specific primers. Transformed Agro-
bacterium was subsequently used to introduce constructs
into rice and barley. The integration of promoter:GUS fusions
Figure 2 Expression of TaPR60 and TaPR9 in different wheat tissues shown by Northern hybridization (a) and quantitative real-time polymerase chain reaction (Q-PCR) (b). Q-PCR was performed using four independent replicates for each tissue. DAP, days after pollination.
Endosperm-specific promoters from rice 469
© 2008 ACPFGJournal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 6, 465–476
in transgenic plants was confirmed by either PCR (for rice) or
Southern blot hybridization (for barley) (Table 1). Southern
blot hybridization gave additional data showing the number
of inserts in transgenic lines of barley. All GUS-expressing
lines showed the same spatial and temporal patterns of
expression after GUS staining. However, the level of expression
differed.
Sixteen rice lines transformed with pMDC164-OsPR602
were selected on hygromycin, and the integration of the
transgene was confirmed by PCR in all lines. However, GUS
activity was detected in only six lines. Six from six selected
barley plants transformed with pMDC164-OsPR602 were
positive in staining for GUS activity. Lines 1, 2, 4 and 5 had
single-copy insertions, whereas two copies of the transgene
were found in line 3 and at least three copies were integrated
in line 6. GUS analyses were performed with all six T0
lines using untransformed wild-type barley and/or barley
transformed with empty vector plasmid as the negative
control. No differences were found between wild-type plants
and plants transformed with empty vector. The profile of
OsPR602 promoter activity was identical in all six independent
T0 lines. The highest level of GUS activity was detected in
line 1.
Integration of pMDC164-OsPR9a was confirmed by
PCR for 11 transgenic rice lines. However, only five T0 lines
exhibited GUS activity. The pattern of GUS expression was
the same in all five lines. Nine transgenic barley lines with
the same construct were selected on hygromycin, and
integration of the transgene was confirmed in all selected
lines by Southern blot hybridization (Table 1). Lines 1, 4, 6, 7
and 9 contained single-copy insertions. Two to three copies
were integrated in lines 2, 5 and 8; line 3 contained more
than five copies of the transgene in the genome. However,
only three transgenic lines (2, 5 and 8) showed GUS activity.
The strongest GUS expression was found in line 2.
The numbers of transgenic lines, transgene copy number
and relative strength of expression are summarized in
Figure 3 Expression of OsPR9a and OsPR602 in different rice tissues, as shown by quantitative real-time polymerase chain reaction. DAP, days after pollination.
Table 1 Information about T0 transgenic lines
Transgenic line
OsPR602:GUS
in transgenic rice
OsPR602:GUS
in transgenic barley
OsPR9a:GUS
in transgenic rice
OsPR9a:GUS
in transgenic barley
Number of putative transgenic lines
selected on antibiotic
16 6 11 9
Number of transgenic lines confirmed
by polymerase chain reaction and
Southern blot hybridization
16 6 11 9
Number of transgene insertions N/A Lines 1, 2, 4 and 5 have
single-copy insertions. Line
3 has two copies. Line 6
contains three copies
N/A Lines 1, 4, 6, 7 and 9 have
single-copy insertions. Line 2
has three copies. Lines 5 and
8 contain two copies. Line 3
has seven copies
Number of transgenic lines with
GUS expression
6: lines 3, 5, 8, 9, 12 and
16; line 8 has the strongest
GUS expression
6: lines 1–6; line 1 has the
strongest GUS expression
5: lines 2, 3, 8, 9
and 11
3: lines 2, 5 and 8; line 2 has
the strongest GUS expression
GUS, β-glucuronidase.
470 Ming Li et al.
© 2008 ACPFGJournal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 6, 465–476
Table 1. All T0 lines, and T1 progeny derived from at least two
independently transformed T0 transgenic lines, were used
for analysis.
Spatial and temporal control of GUS expression by the
OsPR602 promoter in transgenic rice and barley
In the transgenic rice lines transformed with pMDC164-
OsPR602, GUS activity was detected in the pistils and some
flower tissues at anthesis. In flowers, it was found in the
rachilla and vascular bundles of the lemma and palea, but
was not detected in the anther and ovary (Figure 4a, A2). No
GUS expression was observed in the leaf blade, sheath, culm,
auricle, ligule, root or rachis. In the T1 caryopsis, the OsPR602
promoter was found to be switched on at 7 DAP. No GUS
staining was detected in any grain tissues before 7 DAP (data
not shown). In the grain collected at 15 DAP, GUS activity
was clearly observed towards the dorsal side of the grain
(Figures 3 and 4), in the aleurone transfer cell layers and the
adjacent starchy cells. The aleurone transfer cell layers retained
GUS activity until 24 DAP. No GUS activity was detected in
the grain of control plants at any stage (Figure 4a, A3).
Following the GUS assay, the samples of rice grain were
embedded in paraffin wax, sectioned and counter-stained
with safranin orange to achieve high contrast and resolution
of cell morphology. The micrographs A5–A10 in Figure 4a
demonstrate the distribution of GUS expression during early
grain filling. The ovular vascular traces formed a large tissue
mass down the dorsal side of the grain, which serves as a
path linking the maternal vascular tissue to the inner side of
the nucellus. In the grain at 9 DAP, GUS was strongly
expressed in the ovular vascular cells, the adjacent pigment
strand and testa, the three to four layers of aleurone transfer
cells and the adjacent starchy cells (Figure 4a, A5 and A6).
Figure 4 Spatial and temporal β-glucuronidase (GUS) expression in rice (a) and barley (b) directed by the OsPR602 promoter. GUS activity in transgenic rice detected in flower (A2) and towards the dorsal side of the grain at 15 days after pollination (DAP) (A4), but not in control flower (A1) and grain (A3). Bars, 1 mm. Histochemical GUS assay in a transverse section of a T1 caryopsis at 9 DAP (A5, A6) and 13 DAP (A7, A8); longitudinal section of rice caryopsis at 15 DAP (A9, A10). Bars: A5, A7 and A9, 800 µm; A6, A8 and A10, 100 µm. GUS activity in barley detected in hand-cut longitudinal (B2) and transverse (B4) sections of transgenic lines, but not in wild-type (B1, B3) caryopsis at 10 DAP. Histochemical GUS assay counter-stained with safranin in a 10 µm thick transverse section of transgenic barley caryopsis at 10 DAP (B5), longitudinal section of wild-type caryopsis at 10 (B7), 16 (B9), 26 (B11) and 30 DAP (B13), and transgenic barley caryopsis at 10 (B6), 16 (B8), 23 (B10) and 30 DAP (B12). Arrows show GUS-stained tissues. AL, aleurone; DVB, dorsal vascular bundle; ETC, endosperm transfer cell; mp, maternal pericarp; NE, nucellar epidermis; NP, nucellar projection; OVT, ovular vascular trace; PS, pigment strand; Ra, rachilla; SEC, starchy endosperm cells; VB, vascular bundle; VT, vascular tissue. Bars, 1 mm.
Endosperm-specific promoters from rice 471
© 2008 ACPFGJournal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 6, 465–476
GUS activity gradually declined and could not be detected in
the ovular vascular trace or pigment strand at 13 DAP, but
remained in other transfer tissues (Figure 4a, A7 and A8).
Figure 4a (A9 and A10) illustrates GUS activity in the posterior
pole of a caryopsis at 18 DAP.
The whole-mount GUS analyses of grain from transgenic
barley plants transformed with pMDC164-PR602 revealed a
spatial pattern similar to that in rice grain; GUS staining could
be seen as two parallel lines in the ventral groove zone of
transgenic T1 caryopses. The activity of the promoter was
detected in the ETCs and a few layers of adjacent starchy
endosperm cells (Figure 4b, B2 and B4). No GUS activity was
observed in control grain (Figure 4b, B1 and B3). No GUS
activity was found in transgenic caryopses before 10 DAP.
GUS expression in ETCs of barley was observed throughout
grain maturation. It started at 10 DAP and finished at 30 DAP
(Figure 4b, B6 and B12). In one of the six barley lines (line 5),
GUS expression was weaker than in the other lines, but the
OsPR602 promoter was still active at 35 DAP and GUS expres-
sion was observed in two to three layers of transfer cells.
A transverse section (B5) through the middle of the barley
caryopsis (line 1) at 10 DAP is shown in Figure 4b. The identities
of the aleurone layers at the endosperm adaxial axis and
modified aleurone cells near the abaxial side were already
established at this stage, and could be distinguished from the
central starchy endosperm cells. The GUS expression is clearly
seen in the transfer cell layer and adjacent starchy endosperm.
The spatial activation patterns of the OsPR602 promoter in
barley matched well with the data obtained for this promoter
in rice, except that the ventral pigment strand and the
vascular bundles down the chalaza pad in barley did not show
GUS expression. GUS activity was not detected in the flowers
or any vegetative tissues of the transgenic barley plants.
The temporal expression patterns of OsPR602 in rice
differed slightly from those in barley. In rice, GUS expression
started 3 days earlier and stopped 6 days earlier than in
barley.
Spatial and temporal control of GUS expression by the
OsPR9a promoter in transgenic rice and barley
The whole-mount GUS staining of a T1 caryopsis of rice at
26 DAP is shown in Figure 5a (A2 and A3). OsPR9a activity
was present in the rachilla, anthers and mature pollen shortly
before pollination (Figure 5a, A1). No activity in the anthers
and mature pollen was observed for the OsPR602 promoter
in either rice or barley. No activity of the OsPR9a promoter
was detected in the vascular tissues of the lemma and palea
or in other plant tissues. The strongest OsPR9a promoter
activity in rice was found in aleurone transfer cell layers
between 5 and 26 DAP. After 26 DAP, the activity diminished,
and no GUS activity was detected later than 35 DAP.
In the transgenic T1 barley caryopsis, the OsPR9a promoter
was active in the ETC layers and the adjacent starchy
endosperm cells (Figure 5b, B3, B4, B6 and B7). This resembles
the pattern observed for the OsPR602 promoter in barley.
The promoter was active at 9 DAP and activity was detectable
until 35 DAP. No GUS staining was found in the pedicels and
anthers of transgenic barley plants before or at anthesis. No
GUS activity was detected in vegetative plant tissues.
Although transgenic lines were generated with different
levels of GUS expression, on the whole, the OsPR9a promoter
was weaker than the OsPR602 promoter.
Discussion
The manipulation of grain size, shape and composition
depends on access to seed-specific promoters, in particular,
promoters that can drive transgene expression during early
endosperm development. An important group of promoters
are those that are active in the ETC. Several ETC-specific
genes have been described in the literature (Hueros et al.,
1995, 1999b; Doan et al., 1996; Gomez et al., 2002; Gutierrez-
Marcos et al., 2004; Muniz et al., 2006), but the cloning and
analysis of only one ETC-specific promoter, from BETL-1 of
maize, has been reported (Hueros et al., 1999a). The activity
of the BETL-1 promoter was tested in maize, but not in other
plant species. A number of sequences were identified
encoding cysteine-rich proteins similar to the products of the
END1 and BETL genes in mRNA from the liquid fraction of the
syncytial endosperm of wheat. Northern blots and quantitative
PCR confirmed that transcription of two of the cloned genes,
designated TaPR60 and TaPR9, is specifically activated in
grain at the end of the cellularization phase, and that the
genes are expressed until the end of grain maturation
(Figure 2). The cloning of wheat promoters is still a slow
process because of the absence of a genome sequence. In
contrast, rice genome databases are available, but ETC-
specific genes from rice have not been reported. Therefore,
ETC-specific promoters from rice were identified and
characterized in rice and barley to evaluate their broad
applicability in cereal biotechnology. Protein sequences
of TaPR60 and TaPR9 were used to identify their rice
homologues, OsPR602 and OsPR9a, respectively, in rice EST
databases. The alignment of the protein sequences derived
from the rice ESTs with proteins from other plants is shown
in Figure 1. Quantitative PCR analysis of OsPR602 and OsPR9a
expression revealed their specific expression in panicles
472 Ming Li et al.
© 2008 ACPFGJournal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 6, 465–476
before pollination and at 11 DAP. This is very similar to the
data obtained for wheat homologues, except that, in rice,
expression was detected in pre-anthesis panicles (flowers)
and expression in both panicles and developing grain strongly
decreased after 11 DAP.
The nucleotide sequences of the coding regions of
OsPR602 and OsPR9a were subsequently used to identify the
translation start sites of their genes. Regulatory sequences
containing promoters and 5′-untranslated regions (5′-UTRs)
were cloned, and the activity of the promoters was analysed
using the stable transformation of rice and barley plants with
promoter:GUS gene fusion constructs.
Surprisingly, the OsPR602 promoter in rice was active not
only in ETCs, but also in transfer cells of maternal tissue.
Indeed, the spatial pattern was similar, but not identical, to
the previously described expression pattern of END1 in barley
and wheat, as shown by in situ hybridization analyses (Doan
et al., 1996; Drea et al., 2005). In rice, the pericarp has four
vascular bundles, one on each of the ventral and dorsal sides,
and one on each side face. The vascular bundle on the dorsal
side is the widest. It contains many conducting elements and
extends to the upper end of the grain (Hoshikawa, 1989;
Krishnan and Dayanandan, 2003). The activity of OsPR602
was found in the dorsal vascular bundle, but was not
detected in the other three narrow vascular bundles of the
grain. In the early stages (7–12 DAP) of rice endosperm
differentiation, the OsPR602 promoter was active in the ovular
vascular trace, the adjacent pigment strand, the adjacent
nucellar epidermis, the three to four layers of ETCs and the
adjacent starchy endosperm cells. Although the activity of the
OsPR602 promoter was not detected in the remaining three
narrow vascular bundles of the pericarp, it was observed in
vascular tissues of the lemma and palea shortly before anthesis
(Figure 4a, A2). The lemma has five swollen vertical ridges
Figure 5 Analysis of β-glucuronidase (GUS) expression in rice (a) and barley (b) directed by the OsPR9a promoter. GUS activity detected in flowers at anthesis (A1), and in uncut (A2) and hand-cut (A3) caryopsis of rice at 26 days after pollination (DAP). Hand-cut transverse and longitudinal sections of wild-type caryopsis at 18 and 10 DAP (B1, B2) and transgenic barley caryopsis at 20 DAP (B3, B4). Histochemical GUS assay and safranin counter-staining in transverse section of wild-type caryopsis at 20 DAP (B5), and in transverse (B6, B8) and longitudinal (B7) sections of transgenic barley caryopsis at 20 DAP. Arrows show GUS-stained tissues. An, anthers; ETC, endosperm transfer cell; Ra, rachilla. Bars, 1 mm (except B8, 0.25 mm).
Endosperm-specific promoters from rice 473
© 2008 ACPFGJournal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 6, 465–476
with a vascular bundle running through each. The palea has
three vascular bundles – one at the centre of the dorsal
surface and one on both sides (Hoshikawa, 1989). GUS
activity was found in all eight vascular bundles at anthesis,
but disappeared at the early stages of grain development.
The OsPR9a promoter showed rather different patterns of
GUS expression in rice flowers. It was active in the rachilla,
anthers and the mature pollen shortly before pollination, but
showed no activity in the ovary or vascular tissue of the
lemma and palea (Figure 5a, A1). GUS expression was detected
in aleurone transfer cells and adjacent layers of starchy cells,
but was not found in transfer cells of maternal tissues of grain
(data not shown).
The activity of the OsPR602 promoter in rice seeds was
similar to that of the barley asi promoter in developing trans-
genic barley seeds, for which gfp expression was observed
specifically in the pericarp, vascular tissue, nucellar projection
cells and ETCs (Furtado et al., 2003). However, in barley, both
the OsPR602 and OsPR9a promoters are active only in the
ETCs and adjacent starchy endosperm cells. GUS expression
could not be detected in either transfer cells of maternal
tissue or in flowers before pollination. A transcription factor
ZmMRP-1, which can specifically activate promoters in ETCs,
has been identified in maize (Gomez et al., 2002). The
expression pattern of GUS for OsPR602 and OsPR9a in flowers
is clearly different from the situation in maize, and could
mean that the transcription regulator from rice has a broader
specificity than its maize orthologue. However, it is more
probable that the OsPR602 and OsPR9a promoters are
activated by two different transcription factors. If this is the
case, it is probable that only the cis-element responsible for
expression in ETCs is conserved between the two species. In
maize, ZmMRP-1 can activate the BETL-1 promoter in vivo and
interact in vitro with the specific cis-element -TATCTCTATCTC-
from the promoter (Barrero et al., 2006). However, this element
was not found in the promoters of OsPR602 and OsPR9a.
Quantitative PCR data for the expression of OsPR602 and
OsPR9a genes, data obtained for their orthologues from
wheat, as well as data published for END1 and BETL (Hueros
et al., 1999b), indicate that transcriptional activation of the
promoters of these genes takes place at the middle of
endosperm cellularization at 3–6 DAP, reaches a maximum
activity at the end of endosperm cellularization at 8–10 DAP,
and the promoter becomes inactive close to the middle of
endosperm maturation at 20 DAP. A 3–5-day difference was
found in the activation of the same promoter in rice and
barley. This can be partially explained by the observation that
endosperm cellularization is complete at 6 DAP in rice, wheat
and maize, whereas, in barley, it ends at 8 DAP (Olsen, 2001).
If it is assumed that the transcriptional activation of OsPR602
and OsPR9a starts at the middle of the cellularization phase
of endosperm development, this could explain the temporal
difference in the initiation time of activation of both promoters
in barley relative to rice. It is interesting that a gradual
increase in GUS activity from the time of promoter activation
was not seen. Strong GUS activity appeared suddenly at
9–10 DAP.
Northern hybridization identified the weak expression of
END1 in the barley endosperm coenocyte at 5–7 DAP, with
strong expression from 9 DAP until 30 DAP (Doan et al.,
1996). In contrast, mRNA levels of BETL genes in maize grain
deceased at 15–20 DAP (Hueros et al., 1999b). The quantita-
tive PCR data obtained for the orthologous wheat genes
matched the maize BETL result. GUS expression in ETCs
of barley was observed during the whole phase of grain
maturation, long after the expected end of the transcriptional
activity of OsPR602 and OsPR9a promoters. Hence, the
detection of GUS at 30–35 DAP is most probably a result of
the stability of mRNA of the transgene and GUS protein in
ETC cells, rather than sustained expression. In most cases,
strong expression of GUS was observed until 20 DAP and it
then slowly diminished, suggesting an end of transcriptional
activity followed by slow degradation of mRNA and protein.
This implies that the OsPR602 and OsPR9a promoters can be
used to express transgenes until approximately 20 DAP, but
thereafter the amount of protein produced may be dependent
on mRNA and protein degradation.
The activity of the promoters was not quantified, as their
specific expression is restricted to a relatively small number of
grain cells, making quantification problematic. The strength
of GUS activity is also dependent on the number of copies
and position of transgene insertions in the genome. Never-
theless, the OsPR602 promoter appears to be several-fold
stronger than OsPR9a in both rice and barley.
This work suggests that the OsPR602 and OsPR9a promoters
are suitable for ETC-specific expression of genes of interest in
barley. As wheat and barley are closely related species, similar
expression patterns can be expected. However, for the
modulation of nutrient uptake in rice, the OsPR602 promoter
would be preferable for transgene expression, as the OsPR9a
promoter is also active in non-target organs, e.g. anthers and
pollen.
These promoters offer the potential to improve the grain
quality by modifying the quality and quantity of nutrient
uptake through the ETC. Improvements in disease resistance
may also be gained by enhancing the efficacy of this tissue as
a barrier to pathogen movement from maternal tissue to the
developing endosperm.
474 Ming Li et al.
© 2008 ACPFGJournal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 6, 465–476
Experimental procedures
Promoter cloning and plasmid construction
Promoters and 5′-UTRs of OsPR602 and OsPR9a were amplified fromrice cv. Nipponbare genomic DNA using the primers shown inTable 2. The proof-reading DNA polymerase Pfx (Invitrogen,Mulgrave, VIC, Australia) was used to minimize PCR-induced muta-tions and to produce blunt-end fragments for directional cloninginto the pENTR-D-TOPO vector (Invitrogen). The cloned inserts weresequenced and subcloned into the pMDC164 vector (Curtis andGrossniklaus, 2003) using recombination cloning. The resulting con-structs were designated as pMDC164-PR602 and pMDC164-PR9a,and were transformed into Agrobacterium tumefaciens strain AGL1by electroporation. The presence of plasmids in selected clones wasconfirmed by PCR using promoter-specific primers (Table 2).
Southern blot hybridization and quantitative PCR
analysis
Transgene integration into rice plants was confirmed by PCR usingGUS-specific primers (Table 2) with genomic DNA from selectedtransgenic lines. Transgene integration in barley plants was con-firmed by Southern blot hybridization. Genomic DNA from selectedbarley lines was digested with EcoRV, which cuts the T-DNA regiononce upstream of the selectable marker gene. The Southern blot wasprobed with the coding sequence of the hygromycin phosphotrans-ferase selectable marker gene.
Quantitative PCR was carried out according to Burton et al. (2004)using the primer combinations shown in Table 2.
Rice and barley transformation
The constructs pMDC164-PR602 and pMDC164-PR9a were trans-formed into rice and barley using Agrobacterium-mediated trans-formation and the method developed by Tingay et al. (1997) andmodified by Matthews et al. (2001). Rice (Oryza sativa L. ssp. Japonicacv. Nipponbare) and barley (Hordeum vulgare L. cv. Golden Promise)were used as donor plants. To prevent the generation of multiple plantsderived from the same transformation event, individual regenerantswere selected from independently transformed and cultured piecesof callus.
The promoter activity was tested in T0 and T1 plants (T1 and T2 grain).
Histochemical and histological GUS assays
GUS activity in transgenic barley plants was analysed by histochemicalstaining using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) (Bio Vectra, Oxford, CT, USA), asdescribed by Hull and Devic (1995). Different plant organs, wholegrain and grain sections of different ages were immersed in a 1 mM
X-Gluc solution in 100 mM sodium phosphate, pH 7.0, 10 mM sodiumethylenediaminetetraacetate, 2 mM FeK3(CN)6, 2 mM K4Fe(CN)6 and0.1% Triton X-100. After vacuum infiltration at ~88 043 Pa for20 min, the samples were incubated at 37 °C until satisfactory stain-ing was observed. Tissues were incubated in 20%, 35% and 50%ethanol, fixed in FAA (50% ethanol, 5% acetic acid and 4% formal-dehyde) and cleared in 70% ethanol. The whole-mount grains werethen observed under a dissecting microscope, and photographswere taken using a Leica digital camera (Leica, North Ryde, NSW,Australia). The grain samples were further dehydrated in 80% and90% ethanol, and then incubated in 95% ethanol with 0.05% ofthe counter-stain safranin orange for contrast. For longitudinal andtransverse sectioning, the tissues were embedded in paraffin wax,sectioned at 9–12 µm, deparaffinized and mounted in DPX mount-ant (Fluka Biochemika, Buchs, Switzerland), as described in Weigeland Glazebrook (2002). The specimens were observed under a com-pound microscope and photographed.
Acknowledgements
We thank Professor U. Grossniklaus for providing us with a
collection of pMDC vectors, Dr R. Burton for cDNA prepared
from rice tissues, and Ursula Langridge for assistance with
growing plants in the glasshouse.
This work was supported by the Australian Grain Research
and Development Corporation (grant no. UA00083 to P.L.),
International Postgraduate Research Scholarship (IPRS) and
Adelaide University Scholarship (AUS).
References
Albani, D., Hammond-Kosack, M.C.U., Smith, C., Conlan, S., Colot, V.,Holdsworth, M. and Bevan, M.W. (1997) The wheat transcriptionalactivator SPA: a seed-specific bZIP protein that recognizes theGCN4-like motif in the bifactorial endosperm box of prolamingenes. Plant Cell, 9, 171–184.
Table 2 A list of primer sequences used in reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (Q-PCR) and promoter cloning
Gene Forward primer Reverse primer
TaGAPdH (Q-PCR) TTCAACATCATTCCAAGCAGCA CGTAACCCAAAATGCCCTTG
TaPR60 (Q-PCR) GCCAGCAAATCCGAAGATAGAT TTCTCCTCCCACAGCTAAACTAGAG
TaPR9 (Q-PCR) AGCCAATCCTCCATTGTACTGC CCAGACAACACAAGAGGCTCAAT
OsPR602 (promoter) CACCACTCAAAACGAGAAAACTCATTG GGCTATTGCTTTAGTATAAAGCAGC
OsPR9a (promoter) CACCGTGTTCTTCAGGGACGAAAATGATACTCC ATTAGATGATTTATAACACAATCTTATCTAC
GUS (RT-PCR) AGTGTACGTATCACCGTTTGTGTGAAC TACGGATGGTATGTGTCCAAAGCGGCGAT
Endosperm-specific promoters from rice 475
© 2008 ACPFGJournal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 6, 465–476
Barrero, C., Muniz, L.M., Gomez, E., Hueros, G. and Royo, J. (2006)Molecular dissection of the interaction between the transcrip-tional activator ZmMRP-1 and the promoter of BETL-1. Plant Mol.Biol. 62, 655–668.
Becraft, P.W. (2001) Cell fate specification in the cereal endosperm.Semin. Cell Dev. Biol. 12, 387–394.
Broekaert, W.F., Cammue, B.P.A., DeBolle, M.F.C., Thevissen, K.,DeSamblanx, G.W. and Osborn, R.W. (1997) Antimicrobialpeptides from plants. Crit. Rev. Plant Sci. 16, 297–323.
Burton, R.A., Shirley, N.J., King, B.J., Harvey, A.J. and Fincher, G.B.(2004) The CesA gene family of barley. Quantitative analysis oftranscripts reveals two groups of co-expressed genes. Plant Physiol.134, 224–236.
Conlan, R.S., Hammond-Kosack, M. and Bevan, M. (1999) Transcrip-tion activation mediated by the bZIP factor SPA on the endospermbox is modulated by ESBF-1 in vitro. Plant J. 19, 173–181.
Curtis, M.D. and Grossniklaus, U. (2003) A Gateway cloning vectorset for high-throughput functional analysis of genes in planta.Plant Phys. 133, 462–469.
Diaz, I., Vicente-Carbajosa, J., Abraham, Z., Martinez, M., Isabel-LaMoneda, I. and Carbonero, P. (2002) The GAMYB protein frombarley interacts with the DOF transcription factor BPBF andactivates endosperm-specific genes during seed development.Plant J. 29, 453–464.
Diaz, I., Martinez, M., Isabel-LaMoneda, I., Rubio-Somoza, I. andCarbonero, P. (2005) The DOF protein, SAD, interacts withGAMYB in plant nuclei and activates transcription of endosperm-specific genes during barley seed development. Plant J. 42, 652–662.
Doan, D.N.P., Linnestad, C. and Olsen, O.A. (1996) Isolation ofmolecular markers from the barley endosperm coenocyte and thesurrounding nucellus cell layers. Plant Mol. Biol. 31, 877–886.
Drea, S., Leader, D.J., Arnold, B.C., Shaw, P., Dolan, L. and Doonan,J.H. (2005) Systematic spatial analysis of gene expression duringwheat caryopsis development. Plant Cell, 17, 2172–2185.
Furtado, A., Henry, R., Scott, K. and Meech, S. (2003) The promoterof the asi gene directs expression in the maternal tissues of theseed in transgenic barley. Plant Mol. Biol. 52, 787–799.
Gomez, E., Royo, J., Guo, Y., Thompson, R. and Hueros, G. (2002)Establishment of cereal endosperm expression domains: identifi-cation and properties of a maize transfer cell-specific transcriptionfactor, ZmMRP-1. Plant Cell, 14, 599–610.
Gutierrez-Marcos, J.F., Costa, L.M., Biderre-Petit, C., Khbaya, B.,O’Sullivan, D.M., Wormald, M., Perez, P. and Dickinson, H.G.(2004) maternally expressed gene1 is a novel maize endospermtransfer cell-specific gene with a maternal parent-of-origin patternof expression. Plant Cell, 16, 1288–1301.
Hood, E.E. and Jilka, J.M. (1999) Plant-based production of xeno-genic proteins. Curr. Opin. Biotechnol. 10, 382–386.
Hood, E.E., Woodard, S.L. and Horn, M.E. (2002) Monoclonal anti-body manufacturing in transgenic plants – myths and realities.Curr. Opin. Biotechnol. 13, 630–635.
Hoshikawa, K. (1989) The Growing Rice Plant. Anatomical Mono-graph. Tokyo: Nobunkyo Press.
Hueros, G., Varotto, S., Salamini, F. and Thompson, R.D. (1995)Molecular characterization of BET1, a gene expressed in theendosperm transfer cells of maize. Plant Cell, 7, 747–757.
Hueros, G., Gomez, E., Cheikh, N., Edwards, J., Weldon, M., Sala-mini, F. and Thompson, R.D. (1999a) Identification of a promotersequence from the BETL1 gene cluster able to confer transfer-
cell-specific expression in transgenic maize. Plant Physiol. 121,1143–1152.
Hueros, G., Royo, J., Maitz, M., Salamini, F. and Thompson, R.D.(1999b) Evidence for factors regulating transfer cell-specificexpression in maize endosperm. Plant Mol. Biol. 41, 403–414.
Hull, G.A. and Devic, M. (1995) The beta-glucuronidase (gus)reporter gene system: Gene fusions, spectrophotometric, fluoro-metric, and histochemical detection. In: Methods in Plant MolecularBiology, Vol. 49. Plant Gene Transfer and Expression Protocols(Jones, H. ed), pp. 125–141. Totowa NJ: Hunsana Press Inc.
Krishnan, S. and Dayanandan, P. (2003) Structural and histochemicalstudies on grain-filling in the caryopsis of rice (Oryza sativa L.).J. Biosci. 28, 455–469.
Lamacchia, C., Shewry, P.R., Di Fonzo, N., Forsyth, J.L., Harris, N.,Lazzeri, P.A., Napier, J.A., Halford, N.G. and Barcelo, P. (2001)Endosperm-specific activity of a storage protein gene promoter intransgenic wheat seed. J. Exp. Bot. 52, 243–250.
Lijavetzky, D., Carbonero, P. and Vicente-Carbajosa, J. (2003)Genome-wide comparative phylogenetic analysis of the rice andArabidopsis Dof gene families. BMC Evol. Biol. 3, 17–28.
Lopato, S., Bazanova, N., Morran, S., Milligan, A.S., Shirley, N. andLangridge, P. (2006) Isolation of plant transcription factors usinga modified yeast one-hybrid system. Plant Methods, 2, 3–17.
Maitz, M., Santandrea, G., Zhang, Z.Y., Lal, S., Hannah, L.C., Salamini,F. and Thompson, R.D. (2000) rgf1, a mutation reducing grainfilling in maize through effects on basal endosperm and pediceldevelopment. Plant J. 23, 29–42.
Matthews, P.R., Wang, M.B., Waterhouse, P.M., Thornton, S., Fieg,S.J., Gubler, F. and Jacobsen, J.V. (2001) Marker gene eliminationfrom transgenic barley, using co-transformation with adjacent‘twin T-DNAs’ on a standard Agrobacterium transformationvector. Mol. Breed. 7, 195–202.
Mena, M., Vicente-Carbajosa, J., Schmidt, R.J. and Carbonero, P.(1998) An endosperm-specific DOF protein from barley, highlyconserved in wheat, binds to and activates transcription from theprolamin-box of a native B-hordein promoter in barleyendosperm. Plant J. 16, 53–62.
Muniz, L.M., Royo, J., Gomez, E., Barrero, C., Bergareche, D. andHueros, G. (2006) The maize transfer cell-specific type-A responseregulator ZmTCRR-1 appears to be involved in intercellular signal-ling. Plant J. 48, 17–27.
Olsen, O.A. (2001) Endosperm development: cellularization and cellfate specification. Annu. Rev. Plant Physiol. 52, 233–237.
Olsen, O.A. (2004) Nuclear endosperm development in cereals andArabidopsis thaliana. Plant Cell, 16, S214–S227.
Onate, L., Vicente-Carbajosa, J., Lara, P., Diaz, I. and Carbonero, P.(1999) Barley BLZ2, a seed-specific bZIP protein that interacts withBLZ1 in vivo and activates transcription from the GCN4-like motifof B-hordein promoters in barley endosperm. J. Biol. Chem. 274,9175–9182.
Potenza, C., Aleman, L. and Sengupta-Gopalan, C. (2004) Targetingtransgene expression in research, agricultural, and environmentalapplications: promoters used in plant transformation. In Vitro Cell.Dev. Biol. Plant. 40, 1–22.
Qu, L.Q. and Takaiwa, F. (2004) Evaluation of tissue specificity andexpression strength of rice seed component gene promoters intransgenic rice. Plant Biotechnol. J. 2, 113–125.
Russell, D.A. and Fromm, M.E. (1997) Tissue-specific expression intransgenic maize of four endosperm promoters from maize andrice. Transgenic Res. 6, 157–168.
476 Ming Li et al.
© 2008 ACPFGJournal compilation © 2008 Blackwell Publishing Ltd, Plant Biotechnology Journal, 6, 465–476
Shrawat, A.K. and Lörz, H. (2006) Agrobacterium-mediated trans-formation of cereals: a promising approach crossing barriers.Plant Biotechnol. J. 4, 575–603.
Sinclair, T.R., Purcell, L.C. and Sneller, C.H. (2004) Crop transformationand the challenge to increase yield potential. Trends Plant Sci. 9,70–75.
Su, P.H., Yu, S.M. and Chen, C.S. (2004) Expression analyses of a rice10 kDa sulfur-rich prolamin gene. Bot. Bul. Acad. Sin. 45, 101–109.
Suzuki, A., Wu, C.Y., Washida, H. and Takaiwa, F. (1998) Rice MYBprotein OSMYB5 specifically binds to the AACA motif conservedamong promoters of genes for storage protein glutelin. Plant CellPhysiol. 39, 555–559.
Thompson, R.D., Hueros, G., Becker, H.A. and Maitz, M. (2001)Development and functions of seed transfer cells. Plant Sci. 160,775–783.
Tingay, S., McElroy, D., Kalla, R., Fieg, S., Wang, M.B., Thornton, S.and Brettell, R. (1997) Agrobacterium tumefaciens-mediatedbarley transformation. Plant J. 11, 1369–1376.
Vicente-Carbajosa, J. and Carbonero, P. (2005) Seed maturation:developing an intrusive phase to accomplish a quiescent state.Int. J. Dev. Biol. 49, 645–651.
Vonwettstein, D. (1993) Genetic engineering and plant breeding,especially cereals. Food Rev. Int. 9, 411–422.
Wang, H.L., Offler, C.E. and Patrick, J.W. (1994a) Nucellar pro-jection transfer cells in the developing wheat grain. Protoplasma,182, 39–52.
Wang, H.L., Offler, C.E., Patrick, J.W. and Ugalde, T.D. (1994b) Thecellular pathway of photosynthate transfer in the developingwheat grain. 1. Delineation of a potential transfer pathway usingfluorescent dyes. Plant Cell Environ. 17, 257–266.
Wang, N. and Fisher, D.B. (1994) Monitoring phloem unloading andpost-phloem transport by microperfusion of attached wheatgrains. Plant Physiol. 104, 7–16.
Weigel, D. and Glazebrook, J. (2002) Arabidopsis: a Laboratory Manual.Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Wu, C.Y., Adachi, T., Hatano, T., Washida, H., Suzuki, A. andTakaiwa, F. (1998) Promoters of rice seed storage protein genesdirect endosperm-specific gene expression in transgenic rice.Plant Cell Physiol. 39, 885–889.
Yanagisawa, S. (2004) Dof domain proteins: plant-specific trans-cription factors associated with diverse phenomena unique toplants. Plant Cell Physiol. 45, 386–391.
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