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ISSN
0582-9879 Acta Biochim et Biophysica Sinica 2004, 36(1): 21-26 CN 31-1300/Q
Expressional Analysis of an EREBP Transcription Factor
Gene OsEBP-89 in Rice
Hui
SHEN and Zong-Yang WANG*
( The State Key Laboratory of Plant Molecular Genetics, Institute of
Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences,
the Chinese Academy of Sciences, Shanghai 200032, China )
Abstract OsEBP-89 gene from rice (Oryza sativa) encodes an
ethylene responsive element binding protein (EREBP) transcription factor.
Northern blot analysis revealed that OsEBP-89 was expressed in root,
stem, seeds, flowers and leaves of rice. Histochemical assay showed that GUS
expressed mainly in phloem of vascular tissues of the root and stem transition
region (RST), basal part of sheath roots, stem node and basal part of
adventitious roots, also in endosperm of seeds in transgenic rice harboring OsEBP-89/GUS
construct (pNSG). A sequence of region from –279 to –97 was found to play
an important role for OsEBP-89 gene's expression though
promoter deletion assay. The possible function of OsEBP-89 gene was
discussed.
Key
words OsEBP-89 gene; rice; EREBP subfamily;
transcription factor; tissue expression
The
transcription factors bind specifically with the ciselements located in
the promoter regulation region of a gene and play important roles in the
regulation of gene transcription. They have been divided into several families,
such as homeodomain, bZIP, zinc finger, HLH, MADS and POU, based on their
polypeptide structure characteristics in DNA binding domain. The APETALA2(AP2)
transcription factor containing AP2 domain which is a conserved 60 amino acid
residue region defined in plant. According to their AP2 domain numbers, the AP2
family is divided into two subfamilies, one is AP2 protein containing two AP2
domains, and the other is ethylene responsive element binding protein (EREBP)
with only one AP2 domain. Generally, the AP2 subfamily members such as
APETALA2, AINTEGUMENTA and GLOSSY15 play regulatory roles in floral organ
specification and flower development [1–3], while the EREBP subfamily members
are mostly involved in responses to environmental factors or stress, such as
plant hormones, low temperature,drought, insects and pathogen invasion [4–8].
The
GUS(β-glucuronidase) reporter gene system, which was widely used in transgenic
plants, is a powerful tool for gene function research. The temporal and spatial
pattern of gene expression in different plant tissues and developmental stages
can be easily identified by using this system [9–11].
OsEBP-89
gene was cloned via
yeast one hybrid system from a rice cDNA expression library by using the 31 bp cis-element
which located in rice Waxy gene’s promoter as the bait. Sequencing
analysis reveled that this gene encode an EREBP transcription factor. This OsEBP-89gene
may has functions in rice endosperm development, and its expression of can be
induced by ACC and 2,4-D[12]. Here, we report the OsEBP-89 gene
expression pattern in different tissues and developmental stages is
characterized by GUS histochemical assay in transgenic plants.
Materials and Methods
Materials
Rice
plants (Oryza sativa subsp. japonica Zhonghua 11 cultivar)
are planted in a green house.
RNA
extraction
Total
RNA from different tissues of rice is extracted using Trizol reagent (Life
Technologies, Gibco BRL Inc.) as described in the reagent kit manual. Total RNA
from immature endosperm in rice seeds is also extracted by cold hydroxybenzene
reagent as described previously [13].
Northern
blot and Southern blot
Experiments
are performed as described in Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Laboratory Press, 2nd ed.).
GUS
histochemical staining and GUS activity quantitative analysis
GUS
histochemical staining were carried out as described in reference [14]. Rice
tissue samples were first carefully sectioned into slices using a shaver and
incubated in icecold PBS. Then, slices were transferred into the GUS assay
buffer and incubated at 37 ℃ for 8–12 h. Pigments were extracted away from
stained tissues with methanol/acetone reagent (3:1). After extensive
washing with PBS, the slices were stored in 50% glycerol (V/V) until
photo documentation. Quantitative analysis of GUS activity was carried out as
previous report [15].
Rice
transformation
Agrobacterium
tumefaciens mediated
transformation and regeneration of rice were performed as described
previously[16].
RT-PCR
3 μg total
RNA was used as the template. RT-PCR was performed with 5 u/ml AMV reverse
transcriptase XL and primers TSS1/TSS3 or TSS2/TSS3 using mRNA selective RT-PCR
kit (TaKaRa Inc.) according to the manufacturer’s instruction, for 45 ℃ 30 min, 1 cycle; 85
℃ 1
min, 58 ℃ 1
min, 72 ℃ 40
s, 30 cycles. Primers in use were following:
TSS1: 5′-CTATTTACCACTCCCGCGTCGC
-3′;
TSS2: 5′-ACGTCTCTCCCGCAGAAAGAAG-3′;
TSS3: 5′-GAAAACGAAGGTAC
TGCCTTCG -3.
The
resulted RT-PCR products were further analyzed by Southern blot.
Results
Transcription
start site of OsEBP-89 gene
Our
previous work predicted that the transcription start site (TSS) of OsEBP-89 gene
was located in position +1 [Fig. 1(A)] based on the mRNA molecular weight and
software analysis. To find out whether this position was right or not, two
primer pairs were designed: TSS1/TSS3 and TSS2/TSS3 (Fig. 1). Total RNA were
extracted and mRNA selective RT-PCR was carried out. The RT-PCR results showed
a 307 bp specific fragment was amplified using primers TSS2/TSS3 but not TSS1/TSS3.
The plasmid clone containing the genomic sequence was used as PCR template for
positive control and the specific amplified fragments were all detected using
these two primer pairs. After that, PCR products were transferred to the nylon
membranes and Southern blotting was carried out using Os-EBP-89 gene
cDNA as probe [Fig. 1(B)]. The results indicated that the prediction TSS
location was possible.
Fig. 1 Transcription start site of OsEBP-89 gene
(A) Partial DNA sequence of OsEBP-89 gene. TSS1, TSS2 and TSS3 are
the three primers used for RT-PCR. (B) The electrophoresis analysis and
Southern blot of RT-PCR products. L1 and L2 are PCR products of primers TSS1
and
TSS3, TSS2 and TSS3 with plasmids as templates. L3 and L4 are RT-PCR
products of primers TSS1 and TSS3, TSS2 and TSS3 with total RNA as templates.
Fig.2 Northern blot of OsEBP-89 gene
R, roots; RST, roots and stem transition region; LS, leaf sheath; L,
leave; In, internode; N, node; P, pedicel; Ea, young ear; F, young flower; E,
embryo; Em, endosperm.
30 μg total RNA was loaded
each lane. 3′ UTR sequence of OsEBP-89 gene
was used as probe.
Northern
blot of OsEBP-89 gene
To
detect the OsEBP-89 expression pattern, total RNA was extracted and
Northern blot was carried out (Fig. 2). The results showed that OsEBP-89 gene
expressed in root, root and stem transition region (RST), leaf sheaths and
leaves in young seedlings 10 d after germination. OsEBP-89 transcripts
were also detected in node, internode, pedicel, young ears and young flowers
also in embryo and endosperm of rice seeds 12–15 DAP (day after pollination),
but OsEBP-89 gene expression was relatively lower in leaves, young ears
and young flowers.
GUS
expressional analysis of transgenic plants harboring pNSG
GUS
activity has been detected to be mainly in intercalary meristem, RST and
endosperm in pNSG (OsEBP-89/GUS, Fig. 3) transgenic rice plants [12]. To
get more information about OsEBP-89 expression pattern and function, we
studied the temporal and spatial pattern of GUS expression in pNSG transgenic
plants.
The
results of GUS histochemical assay showed OsEBP-89 expression in RST in
seedlings 3, 5 and 10 d after seed germination [Fig. 4(A), (B) and (C)]. To
valuate the GUS activity in root, RST and leaf, these tissues were sampled from
10-d seedlings for GUS activity quantitative assay. The data showed that the
GUS expression level in root was similar to that in RST, but higher than in
leaf for about 2.5–3 folds [Fig. 6 (1)]. During all the stages of vegetable
growth, GUS was detected in bundles of node region [Fig. 4(D) and (E)], basal
part of adventitious root, RST, and basal part of leaf sheath or pedicel. GUS
staining was clearly found in the phloem part of the vascular bundles but not
in the xylem [Fig. 4(D), (O) and (P)]. We also found an interesting phenomenon:
the phloem tissue nearer to the RST or node was more easily detected by the GUS
staining [Fig. 4(I), (J), (L), (M), (Q) and (R)]. No GUS staining was detected
in the phloem part of root far away from RST [Fig. 4(K)], stem far away from
the node [Fig. 4(N)], and leaves far from the sheath [Fig. 4(S)]. Besides, GUS
staining was detected in filaments, microspores and basal part of ovary [Fig.
4(F)], and also in sacutellum of embryo and endosperm [Fig. 4(G) and (H)]. The
results were consistent with those of Northern blot but with more detailed
information.
Fig.
3 Schematic diagram of pNSG and promoterdeletion structures
N, NcoI; P, PstI; Sp, SphI; R, RsaI; H, HeaIII;
Sc, SacI; S, SalI; TSS,
transcription start site; GUS, GUS reporter gene; Nos, Nos Poly-A.
Fig.4 Histochemical analysis of transgenic rice harboring
plasmid pNSG
Longitudinal sections of seedlings after germination 3 d (A), 5 d (B) and 10 d (C); (D) Stem
node transverse section (100 ×). Red (stained with
safranine T) shows parenchyma while blue shows GUS expresses in vascular
tissue; (E) Longitudinal sections of stem node before flowering; (F) Rice
flower(100 ×); (G) Longitudinal sections of
seed and (H) Longitudinal sections of embryo (200×);
(I) Transverse section of root and stem transition region (100×); Transverse section of root near (J) and far away (K)
from root and stem transition region (200×); (L) Transverse
section of stem very close to the node (200×); Transverse section
of stem near (M) and far away (N) from node (200×);
(O) and (P) A enlarged transverse section of vascular tissue from (I) and (L)
separately, shows GUS expressed specifically in phloem (400×); (Q) Transverse section of sheath near the root and
stem transition region (200×); Transverse section
of leave near (R) and far away (S) from sheath (200×). R, root; Co, coleoptile; RST, root and stem transition
region; LS, leave sheath; Vt, vascular tissue; N, node; In, internode; Pc, pith
cavity; At, anther; Ov, ovary; Fi, filament; E, embryo; Em, endosperm; Sc,
sacutellum; AR, adventitious root; Bu, bundles; P, phloem; X, xylem; Pi, pith;
V, vascule; Pa, parenchyma; A, aerenchyma; Ac, air cavity; C, collenchyma.
GUS
expression in transgenic rice endosperm was observed by Yang et al.
[12]. To understand expression variations of OsEBP-89 gene during the
seed maturation, 5–7 seeds with different DAP were obtained and GUS activity in
each seed was quantitatively analyzed. The results showed that GUS expression
started at 3 DAP and reached the highest level at 6 DAP, then decreased. After
20 DAP, the expression level changed very little (Fig.5).
GUS
expressional analysis of transgenic plants harboring several promoter deletion
structures
To
identify which region in OsEBP-89 promoter affecting the gene
expression, reporter plasmids with partial deletion as pNSG, pPSG, pSphSG,
pRSG, pHSG, pNRG and pΔNRG, were constructed (Fig. 3).
Rice
was transformed by these different plasmids using Agrobacterium tumefaciens mediated
method. GUS staining was detected in calli, RST and endosperm of pNSG, pPSG,
pSphSG and pRSG transgenic rices, but not in pHSG, pNRG and p.NRG transgenic ones (Fig. 6). When
these samples were incubated in GUS assay buffer at 37 ℃ longer, GUS
staining was observed in a small part of the pHSG and p.NRG transgenic plants but still none in pNRG
transgenic plants (data not shown). The GUS activities in roots, RST and leaves
from all the transgenic seedlings were quantitatively analyzed. The results
indicated that some upstream elements from RsaI might affect the level but not
the tissue-specificity of OsEBP-89gene expression. The RH fragment (–279
to –97) might play an important role in OsEBP-89 gene expression because
the deletion of this fragment caused obvious changes in the expression of this
gene.
Fig. 5 GUS activity in rice seeds harboring plasmid pNSG
during maturation
The transgenic line code is pNSG-5 (1). DAP, days after pollination; each
column indicates the mean activity and the bar shows the standard deviation.
Fig. 6 GUS activity in rice plants harboring the promoter
deletion structures
Each three columns group indicates the mean activity of roots, RST and
leaves espectively, the bar shows the standard deviation.
1, pNSG; 2, pPSG; 3, pSphSG; 4, pRSG 5, pHSG; 6, pNRG; 7, p.NRG.
Discussion
The
tissue or organ specific gene expression of some transcription factor might
harmonize the plant development process. This situation makes it possible to
regulate different target genes by interacting with different transcription
factors. For example, Finkelstein et al. [17] reported an EREBP
transcription factor gene, ABI4, which expressed highly in legumens but
weakly in flowers, leaf buds and seedlings in Arabidopsis thaliana. He
deduced that ABI4 might have functions in all the vegetable and
generative tissues. OsEBP-89 is also an EREBP transcription factor gene
cloned by yeast one hybrid system in rice cDNA expression library.
Our
results showed that the OsEBP-89 gene was expressed not only in
endosperm but also in phloem of vascular bundles with its expression level
relatively higher in tissues nearer to the RST, node and intercalary meristem.
It can be inferred that OsEBP-89 gene might have very important
functions in starch accumulating of endosperm cells, as well as in rice stem
tissue development. Raskin and Kende [18] reported that deepwater rice could
grow out of the flood submergence quickly. Its stem elongation was caused
mainly by the endogenesis ethylene, which stimulated the cell division of
intercalary meristem of the stem. It was reported that the expressions of EREBP
transcription factors could be induced by ethylene [4]. Our previous work also
showed OsEBP-89 gene expression could be induced by ethylene [12]. So it
can be deduced that the OsEBP-89 gene might have functions in water
stress. During the development of ovary, ovarian wall gradually changed into
pericarp, and it had been reported that there is a circle vascular bundle
within the pericarp of rice. So assimilate could be transported to aleuronic
cells via phloem tissue of the vascular bundles though a structure named as
SE-CC-PS (sieve element-companion cells-pigment strand). The nutrition was
further transported into the endosperm to help the development of seeds
[19,20]. Since OsEBP-89 gene was expressed mainly in the endosperm and
phloem, it can be believed that the OsEBP-89 gene may have functions in
rice seed development by regulating the phloem mediated transportation of
assimilates?
Our
research results showed that the promoter region of OsEBP-89 gene was
involved in the phloem specific expression. The promoter deletion assay
indicated the RH fragment (–279 to –97) might play an important role in OsEBP-89
gene expression. We analyzed the sequence within the RH fragment by
computer-based comparison and did find several elements which were reported to
be involved in the phloem-specific expression, including CCA/TTG repeat (–195
to –185, ACCTGCAACC; –182 to –170, ACCAACTTTTGCCA) [21], ASL box [–160 to –150,
GCAAC(N)6GCAGA ] and GATA motif (–111 to –100,
GATCGGGAGATA) [22]. These elements were all located in RH fragment which was
consistent with that of our previous experiments. However, further work was
needed to evaluate these possibilities.
Acknowledgements
We
thank Mr. Lin-Sheng AN for his big help in rice culturing.
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Received: August 12, 2003 Accepted: November 13, 2003
This work was supported by the grants from the High Technology Department
Program of China(No.2002AA2Z1003) and the Major State Basic Research
Development Program of China (973 Program) (No. G1999011604)
* Corresponding author: Tel, 86-21-64042090-4423; Fax, 86-21-64042385;
E-mail, [email protected]