Báo cáo khoa học: A new rice zinc-finger protein binds to the O2S box of the a-amylase gene promoter pptx

A new rice zinc-finger protein binds to the O2S box of the a-amylase
gene promoter
Rihe Peng
1
1
, Quanhong Yao
1
, Aisheng Xiong
1
, Huiqin Fan
1
, Xian Li
1
, Youliang Peng
2
, Zong-Ming Cheng
3
and Yi Li
4
1
Shanghai Key Laboratory of Agricultural Genetic and Breeding, Agro-Biotechnology Research Center, Shanghai Academy of
Agricultural Sciences, China;
2
Department of Plant Pathology, Chinese Agricultural University, Beijing,
2
China;
3
Department of Plant
Sciences, University of Tennessee, Knoxville, USA;
4
Department of Plant Science, University of Connecticut, Storrs, USA

3
A putative transcription facto r, named RAMY, that binds
to the 20-bp O2S sequences of the regulatory region of the
Amy2 gene promoter has been identified using the yeast one-
hybrid system from a rice library. The full length RAMY
cDNA clone encodes a 218-amino acid protein and is
homologous to the late embryogenesis-abundant protein
(LEA5). In vitro mutagenesis and electrophoretic mobility
shift a ssays confirmed that RAMY can bind with O2S spe-
cifically through an unusual zinc finger with a CXCX
4
CX
2
H
consensus sequence. Low levels of RAMY mRNAs were
detected in rice leaves and roots by Northern blot hybrid-
ization. The plant hormone gibberellin (GA) induces
expression of bo th RAMY and Amy2 genes, as performed by
Northern blot hybridization,
4
buttheincreaseinRAMY
mRNA level occurs prior to that of the Amy2 mRNA level
in the GA-treated aleurone tissues. These data suggest that
RAMY may act as a trans-acting protein and is probably
involved in the GA-induced expression of the r ice a-amylase
gene.
Keywords: rice zinc-induced protein; O2S box; yeast one-
hybrid system.
Cereal a-amylase genes have been one of the primary
systems for exploring t he molecular mechanisms involved in

hormone-regulated gene expression in plants
5
.Duringger-
mination of cereal grains, the embryo releases GA to the
aleurone layer, where it induces the transcription of
a-amylase genes [1].
Functional analyses of a-amylase gene promoters using
transient expression assays with reporter genes have shown
both GA and abscisic acid (ABA) may interact with
transcriptional regulatory proteins or transcription factors
that bind to a short nuclear nucleotide sequence referred to
as the GA response element (GARE) [2–4]. Lanahan et al.
[5] has demonstrated that GARE mediates the hormonal
control of transcription in the promoter of the low-PI gene,
Amy32b. At least three other distinct regulatory elements
have been found to be necessary for high-level a-amylase
gene expression regulated by GA. A closely associated
group of elements is composed of an opaque-2-like protein
binding seq uence (O2S), a sequence e lement with an
enriched pyrimidine nucleotide motif (the pyrimidine
box), the GARE, and box I ( TATCCAT) [6]. Using
quantitative transient expression assays, the most import-
ant e lements h ave b een found to be GARE and O 2S;
mutation or deletion of either GARE or O2S resulted in
lower GA-induced transcription [4,5]. Rogers and Rogers
[4] found that both GARE and O2S functioned only when
positioned in one orientation with respect to each other and
with respect to the TATA box, and when the distance
between them was relatively short. In searching for factors
that interact with the sequences and regulate a-amylase

gene expression, a Myb protein, GAMyb (GA-responsive
Myb protein), was isolated that may specifically bind to a
portion of the G ARE box [7]. GAMyb is able t o activate
the expression of a-amylase and other GA-regulated genes
[8]. A zinc-finger protein has been identified by South-
western screening with baits containing a GARE box and
has been found to repress the expression of a-amylase and
other g enes [9]. In Arabidopsis, at least three proteins, SPY,
RGA and GAI, are thought to negatively regulate GA
responses [10–12]. However, although the O2S box is
another important element for controlling the level of
transcription in a-amylase gene, little is known about the
regulatory proteins or transcription factors that bind to the
O2S sequence. Our interest in the mechanism of seed
germination and development in rice p rompted us to
search for the O2S binding protein. Using the yeast one-
hybrid system, we screened the rice cDNA libraries using
O2S-containing baits ATTGACTTGACCGTCATCGG
from the low pI amy54 promoter [13]. We have isolated
a cDNA clone, RAMY, which encodes a protein that
contains a zinc-finger. Our experimental data indicate that
RAMY protein binds specifically to the O2S elemen t. We
have also determined the importance of the amino acids
within the binding domain of RAMY protein and analyzed
the time course for the induction of RAMY a nd a-amylase
mRNA by GA.
Correspondence to Q. Yao, Shanghai Key Laboratory of Agricultural
Genetic and Breeding, Agro-Biotechnology Research Center, Shang-
hai Academy of Agricultural Sciences, 2901 Beidi Road, China.
Fax: + 86 021 62209988, Tel.: + 86 021 62209988,

E-mail:
Abbreviations: GST, glutathione S-transferase; O2S, opaque-2-like
protein binding sequence; GA, gibberellin; ABA, abscisic acid;
GARE, GA response element.
(Received 30 January 2004, revised 28 A pril 2004,
accepted 19 May 2004)
Eur. J. Biochem. 271, 2949–2955 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04221.x
Materials and methods
Strains and plasmids
Saccharomyces cerevisiae strain EGY4 8 ( MATa,his3trp1
ura3-52 leu::pLeu2-LexAop6) was used as the host strain
for yeast transformations. Escherichia coli strain MC8 (thi
–
,
trp
–
,ura
–
,leu
–
,his
–
) was used to rescue cDNA library
plasmids from yeast DNA preparations. The bait plasmid
pLGD-265 UP1 is a URA-marked
6
E. coli–yeast shuttle
plasmid carrying a lacZ reporter gene under the control of
the CYC1 minimal promoter [14].
The rice cDNA library was constructed in pPC86 vector,

which is a marked
7
yeast expression plasmid containing a
GAL4 activating domain under the control of the yeast
ADC1 promoter [15]. The cDNA was derived from poly(A)
RNA i solated from 15-day-old rice (Oryza sativa L.,
genotype IR36) seedlings grown at 25 °C in a greenhouse.
Yeast one-hybrid screen
A yeast one-hybrid screen was performed to isolate genes
encoding proteins that associated with the O2S box
ATTGACTTGACCGTCATCGG in the Amy2 gene pro-
moter [12]. To construct bait plasmid, the cis-element
containing three copies of O2S was synthesized by PCR
using two primers: Amyb1: 5¢-ACCCTCGAGGTCGA
CGGTATCGATAAGCTTGATTGACTTGACCGTCA
TCGGATTGACTTGACCGTCATCG-3¢,Amyb2:5¢-CA
GGATCCATCACGACAGTCAGTGCCGATGACGG
TCAAGTCAATCCGATG-3¢ (the PCR conditions were:
94 °C, 20 s; 58 °C30s;72°C, 30 s; 25 cycles). Following
PCR, the 110-bp fragment isolated from PAGE was
digested with restriction enzymes, and inserted into the
XhoI/BamHI sites of the vector pLGD
8
-265 UP1.
The lithium acetate protocol was used for yeast transfor-
mation [16]. An overnight culture of yeast cells (0.5 mL) were
inoculated into 50 mL fresh YPD medium and grown for
4 h. Yeast cells were centirfuged at 5000 g for 8 min, and the
pellets washed once with 20 mL sterile distilled water, and
then with 10 mL Tris/EDTA/LiAc (100 m

M
LiAc in Tris/
EDTA). Finally, the cell pe llet was resuspended in 0.5 mL
Tris/EDTA/LiAc. An aliquot of 50 lL yeast cells, 1 lg
plasmid DNA, 50 lg salmon carrier DNA and 300 lL Tris/
EDTA/LiAc containing 40% (w/v)
9
PEG3350 was added to
each tube, and the mixtures were incubated for 30 min at
30 °C with shaking (150 r.p.m.). After heat shock at 42 °C
for 15 min, the collected yeast cells were resuspended i n Tris/
EDTA buffer and plated on selective yeast media.
The total DNA from each positive yeast clone was
isolated according to the method of Robzyk and Kassir [17];
DNA cloning was performed using to standard procedures
[18]. The DNA extracted from yeast cells was electroporated
into E. coli strain MC8 [19]. The transformants were
selected on M9 minimal medium containing a ll amino acids
except tryptophan (M9-TRP), thus selecting pPC86-con-
taining colonies. The plasmid DNA was re-introduced into
a yeast reporter strain to confirm the b-galactosidase
activity. Two oligonucleotide primers GAL4 5¢-GGA
TGTTTAATACCACT-3¢ and TAD4 5¢-TTGATTG
GAGACTTGACC-3¢ derived from the DNA sequence
flanking the GAL4 activating domain and the ADC1
terminator region in the pPC86 vector, respectively, were
used to amplify the rice cDNA inserted into vector pPC86.
PCR was carried out for 30 cycles according to the
following protocol: 94 °C for 30 s, 42 °C f or 45 s, and
72 °C for 3 min. The DNA sequence analysis of the

recombinant plasmids was performed with the model 373
ABI automatic sequencer.
Southern–Northern blot analyses
Extraction of plant DNA and Southern blotting analysis
were p erformed b ased on the m ethod of Bringloe [20].
Total RNA was prepared from rice (O. sativa L. IR36);
leaves and roots were treated with 10 lMGA,and
control seedlings were treated with water by the standard
method
10
[18].
In order to examine the time course of the GA-induced
RAMY and Amy2 gene expression, steady state levels of
their transcripts in the GA-treated aleurone tissues were
determined with Northern blot hybridization. De-embryo-
nated rice half-seeds were de-husked, sterilized with 10%
(v/v) commercial Clorox, and treated with 95% (v/v)
ethanol for 30 s to remove the outer wax layer. After
rinsing with water, the rice de-embryonated half-seeds
(20 g) were submerged in seed buffer (20 m
M
calcium
chloride, 20 m
M
sodium succinate, pH 5.2) in cell culture
dishes. After 10 h of incubation in seed buffer, appropriate
amounts of GA were added to a final concentration of
10 l
M
. Isolated aleurone tissues were incubated with GA

for2,4,6,8,10,12or14h.
RNA was separated by gel electrophoresis an d trans-
ferred to a H ybond N
+
nylon m embrane ( Amersham-
Pharmacia). Hybridization was performed at 65 °Cin
5· NaCl/Cit, 10% (w/v) dextran sulfate, 0.5% (w/v) SDS
and 0.1 mgÆmL
)1
denatured salmon sperm DNA. Filters
were washed twice (each for 15 min) at 65 °Cin2· NaCl/
Cit, 0.1% SDS, and once in 0.1· NaCl/Cit, 0.1% SDS at
65 °C for 15 min.
Production and analyses of GST–RAMY fusion protein
The GST–RAMY fusion protein was generated by
inserting RAMY gene open reading frame between the
sites of BamHI and SacI in the vector pALEX [21]. The
primers used were RAMYZ (5¢-AGGATCCATGGCT
CTCGCTCTCTCCACC-3¢)andRAMYF(5¢-AGA
GCTCAGTGGTGGTGGTGGTGGTGCACTCGGGT
ACGTGGTGAAAC-3¢). Mutated RAMY polypeptides
were produced by in vitro mutagenesis u sing the follow-
ing primers: C182SZ (5¢-CGTGTGGGCTGGATGCT
CTCCT-3¢), C182SF (5¢-GAGGAGAGCATCCAGCCC
AC-3¢), C184SZ (5¢-GTGGGCTGGATGCTCTGCTCG
TCTG-3¢), C184SF (5¢-GCAGACGAGCAGAGC ATC
CAGC-3¢), C189SZ (5¢-CTGCTCGTGTGCTGGTTCTT
CGTCCAC-3¢), C189SF (5¢-GAGGTGGACGAAGAAC
CAGCACACG-3¢), H192AZ (5¢-TGCTGGTTCTTCGT
GCACCTCTGCTGTAAC-3¢), and H192AF (5¢-GTGAG

GCCCTGCTCGCTGTTACAGCAGAGGTGC-3¢). All
mutant genes were cloned into a pUC18 vector and
sequenced for confirmation. The resultant mutant RAMY
genes were then inserted into the BamHI/SacIsitesofthe
vector pALEX.
2950 R. Peng et al. (Eur. J. Biochem. 271) Ó FEBS 2004
The resulting GST–RA MY construct was used to trans-
form E. coli BL21. Transformants were used to inoculate
50 mL cultures of LB/ampicillin and were grown overnight
at 37 °C and harvested by centrifugation. Cell pellets were
resuspended in 9 mL phosphate buffer (pH 7.4), 20 m
M
imidazole was added, and the bacteria were lysed by
sonication. After centrif ugation at 8000 g
12
for 10 m in at
4 °C, the GST–RAMY fusion protein was isolated using
HiTrap chelating columns according to t he manufacturer’s
instructions (Amersham-Pharmacia). SDS/PAGE a nd
Coomassie blue staining were used to determine the purity
of the protein. Protein concentrations were determined
using a protein assay kit (Bio-Rad). Mutant GST–RAMY
fusion protein
13
was expressed and purified in the same way as
for the GST–RAMY fusion protein.
The mutant O2S box was synthesized by PCR using the
primers mAmyb1 (5¢-ACCCTCGAGATTGAGCTAGCC
GTCATCGGATTGAGCTAGCCGTCATCG-3¢)and
mAmyb2 (5¢-CAGGATCCGTCAGTGCCGATGACG

GCTAGCTCAATCCGATG-3¢) The PCR conditions
were: 94 °C, 20 s; 58 °C30s;72°C, 30 s for 25 cycles.
The core binding sequence CTTGA in the conserved O2S
domain was replaced by GCTAG. A gel retardation assay
was carried out as described by Jensen [22]. The XhoIand
BamHI fragment containing three copies of the O2S box
was labelled using a random primer method with the wild-
type or mutant O2S motifs as competitors. One nanogram
of labelled O2S DNA fragments, competitor DNA, 2.5 lg
poly(dI–dC) and 10 lg GST–RAMY binding protein were
mixed in 25 lL of DNA binding buffer [5 m
M
Hepes
pH 7.5, 2 m
M
MgCl
2
,0.2m
M
dithiothreitol, 1 m
M
CaCl
2
,
2% (w/v) g lycerol
14
]. The mixture was incubated for 20 min
at room temperature and loaded onto a 6% polyacrylamide
gel. After migration, the gel was fixed in 5% (w/v) glycerol,
5% (w/v) methanol, and 5% (w/v) acetic acid

15
. Labeled
DNA was then transferred to Whatman paper
16
,driedand
autoradiographed.
Results
Screening for rice cDNA encoding the O2S binding
protein
To isolate genes whose products bind to the O2S domain
(ATTGACTTGACCGTCATCGG) in the Amy54 gene
promoter, two plasmids were used in the yeast one-hybrid
system. The plasmid pPC86 contained a rice cDNA
library to express GAL4–cDNA fusion proteins, and
plasmid pLGD
17
-265UP1wasusedasthebaitwithan
insertion of three copies of the O2S domain at the 5¢ end
of the CYC1 mini-promoter region. Following transfor-
mation of the URA-marked plasmid pLGD-265 UP1
containing the O2S domain and the TRP-marked pPC86
plasmid carrying rice cDNA library
20
into the yeast cells
and selection on selective medium for 2 days, a pproxi-
mately 5 · 10
6
yeast transformants were overlaid onto
SC-TRP-URA X-gal medium using nitrocellulose filters.
In the first round of selection, 31 positive (blue) colonies

were selected. To verify the true positive clones after the
first round selection, total yeast DNA was extracted and
transformed into the E. coli strain MC8. Following
selection for the E. coli strain MC8, 14 individual
transformants were positive (blue) on medium containing
X-gal.
Nucleotide sequence and predicted amino acid sequence
of rice RAMY cDNA
Using GAL4 and TAD4 primers, sequencing analyses of
both strands of all 14 cDNA fragment
21
revealed that they
contained identical, o verlapping sequences en coding the
same protein. The complete nucleotide s equence encoded a
predicted protein of 218 amino acids (Fig. 1).
A search in a number o f random protein d atabases
22
revealed that there is n o significant homology b etween
Fig. 1.
32
The rice RAMY cDNA sequences
and the predicted product of its longest ORF
(GenBank accession no. AY072712). The
putative DNA binding domain is underlined,
and the putative nuclear localization signal
is double-underlined.
RAMY 60 DGSSSSA AREVSWVPDPVTGHYRPSNFAGGRRRRPPRRPPRP 101
G.max
LEAS 59 DTRDGSK AYSTDWAPDPVTGYYRPINHTPEIDPVELRHRLLR 100
N.tabacum

LEA5 51 KWEESS KKTTSWVPDPVTGYYRPESHAKEIDAAELRQMLLN 91
G.hirsutum
LEA5 49 AMKESSSSETRAYSSAWAPDPVTGYYRPENCGAEIDAAELREMLLN 94
V.radiata
ARG2 52 KSGEEKVR- GGEKVSWVPDPVTGYYRPEN-TNEIDVADMRATVLG 94
A.thaliana
ARG21 53 KGVEES TQKISWVPDPKTGYYRPETGSNEIDAAELRAALLN 203
H.vulgare
G3 59 REAEKA AADSSWVPDPVTGHYRPANRSSGADPADLRAAHLG 100
Fig. 2. Alignment of the conserved domain of RAMY with some related proteins. RAMY is compared with the LEA5 p roteins from G. max
(accession no. AAB38782), N. tabacum (accession no. AAC06242) and G. hirsutum (accession no. P46522), the ARG2 proteins from V. radiata
(accession no. P32292) and A. thaliana (accession no. AAC19273), and with the G3 protein from H. vulgare (accession no. CAA55482).
Ó FEBS 2004 A new zinc-finger protein from rice (Eur. J. Biochem. 271) 2951
RAMY with other proteins deposited in the databases.
However, the N-terminal half of RAMY is homologous
to the LEA5 proteins from Glycine max (GenBank acces-
sion no. AAB38782), Nicotiana tabacum (accession no.
AAC06242) and Gossypium hir sutum (accession no.
P46522), the ARG2 protein encoded by cDNAs isolated
from Vigna radiata (accession no. P32292) [23] and Arabid-
opsis thaliana (accession no. AAC19273), and the G3
protein encoded by Hordeum vulgare (accession no.
CAA55482) [24] (Fig. 2). The C-terminal half of RAMY
contained a motif with Cys and H is residues similar to the
zinc finger C3H
23
(Fig. 1, DNA binding domain).
Using the 720-bp full-length RAMY cDNA as a
probe for hybridization of rice genomic DNA that had
been digested separately with HindIII, XbaI, and BamHI,

we observed only one DNA band that hybridized with
the probe from each of the digested DNA samples
(Fig. 3). Because there are no HindIII, XbaI, and BamHI
sites within the 720-bp cDNA, a single band observed in
the Southern blot hybridization experiment suggests a
single copy of the RAMY gene is present in the rice
genome.
RAMY contains a novel zinc finger
Using the yeast one-hybrid system, the GAL4–RAMY
fusion protein exhibited a strong transcriptional activation
function in yeast cells. The GAL4–RAMY fusion protein
24
bound to the cis-element in bait pLGD
25
-265UP1, and
induced transcription of the LacZ reporter gene.
To examine whether RAMY protein binds to the O2S
sequence directly and specifically, we performed an elec-
trophoretic mobility shift assay experiment. As shown in
Fig. 4A, the presence of the purified GST–RAMY protein
resulted in a mobility shift of the
32
P-labelled O2S DNA
Fig. 3. Southern blot analysis of Oryza sativa genomic DNA. Total
genomic D NA (1 0 lg per lane) was digested with the restriction
enzymes, HindIII (H), XbaI(X)andBamHI (B). The positive control
(CK) was pPC86 (RAMY) DNA digested with SalI. Full-length
RAMY cDNA was used as the probe. The m olecular marker (in k b)
was kDNA digested with EcoRI and HindIII.
Fig. 4. Characterization of the DNA binding affinity of RAMY

recombinant protein to the Amy2/O2S sequences. (A) The [a-
32
P]dATP
labelled O2S probes were incubated in the presence or absence of GST
or GST–RAMY. Lane 1, purified GST; lanes 2–5, GST–RAMY.
Binding is competed by the same length of fragment containing three
copies of unlabelled Amy2/O2S sequence; lane 2, 50 · competitor
DNA; lane 3, 10 · competitor DNA; lane 4, 5 · competitor DNA;
lane 5, 1 · c ompetit or DNA. (B) Comparison of DNA b inding pref-
erences of RAMY protein to the mutants (M) and the wild-type O2S
(W). Lane 1, Purified GST; lane 2, GST–RAMY; lanes 3–6, EMSA by
preincubating 25-fold (25 ·) or 100 fold (100 ·)excessamountsof
unlabelled DNA fragments. F, free probe; S, shift probe.
2952 R. Peng et al. (Eur. J. Biochem. 271) Ó FEBS 2004
fragment on the gel, suggesting that the GST–RAMY
protein binds directly to the O2S sequence. Because the
purified GST alone did not produce such a mobility shift,
the shift must be caused by RAMY protein. Furthermore,
the unlabelled O2S D NA competed w ell with t he
32
P-
labelled O2S DNA but nonspecific DNA or mutant O2S
DNA did not (Fig. 4B), demonstrating that the binding of
RAMY to the O2S is specific.
We per formed a n in vitro mutagenesis experiment t o
identify amino acid residues of RAMY protein that are
important for the DNA binding. In the C-terminal region
of RAMY, there are three Cys and one His residues.
Figure 5
2626

shows that mutagenesis of Cys182 severely
reduced binding activity of RAMY to the O2S DNA
sequence. Furthermore, mutagenesis of Cys184, Cys189 or
His192 abolished the binding o f the protein to the O2S
sequence completely.
RAMY mRNA accumulation in GA-treated tissues
To determine whether the expression of the RAMY gene is
induced by GA, and the possible relationship between the
expression of the RAMY gene and the Amy2 gene, we
performed a Northern blot hybridization e xperiment. As
showninFig.6A,RAMY was expressed at low levels in
leaves but almost not at all in roots
27
. However, in both roots
and leaves, expression of RAMY was significantly induced
by exogenous GA. We also used aleurone tissues to study
the time course of the GA-mediated induction of RAMY
and Amy2 expression. Figure 6B shows that RAMY
mRNA was observed 4 h after the GA treatment, but an
increase in Amy2 mRNA was seen only after 10 h of the
GA treatment. The level of RAMY mRNA reached its
maximum 10 h after the GA treatment and returned to its
basal value within 14 h. In contrast, Amy2 mRNA reached
a maximum at 14 h after the GA treatment (Fig. 6B). These
data suggest that RAMY could act as a r egulatory o r
transcription factor for the expression of a-amylase genes.
Discussion
We have cloned a gene, RAMY, from a rice cDNA library
using O2S-contain ing baits with the yeast one-hybrid
method that encodes a zinc-finger protein

28
. We believe that
the O 2S-box binding domain is present in the protein. This
conclusion is supported by the observations that the RAMY
Fig. 5. DNA binding is mediated by the zinc finger domain. (A) SDS/
PAGE showin g the purity of the recombinant GST-fusion proteins
(lanes 2–6). The GST-fusion proteins were over-expressed in E. coli
BL21 (DE3), extracted under non denaturing conditions and purified
by affinity chromatography. (B) G el m obility assay with the purified
GST–RAMY fusion protein (lane 2) or GST-mutant RAMY fusion
protein (lanes 3–6), showing RAMY binding to DNA a nd the effects
of mutations within the RAMY DNA-binding domain on the ability
of the purified fusion protein G ST–RAMY to bind a prob e contain ing
three copies of the Amy2/O2S region.
Fig. 6. RAMY transcript levels in a rice plantsubjectedtoGAtreat-
ment. (A) GA induced RAMY mRNA accumulation in leaves and
roots. L, leaves; L + GA, leaves treatedwithGA;R,roots;R+GA,
roots treated with GA. (B) Induction of RAMY protein and a-amylase
(GenBank accession no. A F411220) by GA. Aleurone tissu e was
incubated with GA for 2, 4, 6, 8, 10, 12 or 14 h. Total RNA samples
were loaded (20 lg per lane), fractionated on a 1.2% formamide/
agarose gel, probed with a
32
P-labelled RAMY probe. 18S, RNA
probed with a
32
P-labelled 18S rRNA probe. a-amy , RNA pro bed
with a
32
P-labelled a-amylase gene probe.

Ó FEBS 2004 A new zinc-finger protein from rice (Eur. J. Biochem. 271) 2953
gene expressed in E. coli produces a protein that binds to the
O2S-box specifically and that repeated yeast one-hybrid
screening of the rice cDNA library with the O2S bait
resulted only in isolation of the RAMY cDNA.
The C-terminal end of RAMY has a motif containing
Cys and His residues reminiscent of a novel C3H zinc finger
motif CXCX
4
CX
2
H and is important in its binding activity
to the O2S domain. Several plant zinc-finger proteins are of
the cluster type with multiple repeated fingers separated by
sequences of different length. The spacing between the
fingers is the main element to determine the specificity of
binding target sequence [25]. However, we only detected one
zinc-finger domain in RAMY, perhaps because the different
structure of the zinc finger motif in RAMY influences the
binding domain. A putative nuclear localization signal
[26,27] sequence is also found in RAMY (Arg90–Arg97),
suggesting that RAMY may be transported into the nucleus
through the nuclear pore complex using its own nuclear
localization signal.
Database searches revealed RAMY was homologous
only to part of the LEA5 (late embryogenesis-abundant)
proteins. LEA5 proteins display a hydrophobic N-terminal
half and a hydrophilic C-terminal half [28]. This family of
proteins is further characterized by a highly conserved motif
of 12 amino acids with the consensus WAPDPVTGYYRP.

RAMY can be grouped into the Lea5
29
familybasedonthe
presence of the canonical 12 amino acid sequen ce motif
(WAPDPVTGYYRP).
Most LEA5-like p roteins a re induced in embryos o r
vegetative tissues by desiccation, ABA or high osmoticum.
The soybean Lea5-like (D-73 like) cDNA accumulates in
desiccating seeds from 20 to 80 days after flowering
30
and in
roots but not in leaves of drought-stressed plants [29,30].
The related gene (Di21)fromArabidopsis displays increased
transcript accumulation in roots and leaves after drought
induction, but is not detected in mature dry seeds of
nonstressed Arabidopsis plants [31,32]. In cotton, the Lea5
transcripts are highly induced in mature leave of water-
stressed plants or in w ater-stressed detached leaves [28].
However, the evidence presented here indicates that RAMY
transcription is induced by GA. The interaction between GA
and ABA may be important in controlling the a-amylase
gene expression.
TheeffectsofGAandABAona-amylase gene
transcription have been established from transient expres-
sion experiments using a-amylase promoter-reporter gene
constructs in aleurone protoplasts [33,34]. Two physically
associated elements are essential: a GA response element
(GARE) regulated by GA and ABA, and an opaque-2
binding sequence (O2S). This is consistent with the hypo-
thesis that protein b inding and interaction between two

separate binding sites are required for high-level transcrip-
tion and proper hormonal regulation [5]. The expression of
GAMyb at the mRNA level is upregulated by GA [7] and
theincreaseintheGAMyb mRNA occurs before that of
Amy21 mRNA after GA treatment. In addition, GAMyb
specifically binds to an Amy21 GARE, and transient
expression experiments have shown that GAMyb activates
transcription of a high-pI a-amylase promoter fused to a
reporter gene in the absence of GA. These results suggest
that GAMyb is a GA-regulated transcription factor
required for transcriptional activation of the high-pI
a-amylase promoter [7]. Similarly, RAMY binds to the
O2S element specifically and RAMY mRNA a lso accumu-
lates prior to the accumulation of the Amy2 mRNA upon
GA treatment. Although, our experimental results suggest
that RAMY is probably a transcription factor for activation
of a-amylase genes, the role of RAMY in t he GA-response
pathway leading to a-amylase gene expression needs to be
established.
Acknowledgements
We would like to thank Dr Qun Zhu for proving us the r ice cDNA
library. This research was supported by China PR Committee of
Science.
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Ó FEBS 2004 A new zinc-finger protein from rice (Eur. J. Biochem. 271) 2955