Role of Conserved Non-Coding Regulatory Elements in
LMW Glutenin Gene Expression
Ange´la Juha´sz1*, Szabolcs Makai2, Endre Sebestye´n1, La´szlo´ Tama´s2, Ervin Bala´zs1
1Applied Genomics Department, Agricultural Research Institute of the Hungarian Academy of Sciences, Martonva´sa´r, Hungary, 2Department of Plant Physiology and
Molecular Plant Biology, Eo¨tvo¨s Lora´nd University, Budapest, Hungary
Abstract
Transcriptional regulation of LMW glutenin genes were investigated in-silico, using publicly available gene sequences and
expression data. Genes were grouped into different LMW glutenin types and their promoter profiles were determined using
cis-acting regulatory elements databases and published results. The various cis-acting elements belong to some conserved
non-coding regulatory regions (CREs) and might act in two different ways. There are elements, such as GCN4 motifs found in
the long endosperm box that could serve as key factors in tissue-specific expression. Some other elements, such as the
AACA/TA motifs or the individual prolamin box variants, might modulate the level of expression. Based on the promoter
sequences and expression characteristic LMW glutenin genes might be transcribed following two different mechanisms.
Most of the s- and i-type genes show a continuously increasing expression pattern. The m-type genes, however,
demonstrate normal distribution in their expression profiles. Differences observed in their expression could be related to the
differences found in their promoter sequences. Polymorphisms in the number and combination of cis-acting elements in
their promoter regions can be of crucial importance in the diverse levels of production of single LMW glutenin gene types.
Citation: Juha´sz A, Makai S, Sebestye´n E, Tama´s L, Bala´zs E (2011) Role of Conserved Non-Coding Regulatory Elements in LMW Glutenin Gene Expression. PLoS
ONE 6(12): e29501. doi:10.1371/journal.pone.0029501
Editor: John R. Battista, Louisiana State University and A & M College, United States of America
Received September 28, 2011; Accepted November 29, 2011; Published December 29, 2011
Copyright:  2011 Juha´sz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: juhasza@mail.mgki.hu
Introduction
Wheat is among the three mayor crops in the world used for
human consumption and animal feeding. Cereals serve as a major
source in calories and proteins in human nutrition. Prolamins, the
storage proteins of wheat play a crucial role in the quality of flour
based end-products. Their allelic structure and expression largely
determine the strength and extensibility of dough made from wheat
flour. Prolamins consisting of monomeric gliadins and polymeric
high molecular weight (HMW-) and low molecular weight glutenin
subunits (LMW-GSs) form a complex protein matrix, called the
gluten. The composition and size of this gluten highly depends on
the expression profile of the different components.
The LMW glutenin subunit gene family is encoded at the Glu-3
loci on the short arm of homeologous chromosomes 1A, 1B and
1D, respectively. Based on their N-terminal sequences they can be
grouped into three main groups. These three main groups were
initially characterized by their mature peptide sequences starting
with either isoleucine (LMW-i-type), serine (LMW-s-type) or
methionine (LMW-m-type) at their N-terminal [1,2,3]. Although
i-type genes are exclusively expressed in the A genome of wheat,
most types are expressed at multiple genomes parallel. If the
number and position of cysteine residues is also considered, 12
distinct groups can be characterized [4]. However, following the
grouping system of Ikeda the homogeneity found in some of the
LMW groups was too low, compared to other groups. If sequences
belonging to the same Ikeda group were aligned some groups
could be further clustered [5]. After a thorough characterisation of
the LMW glutenin sequences some specific short polypeptide
motifs positioned either close to the N-terminal region or mostly in
the C-terminal region have been identified. Using these charac-
teristics several further LMW glutenin gene types, as well as some
sub-types could be distinguished (Table 1). (Alignment of deduced
amino acid sequences of each LMW glutenin gene types is
presented in Supplemental material File S1). Storage protein genes
are specifically transcribed in developing wheat seeds, and their
expression is controlled primarily at the transcriptional level [6,7].
The synthesis and deposition of seed storage proteins (SSPs) is
subject to strict spatial and temporal regulation, which occurs
specifically only in the endosperm during the seed development.
Their expression sensitively depends on the availability of nitrogen
and sulphur in the grain [8] and shows similar profiles, indicating a
remarkable conservation in the machinery responsible for the
maturation gene expression programmes. Common regulatory
elements, such as cis-acting elements and trans-acting transcription
factors observed in monocot SSP genes have already been
reviewed [9,10,11]. The 2300 element or endosperm box is
conserved at around 300 nucleotides upstream of the transcrip-
tional start site in most of the cereal storage protein families
[12,13,14]. The endosperm box is approximately 30 nucleotides
long and is considered to be primarily responsible for the
endosperm-specific expression. It consists of two cis-elements, the
prolamin box (P-box) and the GCN4-motif [15]. The GCN4
motif, also known as the nitrogen element (N motif) was originally
found to be similar to the GCN4-binding motif in yeast [16].
Altogether seven motifs have been identified in storage protein
gene promoters in Poaceae [10]. Based on these results most of the
Poaceae SSP gene promoters are characterized by one GCN4 motif,
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two P-box, one Skn-1 and one GCAA motifs. The Skn-1 like motif
is a half site of the palindrome sequence recognized by the Storage
Protein Activator transcription factor (SPA) and was originally
identified in C. elegans. It also has been identified in storage protein
gene promoters of other cereals [10,17,18]. The GCAA motif is a
highly conserved DNA sequence in the promoters of storage
protein genes found in rice [19] and maize [20], thereby implying
the possibility of their controlling role in transcription. Next to
these elements some other motifs, such as the motif weakly similar
to the maize Opaque 2 (O2) bZIP recognition site have been
described. Alongside these elements, the AACA/TA motif, ACGT
motif and CCAAT motif was also suggested as being involved in
the prolamin gene expression regulation [15,19].
Based on our current knowledge there are three main
transcription factor families involved in the endosperm-specific
transactivation of the prolamin genes, the bZIP, the DOF and the
MYB transcription factors. Basic leucine zipper (bZIP) TFs have
broad binding specificity, binding not only to the ACGT core
sequences, but also to the GCN4 core (TGAGTCA) in wheat
prolamins or to the O2 recognition site in maize (TCCACG-
TAGA) [21]. Active function of bZIP transcription factors is highly
dependent on N availability. These transcription factors are stably
expressed upon amino acid starvation, and degrade as the amino
acid level reaches the demanded level in the cells [22]. The
Prolamin-box is recognized by plant-specific DOF transcription
factors that contain one zinc-finger domain. The wheat prolamin
box binding factor (WPBF) trans-activates the storage protein genes
via binding to the AAAG core motif in their promoter sequence
[23]. Plant MYB transcription factors represent a large family of
proteins which include the conserved MYB DNA-binding domain
in one, two or three copies. Its members, the R2R3-MYB factors,
are involved among others in regulation processes related to
flowering and seed development [24]. The AACA/TA motif is the
core binding site of HvGAMYB, OsMYB5 and TaMYB
transcription factors in barley, rice and wheat respectively
[25,26,27].
Transcriptional regulation and the number and composition of
cis-acting elements show some variation in the different prolamin
gene families. Sulphur rich prolamins (LMW glutenins, gamma-
and alpha-gliadins), as well as sulphur poor omega-gliadins,
possess similar regulatory system. The TATA box sequence
CTATAAA is highly conserved in all the prolamin genes and is
positioned around 260 nucleotides upstream of the start codon.
The presence of the bipartitate endosperm box at the position
2300 is also characteristic of most of the prolamin gene families,
except for the promoters of HMW glutenins. Some prolamin
genes contain additional complete or partial endosperm boxes.
The number and position of these additional elements shows some
differences among the gene families. Shewry and co-workers have
reviewed these endosperm boxes and based on their opinion the
additional complete or partial binding sites do not seem to be
required for the initial promoter activity and are not present in
gamma or omega-gliadin promoters [9]. Basic studies related to
the transcriptional regulation of LMW glutenin genes have been
published after the isolation of the LMW glutenin LMWG1D1
gene [28]. Two prolamin boxes, a CCAAT-motif and a TATA-
box were identified in the first 300 nucleotides upstream of the
start codon [28]. Based on similarities to other cereal storage
Table 1. Classification of the LMW glutenin genes types according to different grouping methods.
LMW glutenin gene
typea N-terminal LMW main typeb
Ikeda
Type/Groupc Locus .300
Reference
accession
IQA ISQQQQAPPFS LMW-i VI/11 Glu-A3 2 X07747
IPP ISQQQQPPPFS LMW-i VI/11 Glu-A3 + FJ447464
IQP ISQQQQQPPFS LMW-i VI/12 Glu-A3 2 FJ876821
IEN IENSHIPGLEK LMW-s II/4 Glu-B3/Glu-D3 + AY542898
MEN MENSHIPGLEK LMW-s - Glu-B3 ? + AY608420
MSP1 MENSHIPGLER LMW-s II/3 Glu-B3 + FJ447463
MSP2 MENSHIPGLER 2 EU369715
MSP3 MENSHIPGLER 2 EU369722
MSG1 METSHIPGLEK LMW-m I/1 Glu-D3 + EU189096
MSG2 METSHIPGLEK 2 EU369734
MSH METSHIPSLEK LMW-m I/2 Glu-B3/Glu-D3 + EU189089
MRC1 METRCIPGLER LMW-m V/10 Glu-D3 + EU189094
MRC2 METRCIPGLER + FJ615311
MDS1 MDTSYIPGLER LMW-m IV/6 Glu-A3 2 FJ549936
MDS2 MDTSCIPGLER 2 FJ549935
MSC1 METSCIPGLER LMW-m IV/8 Glu-A3/Glu-D3 + EU189091
MSC2 METSCIPGLER IV/9 2 AY994358
MSS1 METSCISGLER LMW-m IV/7 Glu-D3 2 AY831795
MSS2 METSCISGLER + DQ457416
MSV METSRVPGLEK LMW-m III/5 Glu-D3 + EU189098
Grouping method a is used in the present study ; method b is reviewed by D’Ovidio and Masci 2004 [65]; method c is based on Ikeda et al., 2002 [4]. The present study
uses a classification method which is based on the thorough comparison of the entire coding region [5]. Sequences longer than 300 nucleotides were used for the
promoter analyses, and they are labelled with a +sign. Shorter sequences or where no sequence information was available for LMW glutenin gene types were labelled
with a - sign.
doi:10.1371/journal.pone.0029501.t001
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protein promoters the 2300 element containing both the
prolamin box and the GCN4-like motif has also been identified
with an additional copy of each motif in close vicinity [15]. These
two copies of endosperm boxes form a so-called long endosperm
box (LEB) [29]. These studies were focusing on the identification
of cis-acting elements as well as basic processes through which the
LMWG1D1 gene is regulated. Papers related to the promoter
analysis of the LMWD1D1 gene describe the mechanisms of
transcriptional regulation only of one of the LMW glutenin gene
types. To better understand the roles of different LMW glutenin
types in the machinery of gluten formation, it is necessary to begin
by assessing the promoter characteristics of this gene family. In this
study, the 59 non-coding regions of most of the LMW glutenin
gene types were characterized. Expressional control of LMW
glutenin genes has been analysed in-silico. Current knowledge on
cis-acting regulatory sequences and possible interacting transcrip-
tion factors attached to these LMW glutenin promoters were
modelled.
Materials and Methods
Data collection and sequence characterization
Plant genomic sequence databases have been screened for
LMW glutenin genes. More than 360 LMW glutenin sequences
have been identified, from which genes with promoter sequences
have been collected and used for further analyses. Altogether 170
LMW glutenin genes have been identified with promoter lengths
between 30 and 2008 nucleotides (for sequences see Supplemental
material File S1). Only promoter sequences over 100 nucleotides
in length were used for further analyses. These sequences included
only the 59 non-coding regions of LMW glutenin genes from
Triticum aestivum L. and most of the i-type, m-type and s-type
sequences identified previously were represented (Table 1) [5,30].
For details of sequence alignment of characterised LMW glutenin
gene types see Supplemental material File S2. Non-coding cis-
acting elements have been identified using published results and
plant promoter databases like PLACE or PlantCARE [31,32].
Promoter profiles were graphically represented using the Promoter
Profiler tool developed by one of the authors. The presence or
absence of the various cis-acting elements was indicated by a 1 or 0
for each accession analysed and was collected into a data matrix.
Accessions belonging to the same LMW glutenin gene type were
aligned and compared using the tools DIALIGN [33] and CLC
Genomic Workbench (CLC Bio). Consensus promoter sequences
were generated using the algorithm of CLC Genomic Workbench
and were used to characterise the promoter types. Conserved
regulatory element regions (CREs) were identified manually after
sequences were aligned.
Method for calculating gene expression based on EST
libraries
Due to the high sequential similarity of LMW glutenin genes,
we did not use clustered ESTs for the apparent error rates of
clustering [34]. A BLAST search with strict parameters was used
instead, and all hits were analysed and judged individually. We ran
the search with the LMW genes as queries, and used the EST
sequences of the selected developing seed libraries representing
80567 ESTs as the search set (the blastn parameters were as
follows: -word_size: 128, -ungapped, -mismatch_penalty: 230). In the
next step, high scoring segment pairs (HSPs) were filtered to count
hits only with 100% identity. All other results were discarded.
Several queries had the same EST hits, as a result of high
sequence homology between LMW genes, so they had to be
compared and classified individually, as a given EST can only
belong to one query sequence. Each EST was assigned to the gene
where it showed the longest aligned length and highest hit identity.
Publicly available EST data from developing wheat seed
libraries of Glenlea (NCBI EST library identifiers #A9H, #A9I
and #A9J), Chinese Spring (#10469, #19547, #10479, and
Mercia (#FIN, #FIQ, #FIT) cultivars and a genotype of
unknown origin published by DuPont (#BSD, #BSE, #BSF,
#BSH, #BSG) have been used for the analyses. All expression
data were in a timeframe of 3 to 30 days after flowering.
Expression data were normalized against total EST hits found in
the single libraries and described as Transcript Per Million EST
values (TPM).
Statistical analyses
Cluster analysis was used to group promoters with similar
profiles. LMW glutenin sequences were characterized based on the
cis-acting elements observed in their promoter regions. The
presence or absence of promoter motifs was denoted with a 1 or
0 in a data matrix. These data were used for K-means clustering
using the Statistica 8.0 (StatSoft Inc.) program package. Prior to
the analysis, the number of clusters were determined using the tree
joining clustering algorithm. The two-way joining clustering
method was used to describe relationships between LMW glutenin
gene types and promoter motif patterns. The aim of this analysis
was to discover promoter motifs or promoter profiles directly
related to the individual LMW gene types.
To be able to compare the abundance of transcripts belonging
to the same gene-, promoter-types or CRE groups in the different
EST libraries a log-likelihood ratio statistic was carried out using
the method of Schaaf et al (2005) [35]. The log likelihood ratio test
(G-test) offers the null hypothesis (all libraries have the same EST
proportion), as well as a straightforward way to proceed when the
null hypothesis is rejected, and the EST proportions in the libraries
are not equal. The latter approach can be adapted to fit the design
of a particular expression analysis using subsets of cDNA libraries.
The G-test procedure assumes the sampling or within-library
variation to be binomially distributed. The use of the G-test on
contingency tables of libraries and specific and non-specific EST
counts can be envisioned as an extension of the use of the Chi-
squared test for the comparison of two libraries. Expression
analyses comparing levels of transcripts among the different
libraries have been carried out. Results were statistically confirmed
using the G-test and represented using the tool of Pavlidis and
Noble (2003) [36].
The function of CREs in gene expression was investigated using
the same statistical methods as used above. Accessions possessing
the same CRE patterns were clustered together. Cis-acting
elements outside the CRE regions were excluded from the
analysis. Expression levels of different CRE groups during the
seed development were charted and significant differences were
calculated using the G tests.
Results
Based on published results and plant promoter databases 24
variants of 12 different SSP gene specific cis-acting promoter
motifs have been identified (Table 2). Three different variants of a
GCN4 like motif and seven different variants of prolamin boxes
have been identified in the LMW glutenin gene promoters. All the
P-box motifs were identified based on sequence identities found in
wheat or other cereal species. P-box7 (TGTAAAAGT) was
originally identified in pea legumins [37]. LMW glutenin gene
type promoters (Figure 1) were described using sequences chosen
after comparing the promoters of each of the types separately.
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Polymorphism identified in the different endosperm boxes,
including the presence or absence of a complete LEB is presented
in table 3. Promoter profiles of individual gene types are presented
in the following section and are discussed based on their main
types, i-, s- and m-type, respectively. Most of the cis-acting
elements identified in the promoter sequences of LMW glutenin
gene family are concentrated in the first 1000 nucleotides
upstream of the start codon.
LMW i-type genes
LMW-i type gene promoters cannot be characterized by the
presence of a LEB; however, they contain an additional EB
around at the 2550 position. Next to these two complete EBs,
there are more P-boxes in the promoters at positions 2112, 2337
and 2411. A bZIP recognition site, SPA bZIP has been identified
around at the position 2650 upstream of the start codon. The
MYB1AT element is missing in the promoter of LMW-i type
genes, except in some genes with an N-terminal of
ISQQQQQQ(Q)PF, which tends to be an inactive form, due to
the inner stop codons in the coding gene sequences. The i-type
genes starting with ISQQQQPPPFS contain a RY core motif at
the position 2139, close to the double TATA box. All of the
promoter sequences contain an AACA/TA motif 2 on the
antisense strand around at the position of 2380. RY core motifs
have been identified on the antisense strand between the positions
of 2877 and 2974. Additional MYB core motifs (AACA/TA
motif 1 and 2) were identified further upstream in the sequences, at
the positions-1037 and -1666.
LMW s-type genes
Mature LMW glutenins starting with a serine belong either to
the MSP, IEN or MEN gene types. MSP genes are exclusively
encoded at the Glu-B3 locus and can be categorised at least into
three different groups based on their sequence characteristics.
Their promoter sequences, annotated as MSP1, MSP2 and MSP3
in this paper, also show some differences. In hexaploid wheat, only
MSP1 type gene promoters were found with a length of over 300
nucleotides and some MSP3 type promoters were identified with a
shorter length. The only difference we could identify among the
MSP type promoters was that these promoters do not possess the
CCAAT motif at the position 210 upstream of the start codon,
except for MSP3 type gene promoters. MSP type genes do not
have a complete LEB at position 2300, however, LEB is
characteristic of both MEN and IEN type genes. MSP1 genes
possess an additional complete EB, located at the position of 2560
accompanied by two or three Skn-1 like elements, and an AACA/
TA motif at 2441. Two additional MYB core motifs have been
identified in the distal promoter region, at 21788 and 21794.
Similar to the i-type sequences the MSP1 type genes also have a
SPA bZIP motif around at the position 2640. G-box like elements
Table 2. Cis-acting elements identified in the promoters of LMW glutenin gene types.
Name DNA binding motif Transcription factor References
(CA)n element CAAACAC bZIP Stalberg et al., 1996 [66]
CCAAT box CCAAT CBF Albani and Robert 1995 [67]
G box like element TGACGT bZIP Menkens et al., 1995 [68]
GCAA motif1 GCAAAAGTG bZIP Takaiwa et al 1996 [19]
GCAA motif2 GCAAAAGTA bZIP present study
GCN4 like motif 1 GTGAGTCAT O2 bZIP Albani et al., 1997 [29]
GCN4 like motif 2 ATGAGTCAT O2 bZIP Mu¨ller and Knudsen 1993 [16]
GCN4 like motif 3 GTGTGACAT O2 bZIP Albani et al., 1997 [29]
AACA/TA motif 1 TAACAA R2R3 MYB Diaz et al., 2002 [25]
AACA/TA motif 2 AACAAA R2R3 MYB Diaz et al., 2002 [25]
MYB1AT core AAACCA R2R3 MYB Abe et al., 1997 [69]
P-box 1 TGTAAAGT PBF DOF Kreis et al., 1985 [70]
P-box 2 TGCAAAG PBF DOF Sugiyama et al., 1985 [71]
P-box 3 TGTAAAG PBF DOF Colot et al., 1987 [28]
P-box4 TGCAAAC PBF DOF Norre et al., 2002 [72]
P-box5 TGCAAAAG PBF DOF Norre et al., 2002 [72]
P-box6 TGGAAAG PBF DOF US Patent Application 20090064374 [76]
P-box7 TGTAAAAGT PBF DOF Shrisat et al., 1989 [37]
RY core site CATGCA ABI3/VP1 Suzuki et al., 1997 [73]
Skn-1 like motif GTCAT bZIP Blackwell et al., 1994 [74]
SPA bZIP GATGACGTGTC O2 bZIP Mena et al., 1998 [38]
TATA-box 1 CTATAAATA TBP Bernard et al., 2010 [75]
TATA-box 2 GTATAAAG TBP Bernard et al., 2010 [75]
TATA like motif TATAA TBP Bernard et al., 2010 [75]
DNA binding motif – recognition sites, Transcription factors – bZIP- basic leuzin zipper, CBF – CCAAT binding factor, O2-bZIP – TF similar to Opaque 2 transcription
factor, R2R3 MYB – MYB transcription factor with two or three binding motif, PBF DOF – prolamin box binding factors of the DNA-binding with one finger domain
transcription factor family, ABI3/VP1 – Abscisic acid insensitive3 or viviparous1 transcription factors, TBP - TATA binding protein. Papers reporting the elements in wheat
or other cereal storage protein gene promoters are presented.
doi:10.1371/journal.pone.0029501.t002
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have been also identified at the positions of 2891 and 21760, in
proximity to one of the MYB recognition sites. All MSP promoter
sequences contain the MYB1AT core element at 234.
The IEN and MEN type LMW glutenins are located at the Glu-
D3 locus and show similar characteristics in their encoding genes.
The promoter regions of IEN type genes show some common
characteristics with the promoter region of MSP type genes, like
the presence of SPA bZIP at the position 2640. Further P-boxes
have been found at positions 2112 and 2406. A single GCN4 like
motif followed by a double Skn-1 like element was identified at
2557 upstream of the start codon. A Skn-1 like motif was found
further upstream in the sequence at 2735 both in MEN and IEN
type genes. This latter group also possesses an ACGT motif at
2193.
The antisense strands of s-type genes also contained some
conserved transcription factor recognition sites. All of the
sequences analysed contain an AACA/TA motif 2 at 2374, and
additional MYB motifs (MYB1AT core) have been found in MSP1
and IEN gene promoters. Anti-sense strands also contained some
RY core elements, conserved at the position of 2193 in IEN type
promoters and 21019 in MSP1 promoters.
LMW m-type genes
LMW m-type sequences are the most diverse group containing
at least seven different gene types. Based on the sequences of their
encoding genes some of them can be further divided into subtypes
(Table 1). MDS1 is a group of pseudo genes, containing inner stop
codons. In some cases, like in groups of MDS types and MSG2,
Figure 1. Promoter regions of the different LMW glutenin gene types. The first 1000 nucleotide region upstream of the transcriptional start
of LMW glutenin gene types is presented through specific examples. Accessions of genes possessing the presented promoter regions are indicated.
Both sense (+) and anti-sense (2) strands are presented. Conserved non-coding regulatory element regions are labelled with rectangles CRE1, CRE2
and CRE3. The identified cis-acting elements are labelled with different colours.
doi:10.1371/journal.pone.0029501.g001
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the promoter sequences found did not exceed 150 nucleotides in
length. Even in these cases the elements positioned closest to the
transcriptional start site (TATA box, CCAAT box and MYB1AT-
core element) were conserved. LEB is not generally characteristic
of m-type genes, except for MRC genes (Table 3).
METRCIPGLER type genes are one of the most abundant m-
type groups, with two subtypes MRC1 and MRC2. Next to the
LEB, their promoters contain an additional complete endosperm
box at 2582 nucleotides upstream of the start codon. Further P-
boxes are found at the positions 2416 and 2182. These P-box
motifs are not followed by GCN4 motifs, but are accompanied
either by a Skn-1 like motif or an AACA/TA motif. A P-box2
motif is also found close to the double TATA box. No conserved
MYB1AT core motif has been found in any of the promoters
belonging to the MRC type genes.
MSV type gene promoters show an entirely different profile
from the rest of the m-type genes. This group does not possess a
complete 2300 element. However, a complete endosperm box (P-
box3 + GCN4 element 2) was found at the position- 565 upstream
of the transcriptional start site. Similar to other m-type promoters
a P-box6 motif is preceding the conserved TATA box. No double
TATA box was identified in these gene promoters, but the
MYB1AT element was present at the position 234. An additional
RY core motif has been identified in the distal region (at 2651) of
these promoters.
Promoters of the MSH type LMW glutenin genes are present in
two different forms, with several common motifs in their
sequences. These promoters contain a MYB core element about
30 nucleotides upstream of the truncated LEB. Further conserved
P-boxes were identified at the positions 2112 (P-box2), 2387 (P-
box3) and 2422 (P-box7), respectively. The MYB1AT motif is
also present at 234. Sequences belonging to METSHIPGLEK
type genes can be divided into two subgroups, MSG1 and MSG2,
from which MSG2 type genes are pseudo genes. The most
distinctive difference found between MSG1 and MSG2 gene
promoters is that MSG2 promoters carry an extra MYB core
element in proximity to the conserved MYB1AT core at position
234. MSG1 genes contain only a truncated element. An
additional EB has been identified at 2421 upstream of the
transcriptional start site. A fifth prolamin box a P-box2 motif is
present 32 nucleotides upstream of the TATA box. Sequences
starting with METSCISGLER can be also divided into two
subtypes. Based on our recent knowledge no promoter information
is available for MSS1 type genes, except for some sequences
originating from Triticum tauschii. Promoters of MSS2 genes possess
an incomplete LEB. Similarly to other m-type gene promoters a P-
box2 motif is positioned at 2113, followed by a double TATA
box1 and a TATA box like motif. MYB1AT core is also present at
234.
METSCIPGLER type LMW glutenin gene sequences can be
also sub-grouped into two distinct groups, from which MSC1
sequences are more common in bread wheat. Some of the MSC2
genes contain inner stop codons. MSC1 type promoters do not
have a LEB. Their 2300 element is also partial, containing a P-
box3 at 2299 and a Skn-1 like element. A second prolamin box
(P-box4) is positioned at 2137 upstream of the start codon. The
position of this P-box is slightly shifted compared to the P-boxes of
the other m-type genes in the relevant regions and overlaps with a
Table 3. Polymorphism observed in the endosperm boxes of LMW glutenin gene types.
LMW glutenin typeLEB LEBcomposition additional EB
2300 element (EB1) EB2
IQA 2 Pbox1 – GCN4 like 1 Pbox5 no data
IPP 2 Pbox1 – GCN4 like 1 Pbox5 Pbox3 - GCN4 like 3
IQP 2 Pbox1 – GCN4 like 1 Pbox5 no data
IEN + Pbox1 – GCN4 like 1 Pbox5 – GCN4 like 3 –
MEN + Pbox1 – GCN4 like 1 Pbox5 – GCN4 like 3 –
MSP1 2 Pbox1 – Skn1 like Pbox 5- GCN4 like 3 Pbox3 – GCN4 like 2-Skn1 like
MSP2 no data no data no data
MSP3 no data no data no data
MSG1 2 Pbox1 – Skn1 like Pbox7 Pbox 7 –GCN4 like 3
MSG2 no data Pbox7 – Skn1 like
no data
MSH 2 Pbox1 – Skn1 like Pbox7
MRC1 + Pbox 1- GCN4 like 1 Pbox 7- GCN4 like 3 Pbox 4 – Skn1 like, Pbox3- GCN4 like 2-Skn1 like
MRC2 + Pbox 1- GCN4 like 1 Pbox7- GCN4 like 3
MDS1 no data no data no data no data
MDS2 no data no data no data no data
MSC1 2 Pbox1 – Skn1 like – no data
MSC2 no data no data no data no data
MSS1 no data no data no data no data
MSS2 2 Pbox1 – Skn1 like Pbox5- GCN4 like 3 no data
MSV 2 Pbox1 Pbox5 Pbox3- GCN4 like 2
EB1 – endosperm box at the position 2300 is the first EB in the Long Endosperm Box, EB2 – second endosperm box in the LEB; +- gene type contains a complete LEB, 2
gene type does not contain a complete LEB; no data – no sequence information is available.
doi:10.1371/journal.pone.0029501.t003
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RY core site at 2139. The antisense strands of m-type sequences
also contain some additional elements. The m-type genes, with a
cysteine at the 5th position of their N terminal domain (MRC,
MSC, MSS) all include an additional AACA/TA motif 2 at the
position-375. This motif is in the neighbourhood of a P-box motif
on the sense strand in most of these genes. This MYB core motif is
absent in the rest of the m-type sequences. The MSV type
promoter sequences include a small group of adjacent motifs at
2772, comprising an AACA/TA motif 2 followed by a Skn-1
motif and a GCN4 like motif.
Expression analysis of LMW glutenin EST sequences
Expression levels of LMW glutenin gene types varied due to the
differences observed in their allelic composition. Some gene types,
like MSS1 or MEN were underrepresented. Significant differences
were identified at p = 0.05 among MRC and MSV type genes in
the Glenlea and Chinese Spring libraries (Figure 2). Tendencies
observed for the rest of the gene types indicate that LMW glutenin
gene expressions follow two different profiles. A group of genes (i-
type genes and most of the s-type genes) show a continuously
increasing level of transcription during development, while the
other set of genes (e.g. m -type genes with cysteine in the fifth
position) take a normal curve distribution in their expression
profile. Their transcriptional maximum is reached at the mid-
phase of the grain filling around at 25 DPA. Allelic effects resulted
in significantly different expression profiles of the same LMW GS
gene type, like in the case of MRC genes in Glenlea and Chinese
Spring. In Glenlea, the MRC2 genes were strongly expressed, and
the level of expression reached the maximum at around 21 DPA.
In cv. Chinese Spring, the other gene type MRC1 was expressed,
and MRC2 was present only in a negligible amount. For the
detailed G test results see supplemental material File S4.
Comparison of the promoter types
A two-way joining cluster analysis was carried out to discover
relationships between LMW glutenin gene types and their
promoter profiles (Supplemental material File S3). There were
motifs whose presence or absence was unique to some of the LMW
glutenin gene types, like the presence of the MYB core motif at the
position 2164 in MRC type gene promoters or absence of the
Skn1 like motif at the position 2278 in MSV type promoters.
However, using the available data set no significant relationship
could be depicted. This suggests that there is a smaller level of
polymorphism detected in the promoters compared to the coding
sequences of the different LMW glutenin types. To reveal
common characteristics in the promoters of the different gene
types a comparative cluster –analysis was carried out using the
promoter motif patterns of the analysed LMW glutenin gene
accessions. The comparison was carried out on a sequence set
containing cis-acting elements located in the 460 nucleotides long
upstream region and resulted in five promoter types (Figure 3).
Characterization of the different promoter groups has been carried
out using the consensus sequences of each promoter group. Based
on the consensus sequences only a few elements seemed to be
conserved in all five groups. The position of the double TATA box
and a P-box2, about 34 nucleotides further upstream of the TATA
Figure 2. Comparison of expression profiles. ChSp, Gl, M and Dup abbreviations are for cDNA libraries originated from developing seeds of
cultivars Chinese Spring, Glenlea, Mercia and an unknown genotype published by DuPont respectively. EST counts are labelled with colours, the
smallest EST values are labelled with white, and the largest EST counts are signed in dark blue. 10 dpa, 20 dpa, etc. represents libraries isolated from
seeds at 10, 20, etc. days after anthesis.
doi:10.1371/journal.pone.0029501.g002
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box1 was highly conserved. Four of the five groups possess a
complete (Prom2 and Prom3) or partial LEB (Prom4, Prom5).
Only two promoter types, Prom1 and Prom5 possessed a
MYB1AT motif at the position 234. Prom2 and Prom3 promoter
types possessed a MYB core motif (AACA/TA motif1) at the
position 2164. These two MYB core motifs were followed by a
prolamin box (P-box4). Another P-box4 motif was found at 2412
in promoter types 2, 3 and 4, respectively. Two of these P-box 4
motifs (in Prom2 and Prom3) are accompanied by Skn-1 motifs.
Promoter types Prom2 and Prom3 are almost identical, except for
a CCAAT motif identified in Prom3 type at 2170.
Prom1 included sequences from the MSV, MSG, MSC and
MSH type sequences. Prom2 and Prom3 consisted of MRC2 and
MRC1 type sequences respectively. The MEN, IEN and i-type
(IAP, IPP and IQP) promoters grouped into Prom4, while Prom5
consisted of MSP1 and MSS sequences.
Using these datasets relationships between promoter profiles
and expression profiles were tested. Based on the results of the log-
likelihood ratio analysis all five promoter types showed significant
changes in the levels of transcripts. For the detailed G test results
see supplemental material File S5. Figure 4 presents expression
profiles of LMW glutenin genes belonging to the same promoter
types in cvs. Chinese Spring, Glenlea and the genotype by
DuPont. As it is seen both in Glenlea and the unknown genotype,
the expression levels of Prom2 type genes were the highest.
However, these types did not express in a high amount in Chinese
Spring, and Prom1 type genes showed the highest expression
levels. Most of the promoter types followed a normal distribution
in time, with a maximum level between 10 and 25 DPA. However,
gene types with promoter profiles Prom4 did not reach their
maximum in the mid-phase of the seed-development, which
indicates a longer operability of the transcriptional apparatus.
LMW glutenin gene promoters were aligned and examined for
regions possessing highly conserved cis-acting elements. Three
conserved non-coding element regions were identified (Figure 1).
The first conserved regulatory region (CRE1) contains motifs up to
2120 nucleotides upstream of the transcriptional site. This set of
cis-acting elements includes the TATA boxes, a CCAAT motif, a
MYB1AT motif, a GCAA motif2, a CAACAC motif and a P-box2
motif. The second group of elements contains motifs between the
2300 and 2420 nucleotides, while the third CRE group contains
promoter motifs between 2500 and 2650. CRE2 included
elements of the long endosperm box surrounded by several Myb
core motifs and some further elements. CRE3 included the region
around at the position 2550 containing another complete
endosperm box, some Skn-1 like motifs and an SPA bZIP motif.
In CRE1 regions three distinct motif patterns were observed,
while in both CRE2 and CRE3 regions there were four distinct
motif clusters to be distinguished. Significant differences were
calculated using the G tests. For the detailed results see
Supplemental material File S6. Based on the comparative analysis,
accessions with the same motif pattern in CRE1 followed a similar
distribution during the seed development, not depending on the
genotypes analysed (Figure 5). If motifs of CRE2 region were also
considered, accessions with the same motif order did not share
similar expression profiles in the different genotypes. However, if
Figure 3. Promoter profiles of consensus promoter types. Prom1, Prom2, Prom3, Prom4 and Prom5 represent groups of accessions
possessing the same promoter profiles. Clustering was made using the K-means clustering method and the promoter motif matrix as input data.
Accessions belonging to the same promoter cluster were aligned and their consensus sequence was used to present characteristic cis-acting
elements. Motifs present on both sense (+) and antisense (2) strands are represented with different colours.
doi:10.1371/journal.pone.0029501.g003
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cis-acting elements in CRE3 were considered accessions belonging
to the same CRE set showed similar expression profile in each
genotype.
Discussion
Based on the present knowledge there are no published results
reviewing the unique expression and transcriptional control of the
LMW glutenin gene family. To understand more detailed aspects
of expression and control of these genes, 59 non-coding regions of
the publicly available LMW glutenin gene sequence have been
analysed. Most of the elements we have found were already
identified in cereal storage protein gene promoters, but were not
discussed in detail in the case of the LMW glutenin genes. The
promoter of the LMW glutenin gene LMWG1D1 was previously
used to describe basic rules of tissue-specific expression in wheat
endosperm [15]. Primary cis-acting elements, such as the prolamin
box and the GCN4-like motif at the position 2300 and the
transcription factors bound to them have been previously
identified. In this study a more detailed analysis has been carried
out, and variants of known elements have been described. We have
identified some other elements which are present in proximity to
each other and seem to be conserved in several gene types.
Characterization of cis-acting elements in LMW glutenin
gene promoters
In vivo footprinting and electrophoretic mobility shift assays
revealed that the P-box within a LMW glutenin gene promoter is
recognised by the WPBF, a member of the DOF transcription
factors [38]. All DOF proteins analysed so far, except for a
pumpkin protein [39], recognized an AAAG motif flanked by
slightly different DNA sequences as the essential sequence element
in DNA-binding assays in vitro [40,41]. This indicates that DNA
sequences bordering the AAAG core-motif have limited impact on
the DOF–DNA interaction [42], although they might result in a
reduced level of expression [43]. The prolamin box taken out of
the context of its native promoter functioned as an active cis-acting
element not only in cultured endosperm cells but also in leaf-tissue
derived suspension cells [44].
Figure 4. Expression profiles of genes with different promoter profiles. Expression profiles are presented for the libraries originating from
the same genotype. TPM - normalised EST counts in transcript per million values. DPA – development stages in days after anthesis.
doi:10.1371/journal.pone.0029501.g004
Figure 5. Effect of conserved noncoding regions on expression levels. Expression levels of accessions belonging to the same CRE type are
presented in cultivars Glenlea, Chinese Spring and the unknown genotype by DuPont for all three CRE regions. CRE1.1, CRE2.1, CRE3.1 etc. represent
groups of accessions possessing the same CRE pattern in the relevant CRE1, CRE2 or CRE3 region, respectively.
doi:10.1371/journal.pone.0029501.g005
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Seven different P-box recognition motifs and three GCN4 like
motif variants have been characterized in the present study. Both
P-box and GCN4 variants are present in different number and
position in the different gene type promoters. A duplicated
endosperm box or long endosperm box might be characteristic of
the LMW glutenin gene promoters, but was also found in some
gamma gliadin gene promoters [45]. Our results indicate that the
LEB motif is characteristic but not common in all members of the
LMW glutenin gene family. It was present in gene types like IEN,
MEN or MRC, while the rest of the LMW GS gene types contain
only partial duplicates, missing either one or both of the GCN4-
like motif elements. In this latter group of promoters, the P-boxes
are usually followed by Skn-1 like motifs. Skn-1 transcription
factors bind to the half sites of the recognition sites of GCN4 type
bZIP TFs. It is quite possible that these two transcription factor
types are competitors for the same recognition site. It is not known
whether they induce the same changes, but they both seem to have
a positive regulatory role [17].
In most of the gene types, complete endosperm boxes have also
been found in other positions. The number of complete EBs and
their position, especially their proximity to other transcription
factor recognition sites might explain some of the differences
observed in the transcriptional levels of these genes (Figure 2). In
some cases, such as in MRC type genes, an additional complete
endosperm box at about 300 nucleotides upstream of the LEB
might have a primary role in the abundant transcription (Figure 1).
However, the highest expression level, which was found in the
group of MSV type genes, could not be related to the presence of
an increased number of complete endosperm boxes. On the
contrary, their 2300 elements are not complete; the promoter
sequences possess only two DOF binding sites (P-box3 and P-
box5) in the LEB region without any bZIP TF binding site in their
vicinity. Similar to the MRC type genes they do contain a
complete EB in the 2550 2580 region. To what extent this
additional EB region is responsible for the differences observed in
their expression level needs to be clarified with experimental data,
even though it is much more likely that additional proximal cis-
acting elements participate in the transcriptional regulation.
Such potential cis-acting elements, like the AACA/TA motifs or
GCAA motifs might have an additional moderating effect on the
transcriptional control of storage protein gene expression.
The GCAA motif is a highly conserved motif found in different
positions in the promoters of cereal storage protein genes, and is a
binding site for several bZIP TFs [19]. This motif overlaps with an
A+T-rich region in rice, was also found in proximity to MYB1AT
core and TATA boxes in one of the LMW glutenin types (MSS2).
The importance of A+T-rich elements for quantitative expression
has been demonstrated in various plant genes [46,47,48]. The
AACA/TA motif was identified as one of the binding sites of MYB
transcription factors in cereals [25,26]. Several different types of
MYB binding sites have been found in the different LMW glutenin
gene promoters close to one of the DOF or bZIP TF recognition
sites (Table 2). The AACA/TA motif 1 is in 20 nucleotides
distance from P-box4 in both MRC type genes and was found at a
30 nucleotides distance in the MSH type genes. A more conserved
AACA/TA motif 2 (at 2375) was found on the anti-sense strand
in about 60% of the gene types. This motif is located at 25
nucleotides distance from a DOF binding site (P-box4), except for
gene types MSC1 and MSS2. These two gene types show a
relatively low level of expression, which might be directly related
to the absence of this motif. The same AACA/TA motif is also at
70 nucleotides proximity to the following GCN4+P-box motif pair
of the LEB region in MRC, MSS, MSP, IEN and i-type genes.
Another highly conserved MYB TF recognition site is present at
34 nucleotides upstream of the start codon. This motif, which was
identified in the promoter of a dehydration-responsive gene in
Arabidopsis thaliana might have a repressing role in the later phase of
endosperm development. The MYB1AT motif is highly conserved
in most of the LMW glutenin gene types, except for the MRC type
gene promoters and some of the i-type promoters.
The CCAAT box is one of the most common elements in
eukaryotic promoters. While many plant genes do not include a
CCAAT motif in their proximal promoter, this seems to be
another shared feature among seed storage protein genes [49].
Genes under the control of promoters containing CCAAT boxes
can be tissue- and/or stage-specific suggesting that patterns of
gene expression are also determined by other cis- and trans-acting
factors. CCAAT boxes are highly conserved within homologous
genes across species in terms of position, orientation, and flanking
nucleotides [50]. They are typically found between 60–100
nucleotides upstream of the transcription start site and are
operating in both direct and inverted orientations [51,52,53,54].
However, in most of the LMW glutenin gene types CCAAT box
motifs are located at 10 nucleotides upstream of the transcriptional
start site.
It seems that only s-type genes (MSP, IEN, MEN) do not
contain this motif. If we consider that transcript levels of these
gene types do not follow a normal distribution during the grain
filling, but seem to reach their expression maximum later, the
absence of this element might have a stabilizing function in the
transcriptional apparatus.
Role of promoter profiles and conserved non-coding
element regions
The composition and distribution of cis-acting elements in
LMW glutenin gene promoters indicate that the position and
order of some of the motifs might be more conserved compared to
other motifs. Some of these conserved sequences are overall
characteristic of storage protein gene promoters in cereals, such as
the 2300 element, while others might be gene family or
chromosome specific.
If promoters based on their motif patterns were grouped, the
expression analysis of their genes showed some statistically
significant differences. Most of the genes followed a normal
distribution in time, with a maximum level between 10 and 25
DPA. The other batch of genes did not reach their maximum
transcript level in the mid-phase and seem to express for a longer
time. Genes with promoter types Prom4 and Prom5 belong to this
latter group, both consisting s-type and i-type LMW glutenin gene
sequences mainly. The Prom1, Prom2 and Prom3 groups consist
mainly genes with m-type sequences. As s-type sequences are
mainly encoded at chromosome 1B and i-type sequences are
expressed exclusively at chromosome 1A, this difference observed
in their 59 non-coding sequences might arise from the evolutionary
differences of their genome progenitor species. In contrast with our
expectations, there were some striking differences in the expression
patterns of genes belonging to the same promoter types depending
on the cultivar analysed. The most obvious difference was seen in
the profiles of Prom2 and Prom3. Both promoter types belonged
to MRC type genes. While in Chinese Spring MRC genes with
Prom2 type promoters were expressed only in insignificant
amounts, this promoter type seemed to be the most abundant
both in cv. Glenlea and the unknown genotype by DuPont. An
opposite trend was observed in the case of the expression of genes
with Prom3 type promoters. Prom3 genes were expressed in high
amounts in Chinese Spring but were found only in low levels in
Glenlea and DuPont. Analysing the allelic composition of the
cultivars Glenlea and Chinese Spring, as well as the genes
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belonging to the Prom2 and Prom3 types, it became clear, that
the difference was rather allelic. Glenlea possess the Glu-D3c allele
which consists mainly of m-type gene sequences, among them
METRCIPGLER type sequences and on the other side Chinese
Spring possess a Glu-D3a allele. One of the differences between
Glu-D3a and c alleles is that while Glu-D3a contains mainly
MRC1 type genes with Prom3 promoter profiles (e.g. FJ615311)
GluD3c possess MRC2 type genes (e.g. EU189094) with Prom2
promoters.
Our results have shown that some gene types share similar
promoter characteristics and possess similar conserved non-
coding sequence regions. Conserved non-coding sequences were
identified in cereals using comparative analysis of different grass
genomes [55]. Cereal genomes exhibit a high level of synteny and
sequence similarity within coding regions, facilitating the
identification of orthologous gene sequences. Despite the
relatively low overall sequence identity among promoter
sequences from cereal genes compared with their coding regions,
numerous expression analyses and transgenic studies involving
promoter-target gene constructs have demonstrated that ortho-
logous genes from different cereal species often share highly
similar patterns of gene expression. Thus, these conserved
patterns of gene expression might be programmed by regulatory
sequences associated with conserved non-coding regions. This
sequence conservation is also characteristic on the prolamins of
Triticeae [56]. It has been reviewed by Shewry et al. (1994) that a
large number of seed proteins have similar sequences to the
proposed ancestral prolamin, which are also present within the
unique regions of the Triticeae cereal prolamins [57]. The earliest
divergence from this sequence was the insertion of repetitive
sequences, to produce the LMW-GS protein and the HMW-GS
protein groups of genes. This early step in prolamin evolution can
be supported by the low level of significant similarities shared in
these prolamin types’ promoter sequences. The ancestral LMW-
GS sequence diverged to give rise to the various sub-groups of
sulphur-rich gliadins, on the ground of small insertion and
deletion events across the gene, whereas the promoter regions
remained relatively similar [56]. A conserved non-coding
sequence survey of LMW glutenins might be a reasonable
method to discover some intra-family characteristics.
As LMW glutenin gene promoters were examined, three
conserved non-coding regulatory element regions (CREs) were
identified. All three CREs contained several different cis-acting
elements, which were conserved in all LMW glutenin gene types.
However, there were elements whose sequences showed some
polymorphisms. If the polymorphism found in the CRE patterns
was related to expression profiles, it became obvious that CRE
regions might have different regulatory roles in the transcription.
Motif patterns in CRE1 and CRE3 resulted in similar
expressional profiles independent from the genotypes analysed.
Cis-acting elements in these regions are responsible for quanti-
tative differences observed among the different LMW glutenin
gene types. Polymorphism detected in the CRE2 region did not
have an impact on the differences observed on transcription level
in the genotypes during the seed filling. CRE2 harbours the LEB
including the N-responsive GCN4-like motif in the 2300
element. This region might play a key role in the tissue-specific
regulation and might have less effect on quantitative differences
observed in transcript abundance. The conserved presence and
function of this region in other cereal and monocot species proves
the existence of a less diverged storage protein promoter,
compared to the polymorphisms detected in the coding sequences
[56].
Model for the transcriptional regulation of LMW glutenin
gene expression
There are several models published to describe basic mecha-
nisms and TF interactions involved in the transcription of cereal
storage protein genes, including that of the LMW glutenin genes
[11,15,29,58]. All models emphasize the importance of the
bipartitate endosperm box, containing a functional GCN4-like
motif and a prolamin box. The bZIP and DOF transcription
factors bound to these elements interact coordinated and play a
crucial role in the regulation of tissue-specific seed-storage protein
gene expression. The transcription factor bound to the GCN4 core
element might require two or more partners to form a functional
unit [43]. The model of Kawakatsu and Takaiwa (2010)
demonstrates the binding of an Opaque2 TF homo-dimer to the
transcription factor recognition sites in maize zein genes [11]. The
effect of these trans-actions was modulated by the presence of
MYB transcription factors, bound to AACA motifs. The AACA
motif was able to suppress the expression of glutelin genes in
vegetative tissues in transgenic rice experiments [59]. The MYB
binding sites were found in the neighbourhood of both P-box and
GCN4 like motifs of rice and barley genes [19,25,43,60,61]. MYB
TFs seem to have a significant role at the beginning of storage
protein expression which is underlined by their expression profiles
compared to bZIP and DOF TFs [25,62,63]. Both MYB and
DOF TFs have been expressed simultaneously at 10DPA in barley
endosperm. Therefore, the pattern of HvGAMYB transcript
accumulation was consistent with the possibility of HvGAMYB
being a regulator of the Hor2 hordein gene [25]. Hammond and
Kosack et al. (1993) described that at 13 days after flowering only
P-box recognition sites were occupied in the 2300 element of
LMW glutenin promoters, but no GCN4 bZIP trans-activation
has been observed at this stage [15]. Expression levels significantly
increased from 18DPA after bZIP transcription factors were
bound to their GCN4-like recognition motifs.
The use of different fragments of the LMW1D1 promoter has
proven the altered effect of several cis-acting elements in the tissue-
specific expression [29]. A significant level of uidA gene expression
in vegetative tissues of transgenic tobacco was observed when a
promoter containing a prolamin box only, without a GCN4-like
motif was used [29]. However, using different lengths of LMW1D1
promoter fragments, including both GCN4-like and Pbox motifs
no GUS expression could be detected in vegetative plant tissues
[64]. A LMW1D1 promoter variant containing the first 938
nucleotides resulted in significantly increased levels of expression
compared to the shorter version of the same promoter. The longer
promoter, which included an additional complete endosperm box
showed a similar developmental onset and an increased level of
GUS activity during seed development in transgenic wheat
experiments [15,64].
Results presented in this paper indicate that the expression of
LMW glutenin genes follow two different patterns. Most of the s-
and i-type genes show a continuously increasing expression
pattern. The m-type genes, however, demonstrate normal
distribution in their expression profiles. We propose that
differences found in their promoter sequences should be related
to the differences observed in their expression (Figure 6A and 6B).
One of the most significant differences is the presence of the
complete long endosperm box in most of the gene types showing
growing expression, while the rest of the genes carry only a
deficient LEB in their promoters. As the deficiency found was
mainly due to the absence of one or both of the GCN4-like motifs,
it might be possible that in the s- and i- types genes, the CRE2
region ensures tissue-specific expression without reduction in gene
expression upon the demanded amino acid levels reached. There
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was another important difference to observe, namely the presence
of an additional SPA bZIP binding site around at 2650
nucleotides (in CRE3) of the promoter types of genes with
continuously growing expression profiles. This motif was fully
absent in the other promoter types. The third most characteristic
difference was the presence or absence of a CCAAT box in the
basal promoter region (CRE1). The CCAAT box is usually
located at around 260–80 nucleotides upstream of the transcrip-
Figure 6. Model for the transcriptional regulation of LMW glutenin gene expression. Intensity of geometric forms marks the frequency of
these elements present in the promoter types or developmental phases. Most important cis-acting elements and the possible TF interactions are
labelled. A - Promoter profile of LMW glutenin genes following a continuously growing expression; B – Promoter profile of gene types following a
normal distribution in expressed transcripts during the seed development; C – model for transcriptional regulation in the early phase and mid phase
of expression. Conserved non-coding sequence regions (CRE1, CRE2 and CRE3) are labelled with open rectangles. Developmental stages are labelled
with DPA.
doi:10.1371/journal.pone.0029501.g006
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tional start site, but was found at the position 210 in the LMW
glutenin gene promoters with maximal expression profiles in the
mid-phase of seed filling. Whether CCAAT box has any direct
effect on this expression pattern is not known, however, as the
CCAAT box does not belong strictly to the basal promoter, it
might function as a repressor during the late phases of
development.
Based on the possible function of the different cis-acting
elements and the potential TF interactions the following model
can be described (Figure 6C). At the beginning of transcription,
elements located in the first conserved non-coding region (CRE1)
might fulfil a basal promoter activity initiating the transcription. At
around 10–13 days after anthesis MYB and DOF transcription
factors might already be expressed and bound to their recognition
sites in LMW glutenin genes (CRE1 and CRE2). The functional
MYB-DOF TF complexes ensure a moderate level of transcrip-
tion. This means that at the earlier phases of seed filling, while
bZIP transcription factors are not expressed yet to trigger
endosperm-specific expression, DOF transcription factors bound
to the prolamin boxes might be responsible for trans-activation of
the storage protein promoters. This basic level of expression might
be moderated with the contribution of MYB transcription factors
as suppressors in other vegetative tissues. These fundamental
processes might be further fine-tuned by the presence of additional
elements, such as CCAAT boxes. As later on, bZIP transcription
factors are bound to the GCN4-like motifs, Skn-1 like motifs and
SPA bZIP recognition sites, the CRE2 region fulfils its tissue-
specific regulator activity. This qualitative function is accompanied
with the quantitative moderator effect of interacting bZIP-DOF
and bZIP-MYB TF complexes in the CRE3 region and gene
expression reaches its maximal level. Based on this model the
position of motifs located in the first 1000 nucleotides upstream of
the transcriptional start determines their primary role as
qualitative or quantitative regulators. Motifs, such as prolamin
boxes or GCN4-like motifs are present both in the tissue specific
CRE2 motif and the distal CRE3 region. Polymorphisms in the
number and combinations of these cis-acting elements can be of
significant importance in the diverse levels of production of single
LMW glutenin gene types, with the help of further elements in the
less conserved promoter regions.
Conclusions
The study presented here was carried out on a statistically
limited sample set, which was due to the limited number of
available promoter sequences. Altogether 164 accessions were
used in the analyses. Some of the gene types, like MSS1, MEN or
IQA were poorly represented. Using this available dataset a model
for the transcriptional regulation of LMW glutenin gene
expression was developed and confirmed with different statistical
approaches. Our results indicate that LMW glutenin gene
expression follows two different patterns. A group of gene types,
mostly belonging to m-type LMW glutenin genes follow a normal
distribution expression pattern, starting to express at around the
tenth day after anthesys. They reach their maximum at around
21DPA and the amount of transcripts continuously decreases
while reaching the full mature status. The rest of the genes, mainly
s- and i-types show a stable level of transcription also after 21DPA.
Their promoter profiles and especially the composition of their
conserved regulatory element (CRE) regions also highlight some
important characteristics, based on which the differences observed
in their promoter patterns can be related to their expressional
profiles.
Later on, these data can be completed with further sequences
when available. However, as this research is an in silico analysis the
results need to be confirmed with wet-lab experiments. Based on
these we suggest to use these results as a guideline, and as a basis
for future in vitro experiments.
Supporting Information
File S1 Fasta files of LMW glutenin gene promoter
sequences retrieved from public databases and used for
analyses. Only sequences longer than 30 nucleotides are
mentioned.
(TXT)
File S2 Alignment file of deduced amino acid sequences
of the different LMW glutenin gene types. Gene accessions
representing the different gene types are the same as presented in
Table 1.
(TIF)
File S3 Results of two-way joining cluster analysis. X
axis represents cis-acting elements, and their position
(in brackets) identified in the LMW glutenin gene
accessions analysed. Y axis represents LMW glutenin gene
types involved in the analysis. Frequency of cis-acting elements in
the individual LMW glutenin gene types are labelled with colours
(from 0 – dark green to 1 – dark red)
(TIF)
File S4 Raw data and G-test results of relationships
between LMW glutenin gene types and their expression-
al profiles carried out based on the methods of Pavlidis
and Noble (2003) [36].
(XLS)
File S5 Raw data and G test results to describe
relationships between promoter types and expressional
profiles
(XLS)
File S6 Motif composition of CRE1, CRE2 and CRE3
regions, expressional data related to CRE groups and G
test result of CRE analysis.
(XLS)
Author Contributions
Conceived and designed the experiments: AJ SM LT. Performed the
experiments: AJ SM. Analyzed the data: AJ SM ES. Contributed reagents/
materials/analysis tools: AJ SM ES. Wrote the paper: AJ SM ES LT EB.
Designed the promoter profiler tool used in the promoter analysis: SM AJ.
References
1. Kasarda DD, Tao HP, Evans PK, Adalsteins AE, Yuen SW (1988) Sequencing
of protein from a single spot of a 2-D gel pattern: Nterminal sequence of a major
wheat LMW-glutenin subunit. Journal of Experimental Botany 39: 899–906.
2. Lew EJL, Kuzmicky DD, Kasarda DD (1992) Characterization of low molecular
weight glutenin subunits by reversed-phase high-performance liquid chroma-
tography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and N-
terminal amino acid sequencing. Cereal Chemistry 69: 508–515.
3. Masci S, Lew EJL, Lafiandra D, Porceddu E, Kasarda DD (1995)
Characterization of low molecular weight glutenin subunits in durum wheat
by reversed-phase high-performance liquid chromatography and N-terminal
sequencing. Cereal Chemistry 72: 100–104.
4. Ikeda TM, Nagamine T, Fukuoka H, Yano H (2002) Identification of new low-
molecular weight glutenin subunit genes in wheat. Theoretical and Applied
Genetics 104: 680–687.
LMW Glutenin Promoter
PLoS ONE | www.plosone.org 14 December 2011 | Volume 6 | Issue 12 | e29501

5. Juha´sz A, Gianibelli MC (2004) Information, hidden in the low-molecular-
weight glutenin gene sequences. In Proceedings of the 8th Gluten Workshop,
Viterbo, Italy, (D. Lafiandra, S. Masci and R.D’Ovidio eds.), The Royal Society
of Chemistry, Cambridge, UK. pp 62–65.
6. Bartels D, Thompson RD (1986) Synthesis of messenger-RNAs coding for
abundant endosperm proteins during wheat grain development. Plant Science
46: 117–125.
7. Sørensen MB, Cameron-Mills V, Brandt A (1989) Transcriptional and pos-
transcriptional regulation of gene expression in developing barley endosperm.
Molecular and General Genetics 217: 195–201.
8. Duffus CM, Cochrane MP (1992) Grain structure and composition. In:
Shewry PR, ed. Barley: Genetics, Biochemistry, Molecular Biology and
Biotechnology 291–317, CAB International, Wallingford, UK.
9. Shewry PR, Halfod NG, Lafiandra D (2003) Genetics of wheat gluten proteins.
In: Hall JC, Dunlap JC, T. Friedmann, eds. Advances in Genetics. Vol. 49
111–184, Elsevier Science, San Diego, USA.
10. Fauteux F, Stro¨mvik MV (2009) Seed storage protein gene promoters contain
conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae. BMC Plant
Biotechnology 9: 126–136.
11. Kawakatsu T, Takaiwa F (2010) Cereal seed storage protein synthesis:
fundamental processes for recombinant protein production in cereal grains.
Plant Biotechnology Journal 8: 939–953.
12. Forde BG, Heyworth A, Pywell J, Kreis M (1985) Nucleotide sequence of a B1
hordein gene and the identification of possible upstream regulatory elements in
endosperm storage protein genes from barley, wheat and maize. Nucleic Acids
Research 13: 7327–7339.
13. Hartings H, Lazzaroni N, Marsan PA, Aragay A, Thompson R, et al. (1990)
The b-32 protein from maize endosperm – characterization of genomic
sequences encoding 2 alternative central domains. Plant Molecular Biology 14:
1031–1040.
14. Kridl JC, Vieira J, Rubenstein I, Messing J (1984) Nucleotide-sequence analysis
of a zein genomic clone with a short open reading frame. Gene 28: 113–118.
15. Hammond-Kosack MCU, Holdsworth MJ, Bevan MW (1993) In vivo
footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat
endosperm. EMBO J 12: 545–554.
16. Muller M, Knudsen S (1993) The nitrogen response of a barley C-hordein
promoter is controlled by positive and negative regulation of the GCN4 and
endosperm box. Plant Journal 4: 343–355.
17. Washida H, Wu CY, Suzuki A, Yamanouchi U, Akihama T, et al. (1999)
Identification of cis-regulatory elements required for endosperm expression of
the rice storage protein glutelin gene GluB-1. Plant Molecular Biology 40: 1–12.
18. Gu YQ, Wanjugi H, Coleman-Derr D, Kong X, Anderson OD (2010)
Conserved globulin gene across eight grass genomes identify fundamental units
of the loci encoding seed storage proteins. Functional and Integrative Genomics
10(1): 111–22.
19. Takaiwa F, Yamanouchi U, Yoshihara T, Washida H, Tanabe F, et al. (1996)
Characterization of common cis-regulatory elements responsible for the
endosperm-specific expression of members of the rice glutelin multigene family.
Plant Molecular Biology 30: 1207–1221.
20. So JS, Larkins BA (1991) Binding of an endosperm-specific nuclear protein to a
maize beta-zein gene correlates with zein transcriptional activity. Plant
Molecular Biology 17: 309–319.
21. Izawa T, Foster R, Chua NH (1993) Plant bZIP Protein DNA Binding
Specificity. Journal of Molecular Biology 230:4: 1131–1144.
22. Kornitzer D, Raboy B, Kulka RG, Fink GR (1994) Regulated degradation of the
transcription factor Gcn4. The EMBO Journal 13:24: 6021–6030.
23. Conlan RS, Hammond-Kosack M, Bevan M (1999) Transcription activation
mediated by the bZIP factor SPA on the endosperm box is modulated by ESBF-
1 in vitro. Plant Journal 19: 173–181.
24. Du H, Zhang L, Liu L, Tang XF, Yang WJ, et al. (2009) Biochemical and
molecular characterization of plant MYB transcription factor family. Biochem-
istry (Mosc) 74(1): 1–11.
25. Diaz I, Vicente-Carbajosa J, Abraham Z, Martinez M, Isabel-La Moneda I,
et al. (2002) The GAMYB protein from barley interacts with the DOF
transcription factor BPBF and activates endosperm-specific genes during seed
development. Plant Journal 29: 453–464.
26. Suzuki A, Wu CY, Washida H, Takaiwa F (1998) Rice MYB protein OSMYB5
specifically binds to the AACA motif conserved among promoters of genes for
storage protein glutelin. Plant and Cell Physiology 39(5): 555–9.
27. Chen R, Ni Z, Nie X, Qin Y, Dong G, et al. (2005) Isolation and
characterization of genes encoding Myb transcription factor in wheat (Triticum
aestivem L.). Plant Science 169: 1146–1154.
28. Colot V, Robert LS, Kavanagh TA, Bevan MW, Thompson RD (1987)
Localization of sequences in wheat endosperm protein genes which confer tissue-
specific expression in tobacco. EMBO Journal 6: 3559–3564.
29. Albani D, Hammond-Kosack MCU, Smith C, Conlan S, Colot V, et al. (1997)
The wheat transcriptional activator SPA: a seed-specific bZIP protein that
recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin
genes. Plant Cell 9: 171–184.
30. Juha´sz A, Gianibelli MC (2006) Structure, characteristics and genetics of low
molecular weight glutenin subunits of wheat. In: W. Bushuk, F. Bekes, C.
Wrigley, eds. Gliadin and Glutenin: The unique balance of wheat quality, ISBN
1-891127-51-9 AACC. pp 171–212.
31. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory
DNA elements (PLACE) database. Nucleic Acids Research 27: 297–300.
32. Lescot M, De´hais P, Thijs G, Marchal K, Moreau Y, et al. (2002) PlantCARE, a
database of plant cis-acting regulatory elements and a portal to tools for in silico
analysis of promoter sequences. Nucleic Acids Research 30: 325–327.
33. Morgenstern B (2004) DIALIGN: Multiple DNA and Protein Sequence
Alignment at BiBiServ. Nucleic Acids Research 32: 33–36.
34. Wang JP, Lindsay BG, Leebens-Mack J, Cui L, Wall K, et al. (2004) EST
clustering error evaluation and correction. Bioinformatics, 20(17): 2973–84.
35. Schaaf GJ, Ruijter JM, van Ruissen F, Zwijnenburg DA, Waaijer R, et al. (2005)
Full transcriptome analysis of rhabdomyosarcoma, normal, and fetal skeletal
muscle: statistical comparison of multiple SAGE libraries. FASEB Journal 19(3):
404–6.
36. Pavlidis P, Noble WS (2003) Matrix2png: A Utility for Visualizing Matrix Data.
Bioinformatics 19: 295–296.
37. Shirsat A, Wilford N, Croy R, Boulter D (1989) Sequences responsible for the
tissue specific promoter activity of a pea legumin gene in tobacco. Molecular and
General Genetics 215: 326–331.
38. Mena M, Vicente-Carbajosa J, Schmidt RJ, Carbonero P (1998) An endosperm-
specific DOF protein from barley, highly conserved in wheat, binds to and
activates transcription from the prolamin-box of a native B-hordein promoter in
barley endosperm. Plant Journal 16: 53–62.
39. Kisu Y, Ono T, Shimofurutani N, Suzuki M, Esaka M (1998) Characterization
and expression of a new class of zinc finger protein that binds to silencer region
of ascorbate oxidase gene. Plant and Cell Physiology 39(10): 1054–64.
40. Yanagisawa S (2002) The Dof family of plant transcription factors. Trends Plant
Science 7: 555–60.
41. Yanagisawa S (2004) Dof domain proteins: plant-specific transcription factors
associated with diverse phenomena unique to plants. Plant and Cell Physiology
45: 386–91.
42. Yanagisawa S, Schmidt RJ (1999) Diversity and similarity among recognition
sequences of Dof transcription factors. Plant Journal 17: 209–14.
43. Wu C, Washida H, Onodera Y, Harada K, Takaiwa F (2000) Quantitative
nature of the Prolamin-box, ACGT and AACA motifs in a rice glutelin gene
promoter: minimal cis-element requirements for endosperm-specific gene
expression. Plant Journal 23: 415–421.
44. Ueda T, Wang Z, Pham N, Messing J (1994) Identification of a transcriptional
activator-binding element in the 27-kilodalton zein promoter, the -300 element.
Mol Cell Biol 14(7): 4350–9.
45. Pisto´n F, Marı´n S, Hernando A, Barro F (2009) Analysis of the activity of a
gamma-gliadin promoter in transgenic wheat and characterization of gliadin
synthesis in wheat by MALDI-TOF during grain development. Molecular
Breeding 23: 655–667.
46. Bustos MB, Guiltinan J, Jordano J, Begum D, Kalkan FA, et al. (1989)
Regulation of beta-glucuronidase expression in transgenic tobacco plants by an
A/T rich,cis-acting sequence found upstream of a french bean beta-phaseolin
gene. Plant Cell 1: 839–853.
47. Czarnecka E, Ingersoll JC, Gurley WB (1992) AT-rich promoter elements of
soybean heat shock gene Gmhsp17.5e bind 2 distinct sets of nuclear proteins in
vitro. Plant Molecular Biology 19: 985–1000.
48. Nietosoleto J, Ichida A, Quail PH (1994) Pf1 – an A-T hook-containing DNA
binding protein from rice that interacts with a functionally defined D(At)-rich
element in the oat phytochrome A3 gene promoter. Plant Cell 6: 287–301.
49. Thompson GA, Larkins BA (1989) Structural elements regulating zein gene
expression. BioEssays 10: 108–113.
50. Mantovani R (1999) The molecular biology of the CCAAT-binding factor NF-
Y. Gene 239: 15–27.
51. Bucher P (1990) Weight matrix descriptions of four eukaryotic RNA polymerase
II promoter elements derived from 502 unrelated promoter sequences. Journal
of Molecular Biology 212: 563–578.
52. Dorn A, Durand B, Marfing C, Le Meur M, Benoist C, et al. (1987) Conserved
major histocompatibility complex class II boxes – X and Y – are transcriptional
control elements and specifically bind nuclear proteins. Proceedings of the
National Academy of Sciences USA 84: 6249–6253.
53. Edwards D, Murray JA, Smith AG (1998) Multiple genes encoding the
conserved CCAAT-box transcription factor complex are expressed in Arabi-
dopsis. Plant Physiology 117: 1015–1022.
54. Mantovani R (1998) A survey of 178 NF-Y binding CCAAT boxes. Nucleic
Acids Research 26: 1135–1143.
55. Morishige DT, Childs KL, Moore LD, Mullet JE (2002) Targeted analysis of
orthologous phytochrome A regions of the sorghum, maize, and rice genomes
using comparative gene-island sequencing. Plant Physiology 130: 1614–1625.
56. Clarke BC, Appels R (1999) Sequence variation at the Sec-1 locus of rye, Secale
cereale (Poaceae). Plant Systematics and Evolution 214: 1–14.
57. Shewry PR, Miles MJ, Tatham AS (1994) The prolamin storage proteins of
wheat and related cereals. Progress in Biophysics and Molecular Biology 61:
37–59.
58. Vicente-Carbajosa J, Moose SP, Parsons RL, Schmidt R (1997) A maize zinc-
finger protein binds the prolamin box in zein gene promoters and interacts with
the basic leucine zipper transcriptional activator Opaque2. Proceedings of the
National Academy of Sciences USA 94: 7685–7690.
59. Zhao WM, Gatehouse JA, Boulter D (1983) The purification and partial amino-
acid-sequence of a polypeptide from the glutelin fraction of rice grains –
homology to pea legumin. FEBS Letters 162: 96–102.
LMW Glutenin Promoter
PLoS ONE | www.plosone.org 15 December 2011 | Volume 6 | Issue 12 | e29501

60. Yoshihara T, Takaiwa F (1996) Cis-regulatory elements responsible for
quantitative regulation of the rice seed storage protein glutelin GluA-3 gene.
Plant Cell Physiology 37(1): 107–11.
61. Yoshihara T, Washida H, Takaiwa F (1996) A 45-bp proximal region containing
AACA and GCN4 motif is sufficient to confer endosperm-specific expression of
the rice storage protein glutelin gene, GluA-3. FEBS Letters 383: 213–8.
62. Diaz I, Martinez M, Isabel-LaMoneda I, Rubio-Somoza I, Carbonero P (2005)
The DOF protein, SAD, interacts with GAMYB in plant nuclei and activates
transcription of endosperm-specific genes during barley seed development. Plant
Journal 42: 652–662.
63. Bevan M, Colot V, Hammond-Kosack M, Holdsworth M, Torres De Zabala M,
et al. (1993) Transcriptional control of plant storage protein genes. Philosophical
Transactions of the Royal Society B: Biological Sciences 342: 209–215.
64. Stoger E, Williams S, Keen D, Christou P (1999) Constitutive versus seed
specific expression in transgenic wheat: temporal and spatial control. Transgenic
Research 8: 73–82.
65. D’Ovidio R, Masci S (2004) The low-molecular-weight glutenin subunits of
wheat gluten. Journal of Cereal Science 39: 321–339.
66. Stalberg K, Ellerstom M, Ezcurra I, Ablov S, Rask L (1996) Disruption of an
overlapping E-box/ABRE motif abolished high transcription of the napA
storage-protein promoter in transgenic Brassica napus seeds. Planta 199:
515–519.
67. Albani D, Robert LS (1995) Cloning and characterization of a Brassica napus
gene encoding a homologue of the B subunit of a heteromeric CCAAT-binding
factor. Gene 167: 209–213.
68. Menkens AE, Schindler U, Cashmore AR (1995) The G-box: a ubiquitous
regulatory DNA element in plants bound by the GBF family of bZIP proteins.
Trends in Biochemical Sciences 20: 506–510.
69. Abe HK, Urao YST, Hosokawa TID, Shinozaki K K (1997) Role of Arabidopsis
MYC and MYB homologs in drought- and abscisic acid-regulated gene
expression. Plant Cell 9: 1859–1868.
70. Kreis M, Forde BG, Rahman S, Miflin BJ, Shewry PR (1985) Molecular
evolution of the seed storage proteins of barley, rye and wheat. Journal of
Molecular Biology 183: 499–502.
71. Sugiyama T, Rafalski A, Peterson D, So¨ll D (1985) A wheat HMW glutenin
subunit gene reveals a highly repeated structure. Nucleic Acids Research 13(24):
8729–8737.
72. Norre F, Peyrot C, Garcia C, Rance´ I, Drevet J, et al. (2002) Powerful effect of
an atypical bifactorial endosperm box from wheat HMWG-Dx5 promoter in
maize endosperm. Plant Molecular Biology 50(4–5): 699–712.
73. Suzuki M, Kao CY, McCarty DR (1997) The conserved B3 domain of
Viviparous1 has a cooperative DNA binding activity. Plant Cell 9: 799–807.
74. Blackwell TK, Bowerman B, Priess J, Weintraub H (1994) Formation of a
monomeric DNA binding domain by Skn-1 bZIP and homeodomain elements.
Science 266: 621–628.
75. Bernard V, Brunaud V, Lecharny A (2010) TC-motifs at the TATA-box
expected position in plant genes: a novel class of motifs involved in the
transcription regulation. BMC Genomics 12;11: 166.
76. US Patent Application 20090064374 - Expression cassettes for seed-preferential
expression in plants.
LMW Glutenin Promoter
PLoS ONE | www.plosone.org 16 December 2011 | Volume 6 | Issue 12 | e29501