From the Laboratorio de Bioquímica y Biología Molecular, Departamento de Biotecnología-UPM, ETS Ingenieros Agrónomos, 28040 Madrid, Spain
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ABSTRACT |
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A barley endosperm cDNA, encoding a
DNA-binding protein of the bZIP class of transcription factors, BLZ2,
has been characterized. The Blz2 mRNA expression is
restricted to the endosperm, where it precedes that of the hordein
genes. BLZ2, expressed in bacteria, binds specifically to the GCN4-like
motif (GLM; 5'-GTGAGTCAT-3') in a 43-base pair oligonucleotide derived
from the promoter region of a Hor-2 gene (B1-hordein). This
oligonucleotide also includes the prolamin box (PB; 5'-TGTAAAG-3').
Binding by BLZ2 is prevented when the GLM is mutated to 5'-GTGctTCtc-3'
but not when mutations affect the PB. The BLZ2 protein is a potent
transcriptional activator in a yeast two-hybrid system where it
dimerizes with BLZ1, a barley bZIP protein encoded by the ubiquitously
expressed Blz1 gene. Transient expression experiments in
co-bombarded developing barley endosperms demonstrate that BLZ2
transactivates transcription from the GLM of the Hor-2 gene
promoter and that this activation is also partially dependent on the
presence of an intact PB. A drastic decrease in GUS activity is
observed in co-bombarded barley endosperms when using as effectors
equimolar mixtures of Blz2 and Blz1 in
antisense constructs. These results strongly implicate the
endosperm-specific BLZ2 protein from barley, either as a homodimer or
as a heterodimer with BLZ1, as an important transcriptional activator
of seed storage protein genes containing the GLM in their promoters.
Hordeins, the major storage proteins of barley seeds, are
prolamins specifically synthesized in the starchy endosperm and are
classified according to their mobility in SDS-electrophoretic gels into
three major classes: B, C, and D, with the B fraction representing
~75% of the total hordein content in most barley cultivars
(cv.)1. All the hordeins are
structurally related, and their genes presumably derive from a common
ancestor by gene duplication and subsequent divergent evolution (1).
The coordinate expression of all hordein genes suggests common
regulatory mechanisms of transcription that should involve both
cis-acting motifs in their promoters and
trans-acting transcription factors (2).
A conserved cis-acting motif that is found in most storage
protein gene promoters of seeds is the endosperm box (EB; Refs. 3 and
4), a bipartite motif located around 300 bp upstream of the translation
initiation ATG codon, that contains two distinct nuclear protein
binding sites: the prolamin box (PB, 5'-TGTAAAG-3'), also called the
endosperm motif (EM), and a GCN4-like motif (GLM, 5'-(G/A)TGA(G/C)TCA(T/C)-3'), which resembles the binding site of the
yeast transcription factor GCN4 (5, 6). These two motifs are present in
B- and C-hordein promoters, whereas only the PB is present in that of
D-hordeins. Functional analysis of a native C-hordein promoter by
particle bombardment of developing barley endosperms (7) have
demonstrated that the GLM is the dominant cis-acting element
and that the PB exerts a silencing effect. However, both GLM and PB
from the promoter of a Hor-2 gene (B1-hordein) are essential
positive elements conferring a high level of transcriptional activity
to the minimal 35S CaMV promoter ( Much of what is known about the genetic and molecular mechanisms
regulating cereal seed storage genes comes from work on maize, where a
bZIP protein, OPAQUE 2 (O2; Refs. 9 and 10), has been shown to bind to
and activate transcription from an ACGT core motif adjacent to the PB
in the promoter of the 22-kDa class of zein genes (11, 12). In wheat
and barley, current knowledge about the transcription factors involved
in the regulation of storage protein genes is not as complete as in
maize although bZIP proteins, such as SPA from wheat and BLZ1 from
barley, have been described in endosperm that are able to interact with
the GLM binding site in prolamin genes. Whereas SPA is seed-specific, BLZ1 is also expressed in other tissues and both have been shown to
transiently transactivate appropriate reporter genes in
planta (8, 13). Recently, the O2 protein has been shown to
interact in vitro with another maize endosperm-specific
factor (PBF) of the Dof (DNA-binding with one
finger) class that recognizes the PB motif in the We describe here the isolation and characterization of a barley
cDNA encoding a novel endosperm-specific bZIP transcriptional activator, BLZ2 (gene Blz2, Barley
leucine zipper 2), that shows a
high degree of sequence similarity to SPA from wheat (13). The BLZ2
protein produced in bacteria binds in vitro to the GLM in
the EB of a B-hordein promoter and transiently transactivates transcription from the GLM in the homologous system, the barley developing endosperm. For maximum transcriptional activation, BLZ2
requires also an intact PB in the proximity of the GLM site. In
addition, BLZ2 activates transcription in the yeast two hybrid-system, where it can form heterodimers with BLZ1.
Plant Material--
Barley (Hordeum vulgare) cv.
Bomi was germinated in the dark, vernalized at 4 °C for
four weeks, and grown in the greenhouse at 18 °C under constant
illumination. Developing endosperms (7, 12, 17 and 21 DAP) and
7-day-old leaves and roots were frozen in liquid N2 and
stored at -70 °C until used for RNA extraction. Developing
endosperms for particle bombardment experiments were collected from
greenhouse plants and used immediately.
Screening of a Barley cDNA Library--
A Northern Blots--
Total RNA from different barley tissues was
isolated essentially as described (21). Blots were probed using the
recommended protocols of the manufacturer for Magna nylon membranes
(MSI). Hybridization was carried out at 65 °C, following standard
procedures (20). A 407-bp fragment (nt 1050-1457, shown in Fig. 1) was used as a specific Blz2 probe. The Northern blots were
subsequently hybridized with a specific probe for a Hor-2
gene (encoding a B1-hordein; Ref. 22) and with a barley 18 S
rDNA-specific probe (23) as a control for sample charge.
Expression of the BLZ2 Protein in E. coli--
To produce the
barley BLZ2 protein in E. coli cells, we used the pT7-7
plasmid (24). To obtain a transcriptional fusion of the Blz2
full-length coding sequence in the ATG-containing NdeI site
of the pT7-7 vector, a 1,230-bp Blz2 cDNA fragment (nt 1-1,230, shown in Fig. 1B) was amplified by the polymerase
chain reaction (PCR) using the following oligonucleotides as primers: (i) LO2FLs
(5'-CCGGGAATTCCATATGGAGCCCGTG
TTC-3'), a forward primer that incorporates EcoRI (double
underlined) and NdeI (single underlined) sites containing
the translation initiation codon (bold) of the longest open reading
frame of the Blz2 cDNA; and (ii) LO2FLas
(5'-TCGGATCCAAGCTTCCTACTGCATCAC-3'),
a reverse primer that added BamHI (single underlined) and
HindIII (double underlined) sites at the 3'-end of the
amplified fragment (stop codon in bold). This PCR fragment was
directionally cloned into an NdeI-BamHI digested
pT7-7 vector and, after sequencing confirmation, the resulting
pT7-7-Blz2 expression plasmid was introduced into the
E. coli BL21(DE3)/pLysS strain. Induction of BLZ2 expression and preparation of E. coli protein extracts were performed
as described previously (8). Cells carrying the pT7-7 vector with no
insert were identically processed as negative controls.
Electrophoretic Mobility Shift Assays (EMSAs)--
The 43-bp
probe containing the EB from the Hor-2 promoter (HOR) and
two mutated versions of it, respectively affecting the GLM (hor1) or
the PB (hor2) described in the corresponding figure, were produced by
annealing complementary single-stranded oligonucleotides that generate
5'-protruding ends. These probes were end-labeled with
[32P]dATP by the fill-in reaction (Klenow
exo-free DNA polymerase; United States Biochemical) and
purified from an 8% polyacrylamide gel electrophoresis (39:1
cross-linking). EMSA experiments were performed essentially as
described previously (8), using 0.5 ng of 32P-labeled probes.
Yeast Strains and LacZ Assays--
The effector plasmids pGBT9
and pGAD424 (CLONTECH), which contain the alcohol
dehydrogenase I (AdhI) promoter fused to the Gal4
DNA binding domain (Gal4DBD; pGBT9 vector) or to the
Gal4 DNA activation domain (Gal4AD; pGAD424
vector), respectively, were used to generate translational fusions with
Blz2 or Blz1 cDNAs or with selected fragments
derived from them. The haploid strain HF7c of Saccharomyces
cerevisiae (CLONTECH), carrying
LacZ (
To test if BLZ2 were a transcriptional activator in yeast, the
full-length Blz2 cDNA (nt 1-1,230, shown in Fig.
1B) was amplified with the LO2FLs and the LO2Flas primers
(see above) and inserted into the EcoRI and BamHI
sites of pGBT9 (pGBT9-Blz2) plasmid. The 5'-terminal coding
region of Blz2 (nt 1-612, shown in Fig. 1B), was
obtained by digestion of the pGBT9-Blz2 with
EcoRI and SspI and inserted into the
EcoRI and SmaI sites of pGBT9. All constructs
were checked by restriction digestion and sequencing. Yeast
transformation was performed by the polyethylene glycol method (25) and
transformants screened for Particle Bombardment in Barley Developing Endosperm--
The
reporter vector was a pUC19-derived plasmid containing the
Particle bombardment was carried out with a biolistic helium gun device
(DuPont PSD-1000) according to Kikkert (29). Gold particles (1.0 µm
in size) were prepared essentially as described by Taylor and Vasil
(30) by mixing 18 µl of gold suspension (60 mg ml Isolation of a Barley bZIP cDNA Encoding the Homeologue of
Wheat SPA--
The presence in wheat and maize of endosperm-specific
bZIP proteins, SPA and O2 (9, 10, 13), that activate transcription through interaction with the GLM or ACGT core, respectively, of the EB
in storage protein gene promoters, led us to search for their barley
counterpart. To isolate such a gene, we used the bZIP coding region of
the ubiquitously expressed barley Blz1 cDNA (8) as a
probe to screen, at moderate stringency, a
Among ten positive clones purified, after screening 5 × 106 plaque forming units, seven were different from
Blz1, and one of them, hereafter Blz2, containing
the longest insert, was selected for further characterization. The
restriction map and the DNA sequence of the insert (1,647 nt) in this
clone appear in Fig. 1, A and
B, respectively. Blz2 is a single copy gene (data
not shown) and encodes a bZIP protein (hereafter BLZ2) that contains 409 amino acid residues and a deduced molecular mass of 44,600 Da. Two
short open reading frames are found upstream of the ATG start codon.
This feature, shared by O2, Blz1, and some other transcription factor genes, may have a role in translation regulation (8, 31).
The BLZ2 protein has a typical bZIP domain (Fig. 1B) with a
basic region followed by leucine heptad repeats. A serine-rich motif
putatively involved in phosphorylation (32), two short stretches rich
in acidic residues that could be involved in transcription activation,
and a putative nuclear localization signal that spans the complete
basic region of the bZIP domain (33) are also found.
The BLZ2 protein is probably the homeologue of wheat SPA (13) because
they share 77.5% identical residues along the whole protein, and
94.8% in their bZIP domains. BLZ2 is also related to the barley BLZ1
protein (34.3% identity over the whole protein; 70.1% in the bZIP
domain), to O2 from maize, coix, and sorghum, to REB from rice, and to
OHP1 from maize and has limited but significant homology with CPRF2
from parsley and RITA1 from rice (see Table I and references of Fig.
2). The phylogenetic dendrogram based on
the comparison of the whole proteins and the sequence alignments that
appear in Fig. 2, clearly indicate that these proteins form a well
defined subfamily of plant bZIPs.
Blz2 Expression Is Restricted to the Endosperm--
The temporal
and spatial pattern of expression of the Blz2 gene was
examined by Northern blot analysis. Total RNA was isolated from
endosperms at different developmental stages, ranging from 7 to 21 DAP,
and from young leaves and roots (7 days after germination). After
hybridization with a Blz2-specific probe, the mRNA was
only detected in the endosperm (Fig. 3).
If BLZ2 were a regulator of hordein gene expression, then one would
expect the occurrence of Blz2 expression prior to and during
the synthesis of the hordein message. To establish this correlation,
the same blot was subsequently hybridized with a probe spanning the
coding sequence of a Hor-2 gene. The Blz2
transcript was already present at 7 DAP, peaking at 12 DAP and
decreasing thereafter (Fig. 3). In contrast, the B-hordein transcripts
were barely detectable at 7 DAP and were still expressed in late
endosperm development (21 DAP). The patterns of Blz2 and
hordein message accumulation are therefore consistent with the
possibility that Blz2 was an activator of the hordein genes.
BLZ2 Binds to the GLM from Hordein Gene Promoters--
To
investigate the involvement of BLZ2 in the regulation of hordein gene
expression, we tested if BLZ2 was capable of binding specifically to
the GLM within the EB sequence from the promoter of the
Hor-2 gene (HOR in Fig.
4A). This DNA fragment was
used because it represents a highly conserved sequence in the
Protein extracts of E. coli, expressing the BLZ2 protein,
were incubated with the 32P-labeled HOR sequence, and its
ability to bind to this probe was tested by EMSAs. As shown in Fig.
4A, two shifted bands appeared when BLZ2 was incubated with
the HOR probe. To further define the motifs within this probe that were
recognized by BLZ2, HOR derivatives containing mutations in the GLM
(hor1) or in the PB (hor2) were tested in EMSAs. Although the BLZ2 was
able to bind to the HOR oligonucleotide, the interaction was abolished
when the GLM 5'-GTGAGTCAT-3' was mutated to 5'-GTGctTCtc-3', as occurs when using the hor1 oligonucleotide (Fig. 4A). However, an
intact PB, 5'-CATGTAAAGTG-3', was not required for the in
vitro binding because mutations in this motif, 5'-gAgGTAAAtTt-3'
(hor2), did not affect the retarded complex formation (Fig.
4B). Specificity of all interactions were assessed by
competition experiments employing 50, 100, and 200-fold molar excess of
cold oligonucleotide probes. In these experiments, HOR and hor2 were
effective competitors, whereas hor1 was unable to compete the BLZ2
binding to HOR or hor2 (Fig. 4, A and B). These
results indicate that BLZ2 binds specifically to the GLM sequence
within the EB of hordein promoters.
BLZ2 Activates Transcription from the GLM of Hordein Promoters in
Bombarded Barley Endosperms--
The functional relevance of the
interaction observed in vitro between BLZ2 and the GLM was
further investigated in vivo by assessing the effect of BLZ2
on transient expression assays in co-bombarded barley endosperms. As
shown in Fig. 5A, GUS reporter constructs were generated containing in their promoters EB variants affected either in the GLM or in the PB, respectively. Developing endosperms (~15 DAP) were transiently transformed by particle bombardment with these reporters alone or in combination with the
Blz2 as effector at a 1:0.5 molar ratio. As represented in Fig. 5B, co-transfection of HOR- BLZ2 Functions as a Transcriptional Activator That Can Form
Heterodimers with BLZ1 in Yeast--
The coexistence of
Blz2 and Blz1 mRNAs in the barley endosperm
prompted us to test if BLZ2 and BLZ1 may form heterodimers in
vivo. We explored this possibility by the yeast two-hybrid system,
using as a "bait" the bZIP domain of BLZ2, fused to the GAL4DBD and
as a "prey" the whole BLZ1 or different fragments of it fused to
the GAL4AD. If these two proteins expressed in yeast do interact, then
the complex becomes a functional transcription factor, which is capable
of binding to the Gal1UAS in the promoters of reporter genes
HIS3 and LacZ, thus activating their transcription.
The constructs prepared are schematically represented in Fig.
6A. The cDNA encoding the
bZIP domain of Blz2 was fused to the Gal4DBD in
the pGBT9 plasmid ("bait" construction) and introduced into
S. cerevisiae HF7c strain. Subsequently, the Blz1
fragments fused to the Gal4AD in the pGAD424 plasmid
("prey"constructions) were introduced into the yeast cells. As
shown in Fig. 6B (lanes 2-5), all strains
co-transformed with cDNA fragments containing the leucine zipper
encoding regions of both proteins activated expression of the
HIS3 and LacZ reporter genes, indicating that BLZ2 interacts through its bZIP domain with BLZ1 in vivo. As
expected, no activation of the reporters was obtained when yeast was
co-transformed with the two pGBT9 and pGAD424 plasmids without inserts
(Fig. 6B, lane 1) or when transformed only with
the "bait" or with the "prey" constructions alone (Fig.
6B, lanes 6 and 7). As a positive control, we used the Blz1 cDNA in the pGBT9 plasmid
(Fig. 6B, lane 8) that had been previously shown
to be an activator in the yeast system (8). The Gal4AD
constructs in lanes 3-5 by themselves did not activate the
Gal1UAS promoter (data not shown).
To assess whether BLZ2 also functions in yeast as a transcriptional
activator, we cloned the full-length or 5'-terminal Blz2 cDNA in the pGBT9 vector and introduced it into S. cerevisiae HF7c strain. As shown in Fig. 6B
(lanes 9 and 10), BLZ2 is a transcriptional activator, and the N-terminal region is sufficient to activate transcription of the reporter genes.
Do BLZ2 and BLZ1 Interact in Bombarded Barley
Endosperm?--
Having established that BLZ2 heterodimerizes with BLZ1
in yeast and that BLZ2, as did BLZ1 (8), mediates transcriptional activation in barley endosperm from the GLM of hordein promoters, we
decided to evaluate the contribution of BLZ2 and BLZ1 to such activation in developing barley endosperms. For this purpose, experiments of co-bombardment were done using the HOR-
As shown in Fig. 7B, BLZ2 and BLZ1, at 0.5:1 ratio to the GUS reporter,
were able to transactivate approximately 3-fold the GUS expression from
the HOR- We have characterized a cDNA clone from barley that encodes an
endosperm-specific bZIP transcription factor (BLZ2) that activates transcription in the homologous tissue (developing barley endosperm) through interaction with the GLM sequence from prolamin gene promoters. In addition, BLZ2 can function as an activator in yeast where it is
capable of heterodimerizing with BLZ1.
The pattern of expression of Blz2 would be consistent with a
putative role in the transcription regulation of those
endosperm-specific genes whose temporal mRNA expression overlap
with that of Blz2. Genes encoding B-hordeins (such as
Hor-2), which account for the major fraction of storage
proteins in barley seeds, follow this expression profile.
The EB of the promoter of a Hor-2 gene is made of two
cis-motifs, PB and GLM, that are well conserved in the
promoters of prolamin genes of the Pooideae grasses, such
are those encoding B- and C-hordeins in barley (22, 36, 37), To investigate the role of BLZ2 in planta, we used transient
expression assays in the barley endosperm tissue where it is naturally
expressed. It is well known that in dicotyledonous hosts the
quantitative or qualitative expression patterns controlled by
monocotyledonous seed-specific gene promoters are frequently altered
(38, 41-43). To avoid these problems, we used a system based on
microparticle bombardment of the homologous developing endosperm that
has been successfully experimented by us and other groups (7, 8, 15,
44). When a reporter gene controlled by the EB from the
Hor-2 promoter fused to the 5'-end of the minimal BLZ2 also behaves as a transcriptional activator in yeast and the
N-terminal region of the protein is sufficient for that activation. The
possibility of protein-protein interactions between BLZ2 and BLZ1, has
also been investigated in the yeast two-hybrid system. These bZIP
proteins are able to interact and, similarly to the vast majority of
factors belonging to this group, the bZIP domain is sufficient to
sustain the dimerization between BLZ2 and BLZ1 (17-19, 45-47).
Considering the potential of BLZ2 to heterodimerize with BLZ1 in
vivo, it is worth noting that such interactions, which are common
in the bZIP family of transcription factors, allow the elaboration of
complex regulatory networks based on the different properties of homo-
and heterodimers in terms of DNA binding and transcription regulation.
Extensively documented examples of such mechanisms exist both in the
animal kingdom and in plants where the presence of different family
members in a given tissue can modify substantially the final regulatory
effect (17, 19, 47, 48).
Transient expression assays were conducted with effector plasmids
carrying Blz2 and/or Blz1 constructs in antisense
orientation to investigate the effects of their depletion. The mRNA
expression of Blz2 in developing endosperm was only
partially counteracted by the antisense approach, and a more effective
GUS reduction was obtained with the Blz1 antisense
construct. However, it was remarkable the effect achieved by
co-transforming with both Blz2 and Blz1 antisense
effectors, which resulted in a dramatic reduction of the basal GUS
activity even at the lowest effector/reporter ratio tested. This
suggests a synergistic effect by a possible BLZ2/BLZ1 heterodimer in
barley endosperm. It is worth noting that the GLM sequences present in
most hordein promoters are of the AP-1 type (AC/GT cores), this being a
constraint for the binding by other plant bZIP factors belonging to the
ATF/CREB group that recognize the ACGT core (49, 50). This observation
together with the data concerning the transcriptional properties
in planta of BLZ2 and BLZ1, strongly support the fact that
both proteins are significant, if not the unique, components of the
machinery that mediates transcription activation through the GLM.
However, we cannot rule out the contribution of other factors to the
endosperm box complex. In this context, interactions with the barley
PB-binding factor (15) could account for major differences in their
mode of action as compared with homo- or heterodimer bZIP formation. We
are currently investigating this possibility.
Barley BLZ2 and wheat SPA show a relevant sequence homology that is not
restricted to the bZIP domain, similar endosperm-specific expression
pattern and similar DNA binding specificities, which suggest that these
genes may be homologous. Thus, our results endorse and substantiate the
observations about the importance of the GLM in the regulation of
storage protein genes and argue for a general conservation of the
endosperm-specific BLZ2/SPA type of bZIP proteins as transcriptional
regulators in cereal seeds of the Pooideae subfamily.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
35S) in bombarded developing
endosperms from barley (8).
300
region of the 22-kDa zein promoter (14). Its barley orthologue (BPBF)
transactivates transcription from the PB element of a native
Hor-2 promoter in co-bombarded barley developing endosperm
(15). With the emerging picture of the EB as a refined
cis-regulatory element whereby several nuclear proteins
interact, it is crucial the identification of the factors that can
participate in this complex and coordinate gene regulation in seeds.
BZIP factors are particularly interesting because these proteins bind
DNA motifs via dimer formation, either as homo- or heterodimers besides
interacting with other regulatory proteins (8, 16-19). More than one
bZIP factor can bind to the same target sequence. The final regulatory
effect of a particular bZIP factor is exerted through the recognition
of a particular cis-motif (G-box, C-box, GLM, etc.) in a promoter and
through its interaction with other regulatory factors (bZIPs and other
types) expressed in a given cell-type at the same time.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAP-II cDNA
library from immature barley (cv. Bomi) endosperms (15)
representing 5 × 106 plaque forming units was plated
after infection of the Escherichia coli strain XL1-Blue
MRF'. The plaques were transferred onto Magna nylon membranes (MSI) by
standard procedures (20) and screened using as a probe a 207-bp
fragment from the barley Blz1 cDNA clone (GenBankTM/EBI accession number X80068; Ref. 8) spanning
most of its bZIP domain. This DNA fragment was 32P-labeled
by the random primed method (Boehringer Mannheim). Prehybridization was
at 55 °C for 2 h in 5× SSC, 5× Denhardt's solution, 1% SDS, and 100 µg/ml salmon sperm DNA. Hybridization was done for 16 h
at the same conditions. The filters were then washed twice for 15 min
at 50 °C in 2× SSC and twice for 30 min at 55 °C in 2× SSC and
0.1% SDS. Using the in vivo excision properties of the Uni-ZAP-XR vector system (Stratagene), the selected clones were excised, according to manufacturer instructions, and recovered in the
pBluescript SK plasmid. DNA sequences were obtained using the ABI PRISM
377 dye terminator sequencing system and the ABI PRISM 377 DNA sequence
analyzer (Perkin Elmer-Applied Biosystems). Analyses of DNA and deduced
protein sequences were done with the GeneBee-Net Ver.1.0 computer facilities.
-galactosidase) and HIS3 (imidazole
glycerol phosphate dehydratase) reporter genes under the control of a
truncated Gal1 promoter that contains
Gal4-responsive elements (Gal1UAS), was used. To investigate if BLZ2 and BLZ1 were able to heterodimerize, the bZIP
domain of Blz2 (nt 538-936, corresponding to amino acids 180-312, shown in Fig. 1B), that was flanked with
EcoRI and BamHI sites by a PCR strategy using as
forward oligonucleotide LO2BZs (5'-AGAATTCAGCTCTT
CCTCATG-3'; EcoRI site underlined) and as reverse primer
LO2BZas (5'-GGGATCCACTGAAAT GGGTCC-3'; BamHI
site underlined), was subcloned into the
EcoRI-BamHI sites of the pGBT9 vector ("bait" construction). The resulting construct was introduced into S. cerevisiae HF7c cells containing the pGAD424 vector carrying each one of the following Blz1 inserts ("prey"
constructions): (i) the full-length cDNA (amino acids 1-391); (ii)
the cDNA region spanning amino acids 195-391; (iii) the region
encoding the bZIP domain (amino acids 195-293); and (iv) that of the
leucine zipper alone (amino acids 225-293).
-galactosidase production (LacZ) by the colony filter lift assay (26) and for growth
in a histidine-depleted agar medium (His
, BIO101).
-glucuronidase reporter gene (27) under the control of a
35S
promoter and fused to the 3'-terminator of the nopaline synthase gene
(3'-nos). In the EcoRI and BamHI sites upstream
of this promoter, the following oligonucleotides were fused: (i) HOR,
composed of the 43-bp sequence of the endosperm box from the
Hor-2 promoter (HOR-
35S); (ii) hor1, containing the HOR
element mutated in the GLM (hor1-
35S); and (iii) hor2, containing
the HOR element mutated in the PB (hor2-
35S). The
35S promoter
alone was used as a control. The effector constructs corresponding to
Blz2 were prepared by cloning its cDNA in the sense or
antisense orientation under the control of the 35S CaMV promoter fused
to the first intron of the maize AdhI gene and followed by
the 3'-nos (35S-I; Ref. 28): for the sense construct, the
Blz2 cDNA, obtained by PCR with the LO2FLs and the
LO2Flas primers, was digested with HindIII and blunted and
then EcoRI-digested and cloned into the 35S-I plasmid; for
the antisense construct, the PCR-amplified fragment was digested with
EcoRI and BamHI and inserted into a 35S-I plasmid
restricted with the same enzymes. The sense and antisense constructs
for Blz1 were prepared as described previously (8).
1)
with 2 µl (2 µg) of Qiagen-prepared plasmid, 25 µl of 2.5 M CaCl2, and 10 µl of 0.1 M
spermidine. In all cases, 150 or 250 ng of the reporter plasmids were
used and the appropriate concentrations of effector plasmids at the
indicated molar ratio. After vortexing for 1 min, the mixture was
incubated on ice for at least 2 min, washed twice with ethanol, and
finally resuspended in 50 µl of ethanol. For bombardment, rupture
disks of 1,100 pounds per square inch (psi) were used and 7 µl of
particles, briefly sonicated, were spotted onto macrocarriers. At a
distance of 7.5 cm from the macroprojectile stopper, developing barley
endosperms (15 DAP) were placed on half-strength MS medium containing
15 g/liter of sucrose and 0.4% phytagel. After bombardment with 154 ng
of gold particles, the endosperms were incubated at 25 °C for
24 h according to Jefferson (27). Blue spots were counted under a
dissecting microscope, and the GUS activity in each assay was expressed
as the mean value of blue spots per endosperm. The histochemical data
were directly correlated with the fluorometrically quantified GUS
activity per mg of protein with a correlation coefficient of 0.96 (data
not shown).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAPII cDNA library
(15) from early developing endosperm (10-15 DAP).
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Fig. 1.
Restriction map and nucleotide and deduced
amino acid sequence of the barley Blz2 cDNA.
A, restriction map of the Blz2 cDNA. The
initiation and termination translation codons are indicated as MET and
STOP, respectively. E, EcoRI; N,
NheI; P, PstI; Ss,
SspI; Xh, XhoI. The EcoRI
and the last XhoI restriction sites derive from the
polylinker of the Uni-ZAP-XR vector. The black box and its
adjacent box with diagonal lines represent the
basic and leucine zipper regions, respectively. B,
nucleotide sequence and deduced amino acid sequence of the
Blz2 cDNA. Amino acid residues of the basic DNA-binding
domain are in bold, the leucine heptad repeats are
circled, and a presumptive serine-rich phosphorylation site
is boxed. Acidic amino acid residues, putatively involved in
activation, are double underlined. Nucleotide sequence
numbers refer to the ATG translation initiation codon. The stop codon
is indicated with an asterisk. The AATAAA polyadenylation
signals are single underlined. Two upstream open
reading frames in the mRNA leader sequence are indicated with a
wavy line.
Comparison between related plant bZIPs
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Fig. 2.
Dendrogram and alignment of the deduced amino
acid sequence of the bZIP domain of Blz2 with those of
related plant bZIP proteins. The basic region is indicated with a
shaded bar, and both the basic region and the leucine
repeats are in bold. Asterisks correspond to
identical residues in the ten sequences compared, and dots
represent conserved substitutions: SPA from wheat (13), O2 from maize
(9, 10), sorghum (SBO2; Ref. 51) and coix (CLO2;
GenBankTM/EBI accession number X78287), OHP1 from maize
(18), REB from rice (52), BLZ1 from barley (8), CPRF2 from parsley
(48), and RITA1 from rice (53).
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Fig. 3.
Northern blot analysis of Blz2
in different barley tissues. Total RNA (15 µg) from
7-day-old roots (R), 7-day-old green leaves (L),
and from 7, 12, 17, and 21 DAP endosperms (E7,
E12, E17, and
E21) were electrophoresed in
formaldehyde-agarose gels, blotted, and hybridized with a
Blz2-specific probe under stringent conditions. The same
filter was subsequently hybridized with a specific probe for the
Hor2 gene (Hor) and with an 18 S rDNA probe
(rib) as a loading control.
300 promoter region of many genes that are exclusively expressed in the
endosperm (34).
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Fig. 4.
EMSAs of the BLZ2 protein with the 43-bp
endosperm box element (HOR) of the promoter of the
Hor2 gene and with its mutated derivatives affected in
the GLM (hor1) or in the PB
(hor2). A, EMSAs with the
32P-labeled HOR and hor1 oligonucleotide probes.
B, EMSAs with the 32P-labeled hor2
oligonucleotide probe. In the two panels,
32P-labeled probe without protein ( ); probe incubated
with 2 µg of protein extracts from bacterial cells transformed with
the pT7-7 plasmid without the Blz2 cDNA insert
(C); probe incubated with 2 µg of protein extract from
bacterial cells transformed with the pT7-7-Blz2
construction (+). 0.5 ng of probes were used in all cases. Competition
experiments were performed by using increasing amounts (50, 100, and
200×) of the indicated unlabeled HOR, hor1, and hor2. Sequences of the
three oligonucleotides, used as probes, are shown at the top
of the panel, with the GLM and PB in bold; identical
residues as in HOR in hor1 and hor2 are represented by dots,
and base mutations are written in lowercase.
35S-GUS and the
Blz2 effector resulted in an increase of about 3-fold in the
GUS activity compared with that driven by the HOR-
35S-GUS alone. The
HOR-
35S-GUS construct gives by itself a higher GUS expression than
that of the control
35S-GUS, probably by the transactivation
elicited by the endogenous barley endosperm factors that bind to the
bipartite EB element. The promoter bearing the mutation in the GLM of
the HOR element (hor1-
35S) renders a lower GUS activity (~20%)
than the HOR-
35S and is not transactivated by the co-bombarded BLZ2.
The hor2-
35S promoter, mutated in the PB, directed a GUS expression
of ~50% of that controlled by the HOR-
35S promoter, both with or
without the BLZ2 effector. These results clearly demonstrate that BLZ2 activates transcription through the GLM in barley endosperm, and that
this activation is also partially dependent on the presence of an
intact PB in the promoter.
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Fig. 5.
Transactivation by BLZ2 in co-bombarded
developing barley endosperms. A, schematic
representation of the reporter and effector constructs used in the
transient expression assays. The effector construct was the
Blz2 cDNA under the control of the 35S CaMV promoter
fused to the first intron of the maize AdhI gene. The
reporter constructs consisted of the GUS gene under the control of the
35S promoter alone or under the control of synthetic promoters
containing the HOR, hor1, and hor2 oligonucleotides (sequences shown in
Fig. 4A) fused at the 5'-end of the
35S. B,
transient expression assays by particle bombardment of developing
barley endosperms (15 DAP) with 150 ng of the indicated reporter
plasmids with or without the Blz2 effector at a 1:0.5 ratio.
GUS activity was detected by biochemical staining and subsequent
counting of blue dots per endosperm and was expressed as
n-fold activation relative to controls without effector.
Standard error of the mean for triplicate independent bombardments,
with the same particle to plasmid suspension ratio, was <15%.
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Fig. 6.
Transactivation by BLZ2 and
interaction between BLZ2 and BLZ1 in the yeast two-hybrid system.
A, schematic structures of S. cerevisiae reporter
genes and effector constructions used. Gal4DBD,
Gal4 DNA binding domain; Gal4AD, Gal4
activation domain; Gal1UAS, Gal4 responsive
elements in a Gal1 truncated promoter. B, growth
of yeast cells containing the corresponding constructions indicated in
Fig. 7A on a minimal His medium and induction
of LacZ (colony lift filter assay). Only cells carrying
"prey" and "bait" interacting proteins (lanes 2, 3, 4, and 5) or activation domains fused to Gal4BD
(lanes 8, 9, and 10) were able to grow without
histidine and to turn blue in the colony lift filter assay for
LacZ induction (+).
35S-GUS construct as reporter and the Blz2 and/or the
Blz1 as effectors, both in the sense and antisense
orientations (Fig. 7A).
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Fig. 7.
Transient expression assays by co-bombardment
of developing barley endosperms (15 DAP) using as effectors both the
sense and antisense Blz2 and Blz1
cDNAs. A, schematic representation of
the reporter and effector constructs used. The effector constructs were
the Blz2 or Blz1 cDNAs in the sense or
antisense orientation under the control of the 35S CaMV promoter fused
to the first intron of the maize AdhI gene. The reporter
construct consisted of the GUS gene under the control of the HOR- 35S
and followed by the 3'-nos. B, transient expression assays
by co-transfection of developing barley endosperms (15 DAP) with 150 ng
of the reporter construct and 1:0.5 ratio of effector in the sense
orientation. For the antisense experiments 250 ng of the reporter and
the indicated reporter:effector ratios (1:0.5, 1:1, and 1:2) were used.
GUS activity was detected by histochemical staining and subsequent
counting of blue dots per endosperm and expressed as n-fold
activation relative to the HOR-
35S control construct without
effector. Standard error of the mean for triplicate independent
bombardments, with the same particle to plasmid suspension ratio, was
<15%.
35S promoter, and no differences in the level of activation
were observed when endosperms were co-bombarded with either one of the
two effectors or with the equimolar mixture of Blz2 plus
Blz1 in the sense orientation. Co-transfections with
Blz2 or with Blz1 antisense constructs decreased the GUS activity obtained with the HOR-
35S-GUS reporter (Fig. 7B). In addition, co-bombardment of endosperms with the
equimolar mixture of both Blz2 and Blz1 antisense
constructs, produced a synergistic effect that resulted in almost the
complete loss of detectable GUS activity, even at the lowest
concentration assayed (Fig. 7B). The GUS enhancement
observed when the effectors were used in the sense orientation and the
drastic decrease when using them in antisense was not an unspecific
effect because GUS fusions to the promoters of the barley sucrose
synthase encoding genes Ss1 and Ss2
(GenBankTM/EBI accession numbers X73221 and X92354; Ref.
35), as well as to the
35S promoter, did not respond either to
Blz2 (Table II) or to
Blz1 (8).
Transient expression analysis in barley endosperm
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
gliadins, and LMW-glutenins in wheat (38, 39) and
-secalins in rye (40). Our results show that BLZ2 binds in vitro in a
sequence-specific manner to the GLM within the EB of a Hor-2
gene promoter and that mutations altering the sequence of the GLM
disrupt the interaction.
35S
promoter (8) was co-transfected with Blz2 as effector, a
3-fold increase in GUS activity was observed at a 1:0.5 ratio. As
expected, mutations in the GLM that avoided in vitro binding by the BLZ2 protein, abolished GUS activation. Mutations in the contiguous PB sequence that did not interfere with the BLZ2 binding in vitro, supported lower levels of BLZ2 activation in
planta (~50%). These results indicate that BLZ2 mediates
transcriptional activation in barley endosperm through specific
interaction with the GLM sequence. An intact PB, recently reported by
us to be recognized by a transcription factor of the DOF class (BPBF;
Ref. 15), is also essential for full transactivation. Moreover, in absence of the exogenous BLZ2 effector, the reporters with mutations, either in the GLM or in the PB promoter sequences, display a much lower
basal GUS activity compared with that sustained by the HOR-
35S promoter. Thus, our results also suggest that a positive relationship between bZIP and DOF transcription factors is necessary for high expression levels to be obtained from the EB of hordein promoters in
barley endosperm. It should be noted that transient expression data
indicate that BLZ2 must not be saturating in barley endosperm or we
would not have seen stimulus upon adding the Blz2 effector plasmid together with the HOR-
35S-GUS reporter. This suggests that
overexpression of BLZ2 in transgenic barley might lead to increased
levels of storage protein gene expression, an agronomic important goal.
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ACKNOWLEDGEMENTS |
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J. Alamillo and I. Aguilar are acknowledged for critical reading of the manuscript. We thank Angeles Rubio for technical assistance in DNA sequencing and J. Garcia and L. Lamoneda for help with the preparation of the manuscript.
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FOOTNOTES |
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* This work was financed by grants DIGCYT Bio94-0404 and DGES PB97-0561 from Ministerio de Educación y Cultura (MEC, Spain).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y10834.
Recipients of Formación Personal Investigator scholarships
from MEC (Spain).
§ To whom correspondence should be addressed. Tel.: 3491-3365707/05; Fax: 3491-3365757; E-mail: pcarbonero{at}bit.etsia.upm.es.
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ABBREVIATIONS |
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The abbreviations used are:
cv., cultivar;
AdhI, alcohol dehydrogenase I;
DAP, days after pollination;
35S,
90-bp minimal 35S CaMV promoter;
EB, endosperm box;
EMSA, electrophoretic mobility shift assay;
Gal1UAS, Gal4 responsive elements in a Gal1 truncated
promoter;
Gal4AD, Gal4 activation domain;
Gal4DBD, Gal4 DNA binding domain;
GLM, GCN4-like
motif;
GUS,
-glucuronidase gene;
HIS3, imidazole glycerol
phosphate dehydratase gene;
LacZ,
-galactosidase gene;
PB, prolamin box;
35S CaMV, cauliflower mosaic virus 35S promoter;
3'-nos, 3'-terminator sequence of the nopaline synthase gene;
bp, base pair(s);
nt, nucleotide(s);
PCR, polymerase chain reaction.
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REFERENCES |
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