First published online June 5, 2003; 10.1104/pp.103.022574
Plant Physiology 132:1475-1488 (2003)
© 2003 American Society of Plant Biologists
DEVELOPMENT AND HORMONE ACTION
Molecular and Biochemical Characterization of VR-EILs Encoding Mung Bean ETHYLENE INSENSITIVE3-LIKE Proteins1
Jae-Hoon Lee and
Woo Taek Kim*
Department of Biology, College of Science, Yonsei University, Seoul
120–749, Korea
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ABSTRACT
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ETHYLENE INSENSITIVE3 (EIN3) is a transcription factor involved in the
ethylene signal transduction pathway in Arabidopsis. Two full-length cDNA
clones, pVR-EIL1 and pVR-EIL2, encoding EIN3-LIKE proteins were isolated by
reverse transcriptase-polymerase chain reaction and by screening the cDNA
library of mung bean (Vigna radiata) hypocotyls. VR-EIL1 and VR-EIL2
share 70% identity and display varying degrees of sequence conservation
(39%–65%) with previously isolated EIN3 homologs from Arabidopsis,
tobacco (Nicotiana tabacum) and tomato (Lycopersicon
esculentum) plants. Gel retardation assay revealed that both VR-EILs were
able to interact specifically with optimal binding sequence-1, the recently
identified optimal binding sequence for tobacco TEIL, with the binding of
VR-EIL2 being more efficient than that of VR-EIL1. Transient expression
analysis using a VR-EIL::smGFP fusion gene in onion (Allium
cepa) epidermal cells indicated that the VR-EIL proteins were effectively
targeted to the nucleus. The fusion protein of VR-EIL2 with GAL4 DNA-binding
domain strongly activated transcription of a reporter gene in yeast cells, and
an essential domain for transcription-stimulating activity was localized to
the amino-terminal acidic region that consists of 50 amino acid residues. In
contrast with what has been previously found in EIN3- and
TEIL-overexpressing Arabidopsis plants, transgenic tobacco seedlings
expressing the VR-EIL genes under the control of cauliflower mosaic
virus 35S promoter did not exhibit a constitutive triple response. Instead,
they displayed a markedly enhanced proliferation of root hairs, one of the
typical ethylene response phenotypes, and increased sensitivity to exogenous
ethylene. In addition, the pathogenesis-related (PR) genes encoding
 -1,3-glucanase, osmotin, and PR1 were constitutively expressed in
35S::VR-EIL lines without added ethylene, and were hyperinduced in
response to ethylene treatment. These results indicate that VR-EILs are
functional in tobacco cells, thereby effectively transactivating the
GCC-box-containing PR genes and enhancing sensitivity to ethylene. The
possible physiological role of VR-EILs is discussed in the light of the
suggestion that they are active components of the ethylene-signaling pathway
and their heterologous expressions constitutively turn on a subset of ethylene
responses in tobacco plants.
The gaseous phytohormone ethylene modulates diverse physiological aspects
of plant growth and development (Yang and
Hoffman, 1984 ; Abeles et al.,
1992). Ethylene also serves as a signaling molecule to elicit
specific changes in gene expression at certain stages of a plant life cycle.
The production of ethylene in plant tissue is normally low, but can be
markedly enhanced by a broad spectrum of developmental and environmental cues,
including seed germination, fruit ripening, leaf and flower senescence, and a
multitude of biotic and abiotic stresses
(Yang and Hoffman, 1984;
Abeles et al., 1992).
After synthesis, ethylene must be perceived and its signal transduced into
the cells to elicit the physiological responses. Progress in understanding the
molecular mechanism of ethylene perception and signal transduction has been
recently made by a combination of molecular and genetic approaches using
Arabidopsis as a model system (for review, see
Kieber, 1997;
Chang and Shockey, 1999;
Bleecker and Kende, 2000;
Stepanova and Ecker, 2000).
The triple response is a well-known response of etiolated seedlings to
ethylene (Yang and Hoffman,
1984). It comprises three distinct morphological changes:
inhibition of hypocotyl elongation, enhancement of radial expansion, and
horizontal stem growth. Alteration in the triple response of etiolated
Arabidopsis seedlings in response to exogenous ethylene was used to isolate
ethylene-related mutants. Among these are the ethylene-insensitive mutants and
the recessive mutant that constitutively displays the triple response in the
absence of exogenous ethylene (Bleecker et
al., 1988; Guzman and Ecker,
1990). Analysis of the mutant genes responsible for these
phenotypes has identified a number of components involved in the ethylene
signal transduction pathway. Ethylene perception is mediated by a family of
membrane receptors, ETR1, ERS1, ETR2, EIN4, and ERS2. Their
predicted translation products are reminiscent of bacterial two-component His
kinases and function as negative regulators
(Chang et al., 1993; Hua et
al., 1995,
1998;
Schaller and Bleecker, 1995;
Hua and Meyerowitz, 1998;
Sakai et al., 1998).
Loss-of-function mutations at the CTR1 locus result in a constitutive
ethylene response, indicating that CTR1 is also a negative regulator of the
pathway (Kieber et al., 1993).
The CTR1 gene encodes a protein with homology to the Raf family of
protein kinases and, hence, a mitogen-activated protein kinase cascade in the
ethylene response pathway has been suggested
(Kieber et al., 1993). CTR1
acts downstream of the receptor and directly interacts with the receptors in a
yeast two-hybrid system (Clark et al.,
1998). Thus, plant cells use a unique combination of a
prokaryotic-sensing system and a eukaryotic-transducing system for the early
step in ethylene signaling. The third gene, ETHYLENE INSENSITIVE2
(EIN2), encodes a novel protein with the amino-terminal 12 integral
membrane domains and shows similarity to the disease-related Nramp family of
metal-ion transporters of mammalian cells
(Alonso et al., 1999).
Epistasis (double-mutant analysis) indicates that EIN3, a positive
regulator, acts at the most downstream position of the ethylene-signaling
pathway. The EIN3 and three related EIN3-LIKE (EIL1,
EIL2, and EIL3) genes were cloned, and their predicted
polypeptides were shown to possess common features for nuclear-localized
transcription factors (Chao et al.,
1997). Solano et al.
(1998) have demonstrated that
EIN3, as well as EIL1 and EIL2, bind to the ethylene-response element (ERE)
present in the upstream region of ETHYLENE-RESPONSE-FACTOR1
(ERF1), an early ethylene-responsive gene encoding the
GCC-box-binding protein. The ERF1 protein, in turn, induces various secondary
ethylene-responsive pathogenesis-related (PR) genes that contain the GCC-box
in their promoters (Solano et al.,
1998). These findings indicate that the nuclear proteins EIN3 and
ERF1 act sequentially for the transcriptional activation of diverse
ethylene-inducible genes in Arabidopsis.
The physiological responses of mung bean (Vigna radiata) seedlings
to ethylene have long been studied, such as triple response and root formation
(Yang and Hoffman, 1984;
Abeles et al., 1992). In
addition, mung bean seedlings have been extensively used for the study of
regulation of ethylene production in response to auxin and environmental and
developmental factors, and ethylene biosynthetic genes are well characterized
(Yang and Hoffman, 1984;
Abeles et al., 1992;
Kim and Yang, 1994;
Yi et al., 1999). However, far
less has been carried out to investigate the mechanism of the
ethylene-signaling pathway and its mode of action in mung bean plants.
Application of ethylene in mung bean seedlings caused a great increase in the
mRNA level of 1-aminocyclopropane-1-carboxylate (ACC) oxidase
(VR-ACO1), the final step of its biosynthetic pathway
(Kim and Yang, 1994;
Kim et al., 1997;
Jung et al., 2000). We are
interested in elucidating the molecular mechanism of the mode of action of
ethylene in regulating its own production. In this respect, isolation and
characterization of mung bean EIN3 homologs would help progress in
the understanding of the ethylene action in mung bean seedlings. Here, we
report the isolation of two full-length cDNAs encoding mung bean EIN3-LIKE
proteins (VR-EIL1 [accession no. AF467784] and VR-EIL2 [accession no.
AF467783]) and analysis of their molecular and biochemical properties.
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RESULTS
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Isolation and Characterization of Mung Bean EIN3 Homologs
To gain more insight into the mechanism of ethylene action in mung bean
vegetative tissues, we proceeded to isolate cDNAs encoding homologs of the
Arabidopsis EIN3. Poly(A)+ RNA was isolated from 4-d-old etiolated
mung bean hypocotyls. After the synthesis of the first strand cDNA from 1
µg of poly(A)+ RNA, PCR was carried out with mixed
oligonucleotides corresponding to the amino acid sequence of QDTTLG as the
upstream primer, of QDKMTA as the downstream primer (see "Materials and
Methods" for sequences), and the first strand cDNA as the template.
These primer amino acid sequences are shown to be highly conserved in
Arabidopsis EIN3 and EILs (Chao et al.,
1997). Total PCR products of about 300 bp in length were
radioactively labeled and used as probes to screen the cDNA library of mung
bean hypocotyls under low stringent hybridization and washing conditions
(Kim and Yang, 1994). Numerous
putative mung bean EIN3 clones were isolated. Subsequent restriction enzyme
mapping and DNA sequencing analyses revealed that these clones could be
divided into two homology classes. Figure
1A shows the restriction enzyme map analysis of pVR-EIL1 and
pVR-EIL2, which contain the longest insert among each class. The pVR-EIL1
clone is 2,150 bp long consisting of a 10-bp 5'-UTR, a 1,827-bp coding
region encoding 609 amino acids, and a 313-bp 3'-UTR. pVR-EIL2 is 2,149
bp long comprising a 1-bp 5'-uncoding region, a 1,866-bp coding region
of 622 amino acids, and a 282-bp 3'-uncoding region
(Fig. 1B). The predicted
molecular masses of the polypeptides encoded by pVR-EIL1 and pVR-EIL2 are 67.0
and 68.4 kD, respectively. Complete sequences for pVR-EIL1 and pVR-EIL2 allow
comparison of the two full-length VR-EIL genes and analysis of their
structural relationship. The overall nucleotide sequence identity between
pVR-EIL1 and pVR-EIL2 is 71%, whereas the coding regions are 73% identical at
the nucleotide level and 70% at the amino acid level. Both mung bean EILs
share 39% to 65% identity at the amino acid level with the Arabidopsis EIN3
and EILs (Chao et al., 1997)
and other plant EIN3 homologs, such as tobacco TEIL
(Kosugi and Ohashi, 2000),
tomato (Lycopersicon esculentum) LeEILs
(Tieman et al., 2001), and
carnation DC-EIL1 (Waki et al.,
2001). Phylogenetic alignment revealed that the VR-EIL1 and
VR-EIL2 proteins are the most closely related to TEIL (65% and 64%,
respectively), whereas the most distantly related to EIL3 (40%) and EIL2
(39%), respectively (Fig. 1C).
As found in other EIN3 homologs, the VR-EIL1 and VR-EIL2 proteins possess an
amino-terminal acidic region and a Pro-rich domain
(Fig. 1B). In addition, five
small clusters of basic amino acids dispersed throughout the protein are also
well conserved in both mung bean EILs. The VR-EIL2 polypeptide has a
carboxy-terminal Asn-rich region that consists of six Asn residues, as
observed in EIN3. However, this poly-Asn repeat is not found in VR-EIL1 and in
any of the Arabidopsis EILs and tomato LeEILs. Both mung bean EIL proteins
contain a single poly-Gln repeat near the carboxy-terminal portion
(Fig. 1B). The Gln-rich region,
along with the acidic and Pro-rich regions, has been proposed to be functional
as transcriptional activation domains
(Chao et al., 1997). Finally,
VR-EIL1 and VR-EIL2 have a Lys residue at positions 245 and 246, respectively,
which is essential for the function of EIN3
(Fig. 1B, indicated by star).
Taken together, these structural conservations suggest that the VR-EIL1 and
VR-EIL2 proteins play a role in the ethylene-signaling pathway in mung bean
plants.
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Figure 1. Structure of the mung bean EIN3 homologs, VR-EIL1 and VR-EIL2. A,
Restriction enzyme map analysis of two mung bean EIL cDNA clones. Solid bars
represent the coding regions, whereas solid lines designate the 5'- and
3'-untranslated regions (UTRs). The positions of the gene-specific
hybridization probes generated by PCR are indicated. The sequences of pVR-EIL1
and pVR-EIL2 have been deposited in the GenBank data base under accession
numbers AF467784 and AF467783, respectively. B, Alignment of the derived
polypeptide sequences of VR-EIL1, VR-EIL2, Arabidopsis EIN3 (AtEIN3;
Chao et al., 1997), tobacco
(Nicotiana tabacum) TEIL (Kosugi
and Ohashi, 2000) and tomato (Lycopersicon esculentum)
LeEIL1 (Tieman et al., 2001).
The conserved amino acid residues are shaded in gray. The amino-terminal
acidic domain (AD), Pro-rich region (PR), and five small basic domains (BDI-V)
are boxed. The poly-Gln and poly-Asn repeats at the carboxy-terminal portion
are marked by dots. Asterisk refers to the Lys residue essential for the
function of Arabidopsis EIN3. The putative nuclear-localized signals are
indicated by solid lines. The arrows depict the primer amino acid sequences
for reverse transcriptase-PCR. Dashes show gaps in the amino acid sequences
introduced to optimize alignment. C, Phylogenetic alignment of EIN3 homologs
from mung bean (VR-EIL1 and VR-EIL2), Arabidopsis (AtEIN3, AtEIL1, AtEIL2, and
AtEIL3; Chao et al., 1997),
tobacco (TEIL1; Kosugi and Ohashi,
2000), tomato (LeEIL1, LeEIL2, and LeEIL3;
Tieman et al., 2001) and
carnation (Dianthus caryophyllus; DC-EIL1;
Waki et al., 2001).
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VR-EILs Bind Specifically to the Conserved Sequence optimal binding
sequence (obs) 1 in Vitro
Kosugi and Ohashi (2000)
carried out a random binding selection analysis and revealed that the obs for
TEIL was A[T/C]G[A/T]A[C/T]CT. This sequence is present in the
5'-upstream region of various ethylene-inducible genes. Among EIN3
homologs, TEIL is the most closely related to VR-EILs
(Fig. 1). Thus, we examined
whether VR-EILs can bind to the consensus sequence. VR-EIL1 and VR-EIL2 were
expressed in Escherichia coli as a fusion protein with
glutathione-S-transferase (GST), and purified recombinant proteins
were used in a gel retardation assay with a 32P-labeled conserved
sequence obs1 (Kosugi and Ohashi,
2000). The full-length 93- to 94-kD GST-VR-EILs gave rise to a
single, discrete DNA-protein complex that migrated more slowly than the free
probe (Fig. 2A). The intensity
of this shifted band increased upon the addition of increasing amounts
(50–100 ng) of GST-VR-EILs. The DNA binding specificity of VR-EILs was
further confirmed by competition binding experiments, which showed that a
50-fold excess of cold obs1 was enough to displace the labeled probe. The
binding capacity of the VR-EIL1 and VR-EIL2 proteins was dramatically reduced
when obsm1, which has A C substitution in the first position of obs1, was
used as a probe (Fig. 2A, lanes
7 and 13), whereas we could not detect any interaction between VR-EILs and
obsm2 that contains GT mutation in the third position of the conserved
sequence (Fig. 2A, lanes 8 and
14). These results imply that VR-EIL1 and VR-EIL2 bind specifically to obs1 in
vitro. The results in Figure 2A
also reveal that the intensity of VR-EIL2-DNA complex is stronger than that of
VR-EIL1-DNA complex, suggesting that VR-EIL2 interacts with obs1 more
effectively that VR-EIL1 does (Fig.
2A, compare lanes 3 and 9, and lanes 4 and 10). To explore whether
VR-EIL1 and VR-EIL2 have a different affinity to obs1, we repeated competition
binding experiments with varying amounts of cold probe. As shown in
Figure 2B, the specific
interaction between VR-EIL1 and obs1 was almost completely abolished by a
10-fold excess of cold probe, whereas VR-EIL2 was still able to bind
significantly to obs1 in the presence of the same amount of cold competitor
(Fig. 2B, lanes 3 and 10). In
addition, the ability of VR-EIL1 to bind to obs1 was significantly reduced by
a 100-fold molar excess of cold obsm1 (Fig.
2B, lane 6). However, the same amount of obsm1 affects the
interaction between VR-EIL2 and obs1 to a much lesser extent
(Fig. 2B, lane 13). Overall,
these results indicate that VR-EIL1 and VR-EIL2 interact specifically with the
conserved sequence obs1, with the binding of VR-EIL2 being more efficient than
VR-EIL1.
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Figure 2. Sequence-specific binding activities of VR-EILs. A, Gel retardation assay
showing full-length GST-VR-EILs binding to obs1, the obs for tobacco TEIL
(Kosugi and Ohashi, 2000). The
radiolabeled probes or varying amounts of cold competitors were incubated in
the presence or absence of the purified recombinant GST-VR-EILs (50 and 100
ng). Lane 1, only the free probe; lane 2, the free probe and GST. Lanes 3, 4,
9, and 10, Radiolabeled obs1 probe; lanes 5, 6, 11, and 12, titration with
varying amounts of cold obs1 as a competitor; lanes 7 and 13, radiolabeled
obsm1 probe; lanes 8 and 14, radiolabeled obsm2 probe. Oligonucleotide probes
(obs1, obsm1, and obsm2) were prepared based on the recent results by Kosugi
and Ohashi (2000). B,
Competition binding analysis. The radiolabeled obs1 probe and purified 100 ng
of recombinant VR-EILs were incubated in the presence or absence of varying
amounts of cold competitors as indicated. Lanes 1 and 8, Without any
competitors; lanes 2 through 5 and 9 through 12, titration with varying
amounts of cold obs1 as a competitor; lanes 6 and 13, a 100-fold molar excess
of cold obsm1 as a competitor; lanes 7 and 14, a 100-fold molar excess of cold
obsm2 as a competitor.
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Targeting of VR-EILs to the Nucleus
Because VR-EILs specifically bind obs1 in vitro
(Fig. 2) and their predicted
sequence contains putative nuclear localization signal
(Fig. 1B), they are expected to
localize to the nucleus. To confirm this, we performed an in vivo targeting
experiment that employed a VR-EIL2-fused soluble-modified green fluorescent
protein (smGFP) as a fluorescent marker in a transient transfection assay. The
smGFP gene was fused to the 3' end of the pVR-EIL2 coding
region in frame under the control of the cauliflower mosaic virus (CaMV) 35S
promoter, and the resulting construct was introduced into onion (Allium
cepa) epidermal cells by the particle bombardment method. Localization of
the fusion protein was then determined by visualization with a fluorescence
microscope. As shown in Figure
3, the control smGFP was uniformly distributed throughout the cell
(Fig. 3A), whereas the
VR-EIL2-smGFP fusion protein was exclusively localized to the nucleus
(Fig. 3B). These observations
support the notion that the putative nuclear targeting sequence of VR-EIL2 is
sufficient and that no additional posttranslational modification may be
necessary for the VR-EIL2 protein to be targeted to the nucleus. We obtained
identical results with VR-EIL1-smGFP fusion protein (data not shown).
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Figure 3. Nuclear localization of the VR-EIL2 gene product. The
smGFP coding region was fused in frame to the 3' end of the
full-length pVR-EIL2-coding region. The construct was introduced into onion
epidermal cells by the particle bombardment method and expressed under the
control of the CaMV 35S promoter. The expression of the introduced gene was
viewed after 12 h by fluorescence microscopy under UV or visible light.
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The VR-EIL Proteins Activate Transcription in Yeast
VR-EIL1 and VR-EIL2 proteins possess an amino-terminal acidic region, a
Pro-rich domain, and a polyGln repeat at the carboxy terminus that might act
as a transcriptional activator motif (Fig.
1). To examine the functional role of these domains, we fused the
coding region of VR-EIL2 and its various deletion mutants to the GAL4
DNA-binding domain expression vector and investigated the behavior of each
construct as a potential transcriptional activator in yeast cells. In the
absence of the GAL4 activation domain, the wild-type VR-EIL2 protein fused to
the GAL4 DNA-binding domain, effectively activating transcription of the
lacZ reporter gene (Fig.
4). This indicates that VR-EIL2 is able to function as a
transcriptional activator in yeast. To identify a minimal transcriptional
activation domain, various deletion mutants of VR-EIL2 were also tested by the
same method. Figure 4 depicts
that deletion of Pro-rich region and carboxy-terminal Gln-rich domain does not
result in loss of transcriptional activation. In contrast, deleting the
amino-terminal acidic domain almost completely abrogated reporter gene
activation. Furthermore, the transcription-stimulating activity was still
apparent when the amino-terminal acidic region (1–50 amino acids) was
fused to the GAL4 DNA-binding domain; the VR-EIL21–50 mutant
protein contained 70% -galactosidase activity compared with that of the
full-length protein (Fig. 4D).
Thus, the amino-terminal 50-amino acid residues of VR-EIL2 play a critical
role in supporting the ability of VR-EIL2 as a transcriptional activator.
Organization and Expression of the VR-EIL Genes
From the results described above, it appears that VR-EILs are nuclear
proteins that specifically interact with obs1 in vitro and possess
transcriptional activation activity. Therefore, we wanted to characterize the
VR-EIL genes in more detail at the molecular level. To assess the
exact copy number of the EIL genes in the mung bean genome, we
constructed gene-specific probes (probes 1 and 2 for each homology class,
Fig. 1A) by PCR based on the
divergent 3'-UTRs of pVR-EIL1 and pVR-EIL2. The genomic DNA isolated
from mature leaves of mung bean plants was digested with EcoRI,
XbaI, or HindIII, and was hybridized with
32P-labeled gene-specific probe 1 or 2 under high stringent
conditions. These hybridizations detected only one major band by those enzyme
digestions, respectively (Fig.
5A). These results imply that the VR-EIL1 and
VR-EIL2 genes are present in a single copy per haploid mung bean
genome. With a general probe derived from the conserved coding region and
under low stringent hybridization and washing conditions, one to two minor
bands were also detected (data not shown). Thus, we could not rule out the
possibility of the existence of additional VR-EIL-related genes in
the mung bean genome.
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Figure 5. Organization and expression of the mung bean EIL genes. A, Genomic
Southern-blot analysis of the VR-EIL genes. The mung bean genomic DNA
(10 µg per lane) was isolated from leaf tissue, digested with
EcoRI (E), XbaI (X), or HindIII (H), and resolved
on a 0.7% (w/v) agarose gel. DNA on the gel was transferred to a nylon
membrane filter. The filter was hybridized to the 32P-labeled
gene-specific probe 1 or probe 2 under high stringent conditions. B, RNA
gel-blot analysis of the VR-EIL genes. Total RNAs (20 µg per lane)
isolated from different parts of 4-d-old etiolated seedlings or 2-week-old
light-grown mung bean plants were resolved on a 1.0% (w/v)
formaldehyde-agarose gel. The gel was blotted onto a membrane filter and the
blot was hybridized to the 32P-labeled gene-specific probe 1 or
probe 2 under high stringent conditions. To examine the effect of ethylene on
VR-EILs mRNA expression, intact etiolated seedlings and light-grown
plants were enclosed in 3-liter jars containing air or air plus 20 µL
L–1 ethylene. After 6 h of treatment, total RNAs
were isolated from different parts of plants and were analyzed as described
above. The blots were visualized by autoradiography. The equivalence of RNA
loading among lanes of the agarose gel was demonstrated by ethidium bromide
staining of rRNA on the gel.
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To examine the spatial and temporal expression pattern of the
VR-EIL1 and VR-EIL2 genes, we monitored the level of
corresponding mRNAs in different mung bean vegetative tissues by northern-blot
analysis. Total RNAs isolated from apical hooks, hypocotyls, and roots of
dark-grown 4-d-old seedlings, or from leaves, stems, and roots of light-grown
2-week-old plants were hybridized with 32P-labeled probe 1 or 2.
The substantial level of the transcripts (2.3 kb) for VR-EIL1 and
VR-EIL2 was detected in every tissue examined of dark- and
light-grown plants (Fig. 5B). However, the relative expression pattern of the two different mRNAs varied in
these tissues, with the VR-EIL1 transcript being predominantly
present in etiolated seedlings. By contrast, the amount of the two mRNAs was
somewhat similar in green plants (Fig.
5B). To investigate whether the activity of the VR-EIL
genes is modulated by ethylene, intact mung bean plants were exposed to 20
µL L–1 ethylene for 6 h, and then total RNAs
were similarly analyzed. As shown in Figure
5B, the steady-state levels of these mRNAs were not significantly
affected by the application of ethylene in dark- and light-grown plants. These
results are in line with the previous observations that the expression of
Arabidopsis EIN3 and tomato LeEILs is not regulated by
exogenous ethylene, and are consistent with the view that the activity of EIN3
and its homologs are possibly subject to control by a posttranslational
mechanism (Chao et al., 1997;
Kosugi and Ohashi, 2000;
Tieman et al., 2001).
Overexpression of VR-EIL1 and VR-EIL2 Enhances Root
Hair Formation in Transgenic Tobacco Seedlings
Chao et al. (1997) produced
Arabidopsis plants overexpressing the EIN3 and EIL1 genes,
respectively, and investigated the effect of overexpression on ethylene
responses in transgenic plants. The etiolated T2 generation
seedlings exhibited characteristics of a constitutive triple response
phenotype in the absence of added ethylene, which were very similar to the
ethylene-treated wild-type and ctr1 mutant seedlings. Similarly, when
tobacco TEIL cDNA was introduced into Arabidopsis under the control of CaMV
35S promoter, the transgenic seedlings displayed a triple response without
ethylene treatment (Kosugi and Ohashi,
2000). These results showed that the enhanced level of EIN3 or
EILs resulted in the constitutive activation of the ethylene-signaling
pathway. On the other hand, Tieman et al.
(2001) constructed tomato
plants that expressed antisense RNA for each of the three LeEIL
genes. Because LeEILs share significant sequence identities, the
antisense expression of one gene conferred the decrease in the expression of
other LeEIL genes. As a result, at least one line from each
anti-LeEIL-transgenic plant showed a high degree of ethylene
insensitivity in many aspects compared with wild-type tomatoes, such as
increased seedling length, reduced leaf epinasty, and delayed flower
abscission in response to ethylene, and inhibition of fruit ripening
(Tieman et al., 2001). The
severity of ethylene insensitivity paralleled the level of reduction in the
total amount of LeEIL transcripts in antisense plants.
The results described above led us to consider the possibility that the
mung bean VR-EILs might be functionally similar to EIN3 or EILs. To examine
this possibility, we established transgenic tobacco plants overexpressing
VR-EIL1 and VR-EIL2, respectively. Tobacco plants were
transformed with binary vectors carrying a fusion of CaMV 35S promoter and
pVR-EILs in the sense orientation by means of the Agrobacterium
tumefaciens-mediated transformation method. For each gene, several
independent primary transformants were obtained based on the resistance
against kanamycin, and transgenic plants were subsequently regenerated and
used for further analyses. The presence of each of the transgenes was
confirmed by PCR analysis (data not shown). In contrast to EIN3- and
TEIL-expressing Arabidopsis, 4-d-old etiolated tobacco seedlings of
the T2 generation did not display a constitutive triple response;
their hypocotyl length was indistinguishable from that of control plants in
the absence of added ethylene (Fig.
6A). However, the VR-EIL-overexpressing seedlings showed
an increased sensitivity to exogenous ethylene, as indicated by more severely
reduced hypocotyl length in comparison with the control seedlings in the
presence of 1 to 50 µM ACC, the immediate precursor to ethylene
(Fig. 6A). A more unique
phenotype specific for both VR-EIL constructs was a marked increase
in root hair formation. As shown in Figure
6B, the proliferation of root hairs was pronouncedly elevated in
dark- and light-grown transgenic lines without ethylene treatment. A closer
inspection revealed that the individual transgenic root had numerous ectopic
hairs, and this phenotype was more exaggerated when treated with 10
µM ACC compared with ACC-treated control roots, suggesting that
transgenic roots are more sensitive to ethylene
(Fig. 6C). Ethylene has long
been suggested to play a role in the initiation of root hairs in various plant
species, including pea (Pisum sativum), fava bean (Vicia
faba), lupine (Lupinus albus), and tulip (Tulipa
gesneriana; Abeles et al.,
1992). Arabidopsis ctr1 mutation confers production of
ectopic root hairs on epidermis cells, which are normally nonhair cells
(Dolan et al., 1994).
Furthermore, Tanimoto et al.
(1995) have demonstrated that
Arabidopsis grown with aminoethoxyvinyl-Gly, an effective inhibitor of
ethylene biosynthesis, or Ag+, an ethylene action inhibitor, shows
a reduced number of root hair, whereas plants grown in the presence of ACC
exhibit ectopic root hair, indicating that ethylene is a positive regulator of
root hair development. Most recently, Cho and Cosgrove
(2002) have shown that
ethylene exerts an unambiguous effect on root hair elongation and mediates
environment-induced root hair initiation (e.g. auxin and root separation from
medium) in Arabidopsis. Thus, the hairy root phenotype of
VR-EIL-expressing segregants is consistent with the notion that
VR-EIL1 and VR-EIL2 are functioning in tobacco seedlings and activate the
ethylene-signaling pathway to turn on a subset of ethylene responses.
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Figure 6. Overexpression of VR-EIL cDNAs in tobacco plants. A, Ethylene sensitivity
of etiolated seedlings of wild type (WT) and VR-EIL-overexpressing
transgenic tobacco lines (VR-EIL1-8 and VR-EIL2-1). Wild-type and transgenic
seedlings of the T2 generation were germinated in the dark in the
absence or presence of different concentrations (1–50 µM)
of ACC at 25°C. Data shown indicate the hypocotyl lengths of etiolated
4-d-old seedlings. The values are means ± SD (n =
60). B, Phenotype of the VR-EIL-overexpressing T2
generation seedlings. The seedlings were germinated in the dark for 4 d
(a–c) or were grown in a growth chamber under a 16-h daylength for 7 d
at 25°C (d–f). a and d, Wild-type plants; b and e, VR-EIL1-8 line; c
and f, VR-EIL2-1 line. C, Enhanced formation of root hairs in
35S::VR-EIL lines. The roots were grown in the dark in the absence
(a–c) or presence (d–f) of 10 µM ACC for 4 d at
25°C. a and d, Wild-type root; b and e, VR-EIL1-8 root; c and f, VR-EIL2-1
root.
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The VR-EIL Genes Effectively Transactivate Various
Ethylene-Inducible PR Genes in Transgenic Tobacco Seedlings
To further investigate the function of VR-EILs in tobacco cells
and to define whether the root hair phenotype of VR-EIL constructs
was due to the activation of the ethylene signaling pathway, we monitored the
expression pattern of various ethylene-responsive genes in transgenic plants.
The PR genes encoding -1,3-glucanase, osmotin, and PR1 were highly
induced after ethylene treatment for 6 h in light-grown 7-d-old wild-type
tobacco seedlings (Fig. 7).
These PR genes are known to have the GCC-box in their promoters
(Ohme-Takagi and Shinshi,
1990; Hart et al.,
1993; Sessa et al.,
1995; Sato et al.,
1996). Moreover, as shown in
Figure 7, high-level
constitutive expression of these PR genes was clearly observed in
VR-EIL1- and VR-EIL2-overexpressing transgenic lines without
ethylene treatment. The levels of PR mRNAs were approximately correlated with
those of the VR-EIL transcripts. Thus, it seems most likely that
VR-EILs are highly functional in tobacco cells, thereby effectively
transactivating the GCC-box-containing PR genes. As the constitutive root hair
formation was the most significant phenotype of VR-EIL-expressing
tobacco plants, we next examined the transactivation activities of VR-EILs in
transgenic root tissue. VR-EIL1 and VR-EIL2 effectively activated the
-1,3-glucanase gene, as evidenced by the constitutive high
level of its mRNA in 35S::VR-EIL roots
(Fig. 7). As expected, ethylene
caused a marked enhancement of the level of tobacco ACC oxidase mRNA
(NG-ACO1, Kim et al.,
1998) in control plants (Fig.
7). However, the steady-state level of this transcript was not
changed in transgenic lines compared with that of wild-type plants.
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Figure 7. Expression pattern of various ethylene-inducible genes in wild-type (WT)
and VR-EIL-overexpressing tobacco plants. Total RNAs (20 µg per
lane) were isolated from 7-d-old light-grown wild-type seedlings and roots
that had been exposed to air or air plus 20 µL
L–1 ethylene for 6 h, and were resolved on a 1.0%
(w/v) formaldehyde-agarose gel. The gel was blotted onto a membrane filter and
the blot was hybridized to the 32P-labeled cDNA probes for
VR-EIL1, VR-EIL2, -1,3-glucanase
(Ohme and Shinshi, 1990),
osmotin (Sato et al.,
1996), PR1 (Kim et
al., 1998), and NG-ACO1
(Kim et al., 1998),
respectively, under high stringent conditions. To investigate the
transactivation activities of VR-EILs, total RNAs (20 µg per lane) were
isolated from 10-d-old green T2 generation plants of various
independent 35S::VR-EIL lines (VR-EIL1-2, VR-EIL1-3, VR-EIL1-4,
VR-EIL1-8, VR-EIL2-1, and VR-EIL2-2), which had been grown without exogenous
ethylene, and were similarly analyzed. The blots were visualized by
autoradiography. The equivalence of RNA loading among lanes of the agarose gel
was demonstrated by ethidium bromide staining of rRNA on the gel.
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Because 35S::VR-EIL1/2 resulted in hypersensitivity to exogenous
ethylene rather than constitutive triple response in tobacco seedlings
(Fig. 6, A and C), we
considered the possibility that ethylene-induced genes are hyperinducible by
ethylene in these transgenic plants. To investigate this possibility, the
transgenic seedlings were incubated with exogenous ethylene and the induction
patterns of genes were examined. As shown in
Figure 8, A and B, the
-1,3-glucanase and osmotin genes were hyperinduced by
ethylene treatment; the mRNA level greatly increased in ethylene-treated
35S::VR-EIL1/2 seedlings compared with ethylene-treated wild-type
plants, confirming that the induction of -1,3-glucanase and
osmotin is dependent on the function of VR-EIL1/2. Thus, transgenic
tobacco plants overexpressing VR-EILs are more sensitive to exogenous
ethylene than wild-type plants, indicating that VR-EILs are functionally
relevant in tobacco cells. On the other hand, the level of ethylene-inducible
NG-ACO1 transcript was almost identical in wild-type and transgenic
seedlings in response to exogenous ethylene
(Fig. 8).
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Figure 8. Induction pattern of -1,3-glucanase, osmotin, and
NG-ACO1 in response to exogenous ethylene in wild-type (WT) and
VR-EIL-overexpressing tobacco plants. A, Total RNAs (20 µg per
lane) were isolated from 7-d-old light-grown wild-type and T2
generation plants of various independent 35S::VR-EIL lines that had
been incubated with or without 20 µL L–1
ethylene for 6 h, and were resolved on a 1.0% (w/v) formaldehyde-agarose gel.
The gel was blotted onto a membrane filter and the blot was hybridized to the
32P-labeled cDNA probes for -1,3-glucanase,
osmotin, and NG-ACO1, respectively, under high stringent
conditions. The blots were visualized by autoradiography. The equivalence of
RNA loading among lanes of the agarose gel was demonstrated by ethidium
bromide staining of rRNA on the gel. B, The relative levels of
-1,3-glucanase, osmotin, and NG-ACO1 transcripts in
wild-type and various VR-EIL-overexpressing tobacco plants in the
presence or absence of exogenous ethylene. Hybridization signals obtained from
the northern-blot analysis of A were quantified with a phosphorimager
(Fuji).
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DISCUSSION
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As ethylene exerts profound effects on such diverse aspects during plant
growth and in the pathogenesis response, its signal transduction pathway and
molecular action mechanism have attracted much interest. The expression of
various development- and defense-related genes is controlled by this simple
gaseous hormone at the transcriptional level. Ethylene-responsive cis-acting
elements and nuclear proteins that specifically interact with these regulatory
regions have been identified in several genes such as the tomato fruit
E4 and E8 genes (Cordes
et al., 1989; Montgomery et
al., 1993; Coupe and Deikman,
1997; Deikman et al.,
1998), the carnation GST1 gene
(Itzhaki et al., 1994;
Maxson and Woodson, 1996), and
the tobacco PRB-1b and osmotin-like PR-5 genes
(Meller et al., 1993;
Sato et al., 1996). These
studies have found two different types of EREs. The study with the aid of
stable deletion analysis of promoters from tobacco chitinase and
PRB-1b genes has defined the GCC-box as an ERE, composed of an 11-bp
sequence (TAAGAGCCGCC) that is highly conserved in the 5'-upstream
region of ethylene-inducible PR protein genes
(Sessa et al., 1995;
Shinshi et al., 1995).
Subsequently, Ohme-Takagi and Shinshi
(1995) have isolated four
different cDNA clones encoding the GCC-box-binding proteins (EREBPs) and have
demonstrated that the accumulation of their transcripts increased in response
to ethylene in various tissues of tobacco plants. More recently, these ERFs
have been found to modulate the GCC-box-mediated gene expression, as
transcriptional activators or repressors in tobacco and Arabidopsis plants,
indicating that ERFs respond to extracellular signals in distinct ways
(Fujimoto et al., 2000; Ohta
et al., 2000a,
2000b). The other kind of ERE
was identified to be responsible for the developmental expression of tomato
E4 and carnation GST1 genes that are induced during fruit
ripening and flower senescence, respectively
(Montgomery et al., 1993;
Itzhaki et al., 1994).
Interestingly, a similar E4- and GST1-like cis-acting
element was also found in the promoter of Arabidopsis ERF1 that
encodes the GCC-box-binding protein
(Solano et al., 1998). Based
on these results, Solano et al.
(1998) renamed the
E4- and GST1-like element as a primary ERE. Furthermore,
they have shown that the Arabidopsis EIN3/EILs family, an essential component
participating in the ethylene-signaling pathway, directly binds to a primary
ERE present in the ERF1 gene. Thus, it seems most likely that there
exists an ethylene signaling sequence in the nucleus from EIN3 to
ERF1 to a variety of ethylene-responsive genes in Arabidopsis. Using
a random binding site selection analysis, Kosugi and Ohashi
(2000) have revealed that the
consensus binding sequence for tobacco TEIL is A[T/C]G[A/T]A[C/T]CT. This
conserved sequence is found to be present in the 5'-upstream regions of
E4, GST1, ERF1, and tobacco NsERF2
(Montgomery et al., 1993;
Itzhaki et al., 1994;
Solano et al., 1998;
Kitajima et al., 2000).
In the present study, we have isolated and characterized two full-length
cDNA clones, pVR-EIL1 and pVR-EIL2, that encode mung bean homologs of the
Arabidopsis EIN3. Both VR-EILs share a high degree of sequence conservation
with previously identified EIN3-LIKE proteins from Arabidopsis, tobacco, and
tomato plants (Fig. 1). Gel
retardation analysis shows that mung bean EILs are able to interact
specifically with obs1, the obs for tobacco TEIL
(Kosugi and Ohashi, 2000).
Competition binding assay suggests that two mung bean proteins have different
binding capacity to obs1, with the interaction of VR-EIL2 being more efficient
than that of VR-EIL1 (Fig. 2B).
Although VR-EIL1 and VR-EIL2 are highly homologous and share various conserved
domains throughout the coding region (see below), VR-EIL2 uniquely possesses
an Asn-rich region at the carboxy terminus that consists of six Asn residues
(Fig. 1B). This domain is also
present in Arabidopsis EIN3, but not in VR-EIL1 and other Arabidopsis EILs and
tomato LeEILs. Thus, it would be intriguing to identify whether a
carboxy-terminal Asn-rich region is functioning in the binding mechanism
between VR-EIL2 and the consensus sequence obs1.
Beside an Asn-rich region, VR-EILs contain several distinct architectural
properties such as an amino-terminal acidic domain, a Pro-rich region, five
small clusters of basic amino acids, and a single poly-Gln repeat near the
carboxy terminus (Fig. 1B). Acidic, Pro-rich, and Gln-rich domains have been indicated to function as
transcriptional activation motifs
(Mitchell and Tjian, 1989;
Chao et al., 1997). To unravel
an essential domain for transcriptional activation, we performed yeast
one-hybrid assay using various deletion mutants of VR-EIL2. The results
demonstrate that the amino-terminal 50-amino acid residues are required to
activate transcription of a reporter gene in yeast cells; the
VR-EIL21–50 mutant protein shows 70%
transcription-stimulating activity compared with that of the wild-type protein
(Fig. 4). In our experimental
conditions, the Pro-rich region and poly-Gln repeat, respectively, contain
only the background level of activity for transcriptional activation in yeast,
suggesting that they are not essential components for the function of a
transcriptional activator. However, we could not rule out the possibility that
those domains may be necessary for full activity of VR-EIL2.
To investigate the physiological role of VR-EILs in more detail,
we constructed transgenic tobacco plants expressing each of these genes under
the control of CaMV 35S promoter. In contrast with what has been previously
found in EIN3- and TEIL-overexpressing Arabidopsis plants,
we observed that 35S::VR-EIL1/2 tobacco lines did not exhibit a
constitutive triple response in the absence of exogenous ethylene
(Fig. 6A). Instead, both
VR-EIL constructs displayed great increase in root hair formation,
and this phenotype was further enhanced in the presence of ACC
(Fig. 6, B and C). In addition,
various GCC-box-containing PR genes, including -1,3-glucanase,
osmotin, and PR1, were constitutively activated in
35S::VR-EIL1/2 plants without ethylene treatment
(Fig. 7). The
VR-EIL-overexpressing lines also show an enhanced sensitivity to
exogenous ethylene as evidenced by following observations: more severely
reduced hypocotyl length and exaggerated root hair formation in comparison
with the control seedlings in response to ACC
(Fig. 6, A and C) and the
hyperethylene-induction of the -1,3-glucanase and
osmotin genes compared with ethylene-treated wild type plants
(Fig. 8). Taken together, these
results support the notion that VR-EILs are functionally relevant in tobacco
cells, which interact with upstream and downstream partners of the tobacco
EIN3 homologs and turn on a subset of ethylene responses.
A wealth of information has been documented that ethylene is closely
associated with the induction of the ACC oxidase gene in climacteric fruits,
resulting in a surge in ethylene production during the ripening process
(Yang and Hoffman, 1984;
Abeles et al., 1992).
Previously, we showed that ethylene markedly stimulated the accumulation of
ACC oxidase mRNA in vegetative tissues of mung bean
(Kim and Yang, 1994;
Jung et al., 2000) and tobacco
(Kim et al., 1998) plants.
Based on these results, it has been proposed that ethylene plays a key role in
regulating ACC oxidase gene expression in vegetative tissues, as it does in
fruit tissue, and that the low basal level of ACC oxidase transcript
constitutively expressed in vegetative tissues is regulated by the endogenous
ethylene present in these tissues (Kim et
al., 1997; Jin et al.,
1999). This autocatalysis reaction by ethylene results from the
transcriptional activation of the ACC oxidase gene
(Park et al., 2001). However,
the results in Figures 7 and
8 reveal that although the
35S::VR-EIL constructs display the phenotype of ectopic root hair
formation, one of the typical ethylene responses, and several
GCC-box-containing PR genes are effectively transactivated in these transgenic
lines, the expression pattern of ACC oxidase transcript (NG-ACO1) was
very similar in transgenic and wild-type tobacco plants. The NG-ACO1
transcript was not constitutively expressed or hyperinduced by ethylene in
transgenic plants. The reason for this unexpected result is not clear at this
moment, but it could be due to the fact that VR-EILs may not be fully
functional in heterologous tobacco cells and, hence, turn on a subset of
ethylene responses. In this regard, it is worth noting that the
VR-EIL lines do not exhibit a constitutive triple response
(Fig. 6A). Alternatively, it is
possible that the EIL family might possess slightly different DNA-binding
specificity (e.g. distinct target sequences and different binding activities)
or differential signal thresholds and sensitivities, resulting in the
activation of different sets of gene expression. For instance,
NG-ACO1 could be more sensitive to TEIL than to VR-EIL1/2 so that
overexpression of VR-EILs did not significantly affect the level of
NG-ACO1 mRNA in transgenic tobacco seedlings. Interestingly, our
computer database analysis suggests that the promoter region of ACC oxidase
from different species, including tomato LeACO1 and LeACO2
(Barry et al., 1996;
Blume and Grierson, 1997),
apple (Malus domestica) GSAO
(Atkinson et al., 1998) and
MD-ACO2 (GenBank accession no. AF015787), mung bean VR-ACO1
(Park et al., 2001), and
Arabidopsis EAT1 (GenBank accession no. X66719) do not seem to have
the consensus sequences for EIN3-binding site or GCC-box. Thus, further
experiments are required to define the ERE present in the 5'-upstream
region of ACC oxidase and the role of ethylene-responsive trans-acting factors
for the regulation of the last step in ethylene production by this simple
signaling hydrocarbon in higher plants.
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MATERIALS AND METHODS
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Plant Materials and Ethylene Treatment
Dry seeds of mung bean (Vigna radiata) were soaked overnight in
aerated tap water. Seedlings were grown on 0.8% (w/v) agar for 4 d in a dark
room at 25°C or for 2 weeks in an environmentally controlled chamber. In
the case of tobacco (Nicotiana tabacum cv Samsun NN) plants,
wild-type and transgenic seedlings were grown on 0.8% (w/v) agar for 4 d in a
dark room or on Murashige and Skoog medium containing 1% (w/v) Suc, B5 vitamin
(12 mg L–1), and 0.8% (w/v) agar (pH 5.8) for 7 d in a growth
chamber under a 16-h light/8-h dark photoperiod at 25°C. For ethylene
treatment, intact plants were enclosed for various periods in 3-liter jars
containing air or air plus 20 µL L–1.
PCR
The first strand cDNA, synthesized from 1 µg of poly(A)+ RNA
isolated from etiolated mung bean hypocotyls, was amplified by PCR using mixed
oligonucleotide primers
(CCGGAATTCCA[G/A]GA[T/C]-[G/A]CIAC[G/A/T/C][T/C]T[G/A/T/C] as the upstream
primer and CGGGATCCGC[G/A/T/C]GTCAT[T/C]TT[G/A]TC[T/C]TG as the downstream
primer). These primer sequences were derived from the conserved amino acid
sequences, QDTTLG and QDKMTA, respectively, which were found to be highly
conserved in Arabidopsis EIN3 and EILs
(Chao et al., 1997). The
amplified PCR products were subcloned into pGEM-T Easy vector system I
(Promega, Madison, WI). PCR was performed in a total volume of 25 µL
containing 1 µL of the first strand cDNA reaction products, 1
µM primers, 10 mM Tris (pH 8.0), 50 mM
KCl, 1.5 mM MgCl2, 0.01% (w/v) gelatin, 200
µM deoxynucleotides, and 2.5 units of Taq polymerase
(Promega). Thirty-five thermal cycles were carried out, each consisting of 1
min at 94°C, 2 min at 48°C to 52°C, and 2 min at 72°C in an
automatic thermal cycler (Perkin-Elmer/Cetus, Norwalk, CT). PCR products were
separated on an agarose gel, and the band was eluted and reamplified by PCR to
increase the amount of DNA for the subsequent subcloning. The various
ethylene-responsive tobacco cDNA clones for expression studies were obtained
by reverse transcriptase-PCR using specific primers constructed based on the
published DNA sequences as described above.
Screening of cDNA Library and Sequencing of DNA
To isolate mung bean cDNA clones encoding EIN3 homolog, the lambda Zap II
mung bean hypocotyl cDNA library (Kim and
Yang, 1994) was screened using the 300-bp PCR products as probes
by an established procedure (Sambrook et
al., 1989). The cDNA inserts containing putative mung bean EILs
were subcloned into Bluescript SK plasmid by in vivo excision of pBluescript
from Zap II vector as described in the protocols by Stratagene (La Jolla, CA).
Sequencing of DNA was performed using the sequenase DNA sequencing kit
according to the manufacturer's manual (U.S. Biochemicals, Cleveland).
Cloning and Expression of VR-EILs
The plasmid pGEX4T-1 (Pharmacia, Uppsala, Sweden) was used for the
expression of GST-VR-EIL fusion proteins. Escherichia coli BL21 (DE3)
strains containing the plasmids were grown at 37°C in 100 mL of 2x
Luria-Bertani medium (10 g of trypton, 10 g of yeast extract, and 5 g NaCl
L–1) supplemented with 1% (w/v) glycerol as an
additional carbon source and 70 µg mL–1
ampicillin. Cells were grown for a further 4 h at 30°C after induction
with 1 mM isopropyl -D-thiogalactoside at 0.6 to
1.0 OD600. The pellet was resuspended in phosphate-buffered saline
containing 1 mM phenylmethyl-sulfonyl fluoride. The suspension was
sonicated on ice with Vibracell sonicator (Sonics and Materials, Danbury, CT),
and Triton X-100 was added to a final concentration of 1% (w/v). Various
fusion proteins were purified by affinity chromatography using glutathione
Sepharose 4B from GST purification modules (Pharmacia).
Gel Retardation Assay
DNA probes and competitors (obs1, obsm1, and obsm2) for gel retardation
assays were produced based on the results of Kosugi and Ohashi
(2000). To reduce nonspecific
DNA-protein binding, purified GST-VR-EIL proteins (50–100 ng) were
preincubated with 0.5 µg of poly (dI-dC) and 0.5 µg of nonspecific DNA
oligonucleotide in 20 µL of binding buffer (10 mM Tris-HCl, pH
8.0, 1 mM EDTA, 1 mM dithiothreitol, 50 mM
NaCl, and 5% [w/v] glycerol) for 10 min on ice. End-labeled DNA probe (0.25
ng) was then added to the reaction mixtures. After incubating for 10 min on
ice, the mixtures were loaded onto an 8% (w/v) nondenatured polyacrylamide
gel. Before loading, gels were pre-run at 10 V
cm–1 for 30 min, and electrophoresis was performed
in 0.5x 54 mM Tris-borate, pH 8.3, and 1 mM EDTA
for 2.5 h. For competition experiments, varying amounts of cold competitor
molecules were preincubated with GST-VR-EILs before addition of the
radiolabeled probe. The gel was dried and autoradiographed.
Subcellular Localization of VR-EILs
The smGFP cDNA was fused in frame to the 3' end of full-length
pVR-EIL coding regions. Transient expression of smGFP fusion constructs was
then performed by introducing the DNAs into onion (Allium cepa)
epidermal cells using the particle bombardment method according to the
manufacturer's protocol (Bio-Rad, Richmond, CA). Fluorescence photographs of
onion cells were taken using a fluorescence microscope (Axiophot; Zeiss, Jena,
Germany) fitted with fluorescein isothiocyanate filters (excitation filter,
450–490 nm; emission filter, 520 nm; dichroic mirror, 510 nm) and color
film (Fuji 400; Fuji, Tokyo). The optimal exposure time was 1 s.
Construction of Various VR-EIL2 Deletion Mutants and Transactivation
Activity Analysis
The deletion mutant constructions were generated using various restriction
sites within the VR-EIL2 cDNA or by PCR. Coding regions for the deleted
proteins were fused in frame to the yeast GAL4 DNA-binding domain expression
vector pAS1-CYH2. The recombinant plasmids were then transformed into
Saccharomyces cerevisiae strain CG1945 as described by the
manufacturer (Clontech, Palo Alto, CA). The transformants were selected by
growth on Trp– synthetic dropout medium at 30°C for 3 d.
The -galactosidase filter-lift assay was performed according to the
manufacturer's protocol (Clontech). -Galactosidase activities were
quantified after growth of yeast strains in liquid culture using chlorophenol
red--D-galactopyranoside as a substrate.
Isolation of Genomic DNA and Southern-Blot Analysis
The mung bean leaf genomic DNA was isolated as described previously
(Park et al., 2001) with
modifications. Each gram of mung bean leaf was pulverized under liquid
nitrogen and was suspended in 3 mL of extraction buffer (8.0 M
urea, 50 mM Tris-Cl, pH 7.5, 20 mM EDTA, 250
mM NaCl, 2% [w/v] sarcosyl, 5% [v/v] phenol, and 20 mM
2-mercaptoethanol). After successive extractions with
phenol/chroloform/isoamylalcohol (25:24:1, v/v), the aqueous phase was
concentrated by ethanol precipitation. The pellet was resuspended in 10
mM Tris-Cl (pH 7.5) and 1 mM EDTA adjusted to a density
of 1.5 g mL–1 by the addition of CsCl and the DNA
was centrifuged overnight at 200,000g. The DNA band was collected,
extracted with water-saturated 1-butanol, precipitated by ethanol, and then
resuspended in 10 mM Tris-Cl (pH 7.5) and 1 mM EDTA.
Mung bean genomic DNA (10 µg per lane) was digested with appropriate
enzymes, separated by electrophoresis in a 0.7% (w/v) agarose gel, and blotted
onto a nylon membrane filter (Amersham, Arlington Heights, IL). The filter was
hybridized to 32P-labeled gene-specific probe 1 and probe 2,
respectively, under high stringent conditions.
RNA Isolation and Northern-Blot Analysis
Total RNAs of mung bean and tobacco plants were obtained by a method
adapted from the established protocols reviewed by Lizzradi
(1983) with modifications as
described previously by Jin et al.
(1999). The total RNAs were
precipitated overnight at 4°C by the addition of 0.3 vol of 10
M LiCl and then precipitated in ethanol. The RNAs (20 µg) were
fractioned by electrophoresis in a 1.0% (w/v) formaldehyde-agarose gel and
were blotted onto a nylon membrane filter. Equal loading of RNA was confirmed
by visualizing the ethidium bromide-stained ribosomal RNA content under UV
light at the end of electrophoresis. The filter was hybridized to various
32P-labeled cDNA probes for mung bean VR-EIL1 and
VR-EIL2, and tobacco -1,3-glucanase, osmotin-like
PR-5, PR1, and NG-ACO1. The blot was washed and
visualized by autoradiography at –80°C. Hybridization signals were
quantified with a phosphorimager (Fuji).
Gene Constructs for Transgenic Plants and Tobacco Transformation
A vector, pBI121, which contained the CaMV 35S promoter and nopaline
synthase terminator, was used for tobacco transformation. pVR-EIL cDNA
fragments containing the complete open reading frame were generated by PCR
using high-fidelity Ex-Tag polymerase (Takara, Kyoto). To facilitate
subcloning of PCR products, forward and reverse primers for each cDNA clone
were tagged by appropriate restriction enzymes (pVR-EIL1,
XbaI/BamHI; pVR-EIL2, BamHI/SacI). The
-glucuronidase reporter gene of pBI121 was eliminated and the tagged
pVR-EIL cDNA fragments were inserted into the corresponding sites of pBI121.
The fusion gene constructs were transferred to Agrobacterium
tumefaciens strain LBA4404 by electroporation according to Shen and Forde
(1989). Leaf discs of tobacco
cv Samsun NN were transformed essentially as described by Horsch et al.
(1985). Transgenic plants were
selected on the Murashige and Skoog medium containing 200 µg
mL–1 kanamycin and 500 µg
mL–1 carbenicillin. The presence of transgene in
the transformants was confirmed by PCR and northern-blot analysis with
32P-labeled gene-specific probe 1 and probe 2, respectively.
Primary transformants were self-fertilized and seeds were collected.
T2 seeds were harvested from individual T1 plants for
further studies.
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ACKNOWLEDGMENTS
|
|---|
We thank Sunglan Chung (Department of Biology, Yonsei University) for her
critical reading of the manuscript.
Received February 21, 2003;
returned for revision April 2, 2003;
accepted April 2, 2003.
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FOOTNOTES
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|---|
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.103.022574.
1 This work was supported by the Plant Diversity Research Center (21st
Century Frontier Research Program of Ministry of Science and Technology
project no. PF 003105–01) and by Korea Science and Engineering
Foundation (Plant Metabolism Research Center, Kyung Hee University, to
W.T.K.). 
*
Corresponding author; e-mail
wtkim{at}yonsei.ac.kr;
fax 82–2–312–5657.
|
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