Plant Physiol. (1999) 120: 473-480
Transgenic Overexpression of the Transcription Factor
Alfin1 Enhances Expression of the Endogenous MsPRP2
Gene in Alfalfa and Improves Salinity Tolerance of the
Plants1
Ilga Winicov2, * and
Dhundy R. Bastola3
Departments of Microbiology and Biochemistry, University of Nevada,
Reno, Nevada 89557
 |
ABSTRACT |
Alfin1
cDNA encodes a putative transcription factor associated with NaCl
tolerance in alfalfa (Medicago sativa L.). The
recombinant protein binds DNA in a sequence-specific manner, including
promoter fragments of the NaCl-inducible gene MsPRP2.
Alfin1 function was tested in transgenic alfalfa under the
control of the 35S promoter in the sense and antisense orientations
with the endogenous MsPRP2 as a reporter gene. Calli
overexpressing Alfin1 were more resistant to growth
inhibition by 171 mM NaCl than vector-transformed controls, whereas calli expressing Alfin1 in the antisense
orientation were more sensitive to NaCl inhibition. Transgenic plants
overexpressing Alfin1 in the sense orientation grew
well. In contrast, the antisense transgenic plants grew poorly in soil,
demonstrating that Alfin1 expression is essential for
normal plant development. Transgenic calli and plant roots
overexpressing Alfin1 showed enhanced levels of
endogenous MsPRP2 mRNA accumulation. However,
MsPRP2 mRNA accumulation was also regulated in a
tissue-specific manner, as shown in leaves of transgenic plants
overexpressing Alfin1. These results suggest that Alfin1
acts as a transcriptional regulator in plants and regulates
MsPRP2 expression in alfalfa.
Alfin1 overexpressing transgenic plants showed salinity
tolerance comparable to one of our NaCl-tolerant plants, indicating
that Alfin1 also functions in gene regulation in NaCl
tolerance.
| |
INTRODUCTION |
Plants and cells adapt to changes in the ionic environment as a
result of salinity and drought through temporal or sustained regulation
of a large number of genes (for review, see Bohnert et al., 1995
;
Ingram and Bartels, 1996; Bray, 1997), but the molecular mechanisms
responsible for this regulation have remained elusive. We have
documented coordinated gene regulation in long-term acquired NaCl
tolerance in alfalfa (Medicago sativa L.) and
rice (Winicov et al., 1989; Winicov, 1991, 1996) and have been
interested in defining a functional role for a putative transcription
factor, Alfin1, in the altered gene expression in NaCl-tolerant alfalfa (Winicov, 1993; Bastola et al., 1998).
A relatively small number of transcription factors have been identified
to date that bind to promoter elements in genes regulated by
NaCl/drought stress (for review, see Ingram and Bartels, 1996; Shinozaki and Yamaguchi-Shinozaki, 1997; Winicov and Bastola, 1997),
and much of the information has been gene specific. A more complex view
of transcriptional regulation is implied by the requirement of a
coupling element for stress regulation of the barley HVA22 gene containing the ABA response element (Shen et al., 1996) and the
combined role of myc and myb transcriptional
activators in ABA- and dehydration-inducible expression of a promoter
region of the rd22 gene (Abe et al., 1997). The potential
interactions of various factors is compounded further in that
transcription factors such as myc and myb belong to extensive multigene
families with tissue-specific expression patterns. Nevertheless, recent reports have shown that ectopic expression of transcriptional activators can result in changes in plant responses to cold
(Jaglo-Ottosen et al., 1998) and disease resistance (Cao et al., 1998)
and changes in metabolic products in plants (Tamagnone et al., 1998)
and cultured cells (Grotewold et al., 1998) by affecting the levels of
expression of endogenous genes, indicating the possibility of testing
the function of individual transcription factors.
Alfin1 cDNA encodes a novel member of the
zinc-finger family of proteins, and its modulation in NaCl tolerance
makes it an interesting target for manipulation in plants. It contains
sequence information for adjacent Cys-4 and His/Cys-3 zinc-finger
domains that appear to bind adjacent G-rich triplet motifs in DNA
(Bastola et al., 1998). It also contains an acidic region
characteristic of DNA-binding proteins that interact with other
proteins (Kakidani and Ptashne, 1988) and therefore is likely to
function as a transcription factor in plants. Alfin1 is
expressed predominantly in roots, appears to be unique or a low-copy
gene in the alfalfa genome, and shows conservation among such diverse
plants as alfalfa, rice, and Arabidopsis (Winicov and Bastola, 1997).
These characteristics, in addition to in vitro binding to promoter
fragments of the root-specific MsPRP2 gene that is also NaCl
inducible (Winicov and Deutch, 1994; Deutch and Winicov, 1995),
suggested that it may have a significant function in plant-root
gene expression and contribute to gene regulation in NaCl tolerance.
To test the functions of Alfin1, we made constructs of the
Alfin1 cDNA in the sense and antisense orientations, driven
by the strong CaMV 35S promoter, transformed alfalfa, and looked for
MsPRP2 expression as a potential reporter for Alfin1
activity in vivo. The antisense transformants demonstrated that normal Alfin1 transcript levels were essential for plant
development in soil. However, antisense transformation only minimally
affected callus growth on control medium. Nonetheless, increased or
decreased Alfin1 expression in the transformed callus
correlated positively with relative growth in NaCl-containing medium in
culture. In addition, we were able to monitor the mRNA levels of the
endogenous alfalfa MsPRP2 gene. In this paper we report that
Alfin1 overexpression in transgenic plants led to
MsPRP2 accumulation in callus and roots, suggesting that
Alfin1 acts as a transcriptional regulator in plants and plays an
important role in MsPRP2 expression in alfalfa. Because
transgenic plants overexpressing Alfin1 also showed improved
NaCl tolerance, comparable to our NaCl-tolerant plant previously
regenerated from cell culture, Alfin1 expression must play
an important regulatory role that can provide enhanced NaCl tolerance
in alfalfa.
| |
MATERIALS AND METHODS |
Plant Material
Alfalfa (Medicago sativa L. cv Regen S) cell lines were
maintained on SH growth medium (Schenk and Hildebrandt, 1972) in
continuous light with and without 171 mM NaCl, as
described previously (Winicov et al., 1989; Winicov and Button,
1991). Because of the autotetraploid genotype of alfalfa, all
experiments were performed with the parent control plant labeled no. 1, which represents the NaCl-sensitive wild type. All transformations were
done with material from this plant or the NaCl-tolerant mutant no. 9, originally selected and regenerated from no. 1 (Winicov, 1991). The
NaCl-sensitive parent and NaCl-tolerant plants regenerated from the
NaCl-tolerant cell cultures (Winicov, 1991) were maintained in the
greenhouse and propagated by cuttings. The influence of NaCl on plant
growth was determined on replicate rooted cuttings of plants
established in Conetainers in perlite and watered daily with
one-quarter-strength Hoagland solution (Hoagland and Arnon, 1938), with
or without the indicated concentrations of NaCl, as described
previously (Winicov, 1991). All plant material was harvested at the
same time of day.
Recombinant Plasmid Construction
The full-length coding Alfin1 clone (pA50) consists of
a 904-bp fragment of Alfin1 cDNA (accession no. L07291) in
pBluescript SK
(Stratagene). It contains a 30-bp 5
-untranslated
leader, a complete 771-bp coding sequence, and 103 bp of the
3-untranslated region, including the translation termination codon
(Winicov, 1993). This cDNA fragment was cloned in the sense and
antisense orientations in the MCS of the binary expression vector
pGA643 (An et al., 1988), as shown in Figure
1.
View larger version (21K):
[in this window]
[in a new window]
| Figure 1.
Schematic representation of Alfin1
sense and antisense constructs used in transformation of alfalfa.
Restriction sites are as follows: E, EcoRI; H,
HindIII; B, BglII; and S,
SalI. BR and BL are T-DNA right and left borders,
respectively (An et al., 1988).
|
|
To generate the sense construct, the 939-bp
HindIII-XbaI fragment from pBluescript SK was
first subcloned in pFLAG (International Biotechnologies Inc., New
Haven, CT), designated as PF-pA50, to gain a restriction site suitable
for cloning the cDNA fragment in pGA643. The 957-bp
HindIII-BglII fragment from PF-pA50 containing Alfin1 cDNA was then ligated to pGA643 in the MCS 3 to the CaMV 35S
promoter to give pGA-sense. This clone was predicted to give the
complete Alfin1-coding transcript, but unlike the endogenous Alfin1 mRNA it carried additional sequences from the vector
in its 3-untranslated region.
To generate the antisense construct (pGA-ATS), the 944-bp
ClaI-XbaI fragment from pA50 (pBluescript SK)
was ligated directly into the pGA643 MCS. Although another
ClaI site has been reported upstream of the MCS in pGA643,
we found that only the ClaI site in the MCS, indicated in
Figure 1, was cut by the enzyme.
The plasmids pGA-sense, pGA-ATS (antisense), and pGA643 (vector) were
propagated in Escherichia coli strain MC1000 (a gift from
Dr. G. An, Washington State University, Pullman) in the presence of
tetracycline. The freeze-thaw method, as described by An et al. (1988),
was used to transform Agrobacterium tumefaciens LBA 4404 (Hoekema et al., 1983) with the recombinant binary plasmid. Transformed colonies were selected on 12 mg/L rifampicin and 6 mg/L
tetracycline. Recombinant transformed colonies were identified by
colony hybridization using the Alfin1 670-bp
EcoRI fragment from pA50 (Sambrook et al., 1989).
Plant Transformation
Alfalfa NaCl-sensitive wild-type parent plant no. 1 (Winicov,
1991) leaves were transformed by A. tumefaciens
cocultivation on SH growth medium, including 2 mg/L 2,4-D and 2 mg/L
kinetin (Schenk and Hildebrandt, 1972), and supplemented with 50 µM acetosyringone (Aldrich) for 30 to 60 min at
room temperature. One of the successful transformations was carried by
cocultivating A. tumefaciens carrying the pGA-ATS with
immature ovaries from the NaCl-tolerant alfalfa IW9 line (Winicov,
1991). After 2 to 6 d on callus medium the explants were
transferred to selection medium (SH medium supplemented with 300 mg/L
carbenicillin and 100 mg/L kanamycin) and incubated for 3 to 4 weeks.
The resistant calli were subcultured on the selection medium on a
monthly basis. Plants were regenerated from the transformed calli on SH
medium (without hormones) supplemented with 100 mg/L kanamycin. Plants
with well-defined shoots and roots were transferred to peat moss and
subsequently to soil.
DNA Extraction and PCR Analysis
Genomic DNA was extracted from 0.5 g of frozen callus or
leaves using DNAzol genomic DNA isolation reagent (Molecular Research Center, Inc., Cincinnati, OH), as described by the manufacturer. PCR
was carried out in a 25-µL total reaction containing 250 ng of
genomic DNA, 1× PCR buffer (50 mM KCl, 10 mM
Tris-HCl, pH 9.0, and 0.1% Triton X-100), 100 µM
deoxynucleoside triphosphates, 0.2 µM each of the forward
(primer common to all PCR analyses in this section = 5 CCA CTA
ATT CGT CCT GCT GG 3) and the reverse sequence primers (Midland
Certified Reagent Co., Midland, TX) (PS for sense [5 CCA GTC CCT CTC
CTG CAT TC 3], PA for antisense [5 GGA CAA GGT GCA ACC TGT GG 3],
and PG for vector [5 AAG TGT GCT TGA GCT CGG TC 3]), and
0.25 unit of Taq polymerase (Promega). The forward sequence
primer was from position 2432 bp and the reverse primer was from 3404 bp for the pGA-vector, 3356 bp for the pGA-sense, and 3359 bp for the
pGA-antisense DNA sequence of the T-DNA right border. This combination
of PCR primers gave 973-, 926-, and 928-bp products, respectively.
The Gene Amp PCR System (model 2400, Perkin-Elmer) was programmed for
an initial denaturing temperature of 94°C for 4 min, a second
denaturing temperature of 94°C for 1 min, an annealing temperature of
62°C for 90 s, and an extension temperature of 72°C for 1 min.
The reaction was carried out for 35 cycles. An additional extension at
72°C followed for 7 min after completion of the final cycle.
RNA Extraction and Blot Analysis
Total RNA was extracted from roots and shoots containing both
leaves and stems from plants grown for 17 d with and without 128 mM NaCl, or callus grown for 1 month with and without 171 mM NaCl, and analyzed under high-stringency hybridization
and wash conditions, as described previously (Winicov and Deutch, 1994;
Winicov and Krishnan, 1996). Northern analysis for Alfin1 was done with the 670-bp EcoRI large fragment from pA50; for
MsPRP2, the probe was the EcoRI fragment from pA9
(Winicov and Deutch, 1994); the constitutively expressed
Msc27 was probed with the PstI fragment (Gyorgyey
et al., 1991); and the 763-bp EcoRI-BglII fragment from pGA643 (the region between the 3 end of the MCS and the
T-DNA left border) was used to detect transgenic Alfin1 expression. Gel-purified fragment probes were labeled with
[32P]dCTP using the random primer-extension
system (DuPont-NEN).
| |
RESULTS |
Alfalfa Calli Transformed with Sense and Antisense
Alfin1
NaCl-sensitive alfalfa cells were transformed with pGA-sense,
pGA-ATS (antisense), and the vector pGA643. Many kanamycin-resistant lines were isolated from independent transformations in three different
experiments. A total of 22 independent transformed lines were obtained
with pGA-sense, 14 independent lines were obtained with pGA-ATS, and
comparable numbers were obtained using the empty vector pGA643. No
consistent differences in cell growth were observed between
transformants of the different constructs, although significant growth
differences could be seen between independently transformed cell lines.
Only transformed calli showing good growth on kanamycin were further
maintained and analyzed. Kanamycin-resistant transformants were
confirmed by PCR to carry the appropriate inserts (data not shown).
The influence of transformation with Alfin1 was measured by
alfalfa callus growth on SH medium with and without 171 mM NaCl, as shown in Table
I. Two NaCl-sensitive cell lines (1,1 and
1,5) were independently initiated in culture. They showed 92% and 84% growth inhibition by NaCl, respectively, as measured by an
NaCl-dependent increase in callus wet weight after 4 weeks of growth.
The 1,1 cells transformed with pGA-sense showed less growth inhibition by NaCl than those transformed by the pGA643 vector alone. In contrast,
1,5 cells transformed with the pGA-ATS appeared to grow somewhat more
slowly on the control medium and were more sensitive to growth
inhibition by NaCl than the pGA643 vector-transformed cells. These
results were consistent with our hypothesis that Alfin1 helps to
maintain cellular functions in our NaCl-tolerant alfalfa. However, none
of the sense transformants was able to grow as well on 171 mM NaCl as on the control SH medium.
Overexpression of Alfin1 in Transgenic Callus
Increases MsPRP2 mRNA Levels
Alfin1 expression was determined in the
pGA-sense-transformed callus by northern analysis of total RNA using
the constitutively expressed Msc27 gene probe to monitor RNA
concentrations in each lane. In Figure 2,
the results show clearly that Alfin1 expression was greatly
enhanced in the S1, S2, S4, and S6 pGA-sense-transformed cell lines
compared with untransformed and vector-transformed cells. Some
variability in the levels of expression was observed between different
transformants, consistent with the prevalent variability resulting from
independent transformation events. Concurrent with the enhanced
Alfin1 expression in the transgenic cells we also found
significantly increased levels of MsPRP2 transcripts. The
levels of MsPRP2 transcripts found in pGA-sense-transformed cells were higher than those found in NaCl-tolerant cells grown in the
presence of NaCl, and we could not detect further NaCl-induced enhancement of the high levels of MsPRP2 mRNA accumulation
in the pGA-sense-transformed callus. Because recombinant Alfin1 was shown to bind to promoter fragments of MsPRP2 in vitro
(Bastola et al., 1998), the enhanced levels of endogenous
MsPRP2 transcripts in callus overexpressing
Alfin1 suggest that Alfin1 regulates alfalfa
MsPRP2 expression in vivo.
View larger version (42K):
[in this window]
[in a new window]
| Figure 2.
Northern analysis of Alfin1 and
MsPRP2 expression in control and transgenic calli from
Alfin1 sense transformants. Lanes 1 and 2, RNA isolated
from untransformed NaCl-tolerant callus grown with or without 171 mM NaCl for 4 weeks; lane 3, RNA isolated from
untransformed NaCl-sensitive callus; lane 4, RNA isolated from
NaCl-sensitive callus transformed with pGA vector (1V); lanes 5 to 9, RNA isolated from NaCl-sensitive callus transformed with
Alfin1 sense construct (S1, S2, S4, and S6 are
independently transformed lines); and lane 9, RNA isolated from
S2-transformed callus grown in 171 mM NaCl. Each lane
contained 10 µg of total RNA. Each blot was hybridized sequentially
with the following probes: Alfin1, the large
EcoRI fragment (Fig. 1); MsPRP2, the
carboxy-terminal and 3-untranslated region fragment (Winicov and
Deutch, 1994); and Msc27, the fragment of a
constitutively expressed alfalfa gene.
|
|
Phenotype of Alfin1 Sense and Antisense
Transgenic Plants
To investigate the molecular and growth characteristics influenced
by Alfin1 numerous plants were regenerated from
pGA-sense-transformed calli and calli transformed with the vector
alone. Three pGA-sense-transformed plants, regenerated from independent
transformations events, were maintained for molecular and growth
studies. All three plants grew well, flowered, and set seed. The sense
transformants appeared normal, although young leaves were somewhat
broader than those from the parent plant and appeared to senesce
somewhat earlier.
Calli transformed with the pGA-ATS construct regenerated shoots
readily, but root development was poor. Treatment of the regenerating shoots with 5 µM naphthalene acetic acid gave some root
development, but none of the dozen plantlets transferred to soil
survived for more than 2 weeks. Only one pGA-ATS-transformed plant
survived in soil for about 6 months, but it remained severely dwarfed
in both root and shoot growth. These results strongly indicated that Alfin1 antisense expression was deleterious to growth and
root formation and that Alfin1 transcripts were necessary
for plant development in soil, although antisense did not have a
similar impact on callus growth in normal SH medium.
Overexpression of Alfin1 in Transgenic Plants
Increases MsPRP2 mRNA Levels in Roots
Three of the primary transformed plants with pGA-sense constructs
were analyzed for tissue-specific expression of the Alfin1 transgene and its putative target gene MsPRP2. Gel-blot
analysis of leaf total RNA from soil-grown plants shown in Figure
3 confirmed that the
pGA-sense-transformed plants showed high levels of Alfin1 mRNA expressed from Alfin1 under the control of the CaMV 35S
promoter, in contrast to the untransformed parent plant. The presence
of the transgene transcripts was demonstrated by probing of the same blot with the BglII/EcoRI fragment of the pGA643
vector, which is adjacent to the 3 end of Alfin1 cDNA and
is apparently transcribed in Alfin1 sense mRNA in the
transformants.
View larger version (50K):
[in this window]
[in a new window]
| Figure 3.
Northern analysis of Alfin1
expression in control and transgenic plants from Alfin1
sense transformants. RNA was isolated from leaves of control and
transgenic plants. Lane Con, No. 1 control NaCl-sensitive parent plant
for all transformations; lanes S1, S2, and S3, plants transformed with
the Alfin1 sense construct and regenerated from
transformed callus; and lane V, vector-transformed plant. Each blot was
hybridized sequentially with the following probes:
Alfin1, large EcoRI fragment (Fig. 1);
pGA-vector, EcoRI/BglII
fragment from pGA643 to show readthrough of the Alfin1
transgene; and Msc27, fragment of a constitutively
expressed alfalfa gene. Each lane contained 10 µg of total RNA.
|
|
Figure 4 shows similar results from the
Alfin1-overexpressing transgenic plants grown in
one-quarter-strength Hoagland solution. The MsPRP2
transcript levels increased in the roots of the
Alfin1-overexpressing plants (Fig. 4). The
vector-transformed plant no. 1 showed somewhat increased levels of
MsPRP2 mRNA in roots, but this level was not maintained in
the presence of NaCl. In fact, the MsPRP2 mRNA levels were
comparable from NaCl-grown control no. 1 and the vector-transformed no.
1 plants. In contrast, the three transgenic plants overexpressing Alfin1 maintained proportionately higher levels of
MsPRP2 mRNA in roots after growing for 17 d on 128 mM NaCl-supplemented one-quarter-strength Hoagland solution. The mRNA profiles from NaCl-tolerant no. 9 plants
are shown for a comparison. Whereas high levels of Alfin1 mRNA were found in both roots and leaves because of the 35S promoter control of the transgene, Alfin1 overexpression had a
negligible effect on MsPRP2 transcript levels in leaves of
transgenic plants grown on one-quarter-strength Hoagland solution. NaCl
treatment did not further enhance the MsPRP2 mRNA levels in
the transgenic plants, as shown in Figure 4. These results support the
Alfin1 functional role in MsPRP2 expression primarily in
roots and indicate that additional tissue-specific factors contribute
to the differences observed in MsPRP2 mRNA levels between
roots and leaves.
View larger version (35K):
[in this window]
[in a new window]
| Figure 4.
Northern analysis of Alfin1 and
MsPRP2 expression in control and transgenic plants from
Alfin1 sense transformants grown in one-quarter-strength
Hoagland solution with or without 128 mM NaCl. RNA was
isolated from roots and leaves of control plants and plants tested for
NaCl tolerance described in Table II legend. Lanes #1, Parent wild-type
control; lanes 1V, control transformed with empty vector; lanes #9,
NaCl-tolerant plant regenerated from NaCl-tolerant callus; and lanes
S1, S2, and S3, parent no. 1 transformed with pGA-sense. The blot was
hybridized sequentially with probes as described for Figure 2. Each
lane contained 10 µg of total RNA.
|
|
Effect of Alfin1 Overexpression on the NaCl-Tolerance
Characteristics of the Transgenic Plants
To determine if Alfin1 overexpression had an effect on
the NaCl-tolerance phenotype of the transgenic plants, we compared the
growth characteristics of the three pGA-sense-expressing transgenic plants with those of the wild-type NaCl-sensitive parent plant (no. 1), vector-transformed plants, and our previously
regenerated NaCl-tolerant plant IW9 (Winicov, 1991). Tolerance was
measured as relative new growth obtained from established transgenic
and control plants that had been cut back to the crown and then treated for 17 d with 128 mM NaCl. As shown in Table
II, one parental control and one
vector-transformed plant died from the NaCl treatment. All
pGA-sense-expressing transgenic plants and our NaCl-tolerant IW9 plants
survived and grew two to three times as well as the parent and
vector-transformed controls. It is important to note that IW9 had
maintained its significant NaCl-tolerant characteristics for more than
9 years after propagation by cuttings in the greenhouse. All three
independently regenerated transgenic plants overexpressing Alfin1 showed growth characteristics similar to or better
than those of our NaCl-tolerant IW9. Vector-transformed controls were as NaCl sensitive as the parent plant. These results are consistent with data from another experiment, which tested tolerance to 171 mM NaCl in plants established for only 1 week.
That experiment showed 14%, 43%, 57%, and 86% survival of no. 1 (parent), sense-1, sense-2, and IW9 plants, respectively, after 9 d of NaCl treatment. The results from both experiments indicate that
Alfin1 overexpression can provide increased NaCl tolerance
in alfalfa.
View this table:
[in this window]
[in a new window]
|
Table II.
Growth properties of Alfin1-sense-transformed
plants on 128 mM NaCl
Multiple rooted cuttings from each plant were established in individual
Conetainers in perlite for 6 weeks and grown on one-quarter-strength
Hoagland solution. All shoots were then cut back to the crown. Growth
was continued from that point on one-quarter-strength Hoagland solution
supplemented with 128 mM (0.75%) NaCl. The newly regrown
shoots were harvested and weighed after 17 d. Data are means ± SD.
|
|
Alfin1 and MsPRP2 steady-state mRNA levels were
determined for the NaCl-treated and control plants at the time of the
harvest described in Table II and are shown in Figure 4. The S1, S2,
and S3 pGA-sense transgenic plants had high levels of Alfin1
and MsPRP2 mRNA in roots, but not in shoots, regardless of
growth in 128 mM NaCl, although some
NaCl-dependent decrease in MsPRP2 mRNA levels is apparent in
the S3 transgenic plant. The MsPRP2 transcript levels appear
to be higher in the pGA-sense transgenic plants than in our
NaCl-tolerant plant no. 9. Although Table II shows significant
differences in the NaCl tolerance of the plants at 128 mM NaCl after 17 d, we did not detect
comparable levels of NaCl inducibility of MsPRP2 mRNA
accumulation (Fig. 4), as had been seen in plants treated with 171 mM NaCl for 7 d (Winicov and Deutch, 1994).
Whether this difference was due to the lower NaCl concentration or to
plant adjustment after a longer time of growth in NaCl will have to be
determined and correlated with levels of MsPRP2 protein accumulation
when plants are grown for prolonged periods in NaCl.
| |
DISCUSSION |
Overexpression of Alfin1 was engineered in transgenic
callus and alfalfa plants under the control of the strong CaMV 35S
promoter. Our previous experiments suggested that Alfin1 was likely to
function as a transcription factor, since we had shown
sequence-specific DNA binding of the recombinant protein in vitro and
specific binding to promoter fragments of the MsPRP2 gene
from alfalfa (Bastola et al., 1998). In this paper we are able to show
in callus and plants overexpressing Alfin1 a concomitant
increase in the endogenous MsPRP2 mRNA levels, indicating
that the Alfin1 gene product regulates MsPRP2
expression in vivo from its normal promoter. These results are
consistent with our prediction that Alfin1 is a transcription factor,
regulating plant gene expression, and acts in a dominant fashion in
overexpressing transgenic plants.
Although Alfin1 was expressed from the 35S promoter in both
roots and leaves, significant MsPRP2 transcript induction
from its natural promoter in the transgenic plants was detected in callus and roots, the tissues in which Alfin1 is primarily
expressed (Bastola et al., 1998). Small differences in
MsPRP2 mRNA induction by Alfin1 overexpression
were observed in leaves of soil-grown plants (data not shown) but not
in plants grown on one-quarter-strength Hoagland solution, suggesting
subtle variation due to the nutritional state of the plants. The
differential response in leaves and roots to high levels of
Alfin1 mRNA could result from the presence of a
transcriptional or posttranscriptional inhibitor of MsPRP2
transcript accumulation in leaves or may indicate the requirement for
additional root-specific transcription factors for high levels of
expression from the MsPRP2 promoter. Additional experiments
should differentiate between these two possibilities. The callus
complement of participating factors in MsPRP2 expression
appears similar to that of the root, because Alfin1
overexpression led to a significant increase of MsPRP2
transcripts in callus culture.
The plant phenotype of pGA-ATS transformants was striking in its
inability to sustain growth in soil, especially since we observed no
substantially altered phenotype in antisense-expressed callus grown on
SH medium. These results suggested a low level of redundancy for Alfin1
function and demonstrated that maintenance of Alfin1
expression was essential for root development and plant growth in soil.
Another function affected by Alfin1 antisense expression
could be root-shoot communication via the vascular system, which
suggests that the Alfin1 protein may regulate other genes in addition
to MsPRP2. On the other hand, overexpression of
Alfin1 showed no major visible phenotype, even though it was inappropriately expressed in the shoot.
Because Alfin1 was first cloned from NaCl-tolerant alfalfa
callus (Winicov, 1993), our demonstration of improved NaCl tolerance in
the transgenic plants overexpressing Alfin1 significantly
associates the product of this gene with improved NaCl tolerance.
However, its relationship to the mutation(s) that allowed the
regeneration of our NaCl-tolerant plants, such as IW9 (Winicov, 1991),
remains unclear. Transgenic plants have been engineered in a number of laboratories to overexpress single genes, which are known to be up-regulated by NaCl/drought stress in prokaryotes or plants with incremental improvements in NaCl tolerance (Tarcyznski et al., 1993;
Kishor et al., 1995; Pilon-Smits et al., 1995; Xu et al., 1996).
However, NaCl tolerance has also been considered to be a quantitative
trait (Foolad and Jones, 1993), and the molecular mechanisms by which
plants could acquire improved long-term NaCl tolerance, involving the
regulation of many genes, are still not understood (for review, see
Winicov and Bastola, 1997; Winicov, 1998). Therefore, the possible
function of transcription factors associated with stress responses has
been of significant interest.
It has been shown that both myc and myb proteins function as
transcriptional activators in the rd22 gene, which is
induced by ABA and dehydration (Abe et al., 1997). Many of the NaCl-
and drought-induced genes are also induced by ABA, and ABA response element-binding proteins have been cloned (Guiltinan et al., 1990). Other genes responding to NaCl/drought stress and cold are induced in
an ABA-independent manner involving the cis-acting DRE
(DNA regulatory element)
(Yamaguchi-Shinozaki and Shinozaki, 1994). Recently, the CBF1 protein
(Stockinger et al., 1997), which has been shown to recognize the DRE,
was shown to function in enhancing freezing tolerance (Jaglo-Ottosen et
al., 1998) in Arabidopsis. These findings suggest that the phenotypic
changes involving altered gene expression and resistance to stress
might be manipulated through the relevant transcription factors.
Transgenic manipulation of Alfin1 expression, therefore, is
of interest because we have demonstrated Alfin1 to be a
regulatory gene that can influence the expression of MsPRP2
in a specific manner. An interesting result of the enhanced
Alfin1 expression in our transgenic plants was the finding
that these plants demonstrated enhanced NaCl tolerance. It is likely
that, as a transcriptional regulator, Alfin1 also influenced the
regulation of other genes in our transformed plants, which could have
contributed to the enhanced NaCl tolerance observed in our transgenic
plants. Table III shows that many of the
genes that have been shown to be up-regulated by NaCl/drought stress
also contain Alfin1-binding motifs in their promoters. At present, we
do not know if any of these other genes are differentially
regulated in our Alfin1-overexpressing plants, but we
might expect to see changes in their expression if Alfin1 had a general regulatory role in NaCl tolerance.
View this table:
[in this window]
[in a new window]
|
Table III.
Alfin1-binding sites found in NaCl/drought
stress-induced promoter sequences
Selection of potential Alfin1-binding sites was made for the
coding strand on the basis of at least two adjacent triplets, one of
which is GTG and the other of which is bordered by a G as defined by in
vitro Alfin1 binding (Bastola et al., 1998). Additional
sites (not shown) were found on the noncoding strand in many of these
gene promoters. Numbers in parentheses indicate accession numbers.
|
|
Future experiments will determine the extent and specificity of plant
gene regulation by Alfin1 and the extent to which enhanced Alfin1 expression could be useful in manipulating plant
growth tolerance of environmental conditions.
| |
FOOTNOTES |
1
This work was supported in part by a Hatch grant
from the Nevada Agricultural Experiment Station, by the National
Science Foundation Experimental Program to Stimulate Competitive
Research, Women in Science and Engineering, and by the National
Research Initiative Competitive Grants Program (grant no. 9401235 to
I.W).
2
Present address: Department of Plant Biology,
Arizona State University, Main Campus, P.O. Box 871601, Tempe, AZ
85287-1601.
3
Present address: Department of Biochemistry and
Molecular Biology/3008EI, University of Nebraska Medical Center, Omaha,
NE 68198-4525.
*
Corresponding author; e-mail winicov{at}asu.edu; fax
1-602-965-6899.
Received November 30, 1998;
accepted February 24, 1999.
| |
ABBREVIATIONS |
Abbreviations:
CaMV, cauliflower mosaic virus.
MCS, multiple
cloning site.
SH, Schenk and Hildebrandt.
| |
ACKNOWLEDGMENT |
We thank B. Mitchell for excellent greenhouse and laboratory
help.
| |
LITERATURE CITED |
Abe H,
Yamaguchi-Shinozaki K,
Urao T,
Iwasaki T,
Hosokawa D,
Shinozaki K
(1997)
Role of Arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression.
Plant Cell
9:
1859-1868
[Abstract]
An G,
Ebert P,
Mitra A,
Ita S
(1988)
Binary vectors.
In
SB Gelvin,
RA Schilperoort,
eds, Plant Molecular Biology Manual, Section A, Chapter 3.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 1-19
Bastola DR,
Pethe VV,
Winicov I
(1998)
Alfin1, a novel zinc-finger protein in alfalfa roots that binds to promoter elements in the salt-inducible MsPRP2 gene.
Plant Mol Biol
38:
1123-1135
[CrossRef][ISI][Medline]
Bohnert HJ,
Nelson DE,
Jensen RG
(1995)
Adaptations to environmental stresses.
Plant Cell
7:
1099-1111
[CrossRef][ISI][Medline]
Bray EA
(1997)
Plant responses to water deficit.
Trends Plant Sci
2:
48-54
Cao H,
Li X,
Dong X
(1998)
Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance.
Proc Natl Acad Sci USA
95:
6531-6536
[Abstract/Free Full Text]
Claes B,
Dekeyser R,
Villarroel R,
Van den Bulcke M,
Bauw G,
Van Montagu M,
Caplan A
(1990)
Characterization of a rice gene showing organ-specific expression in response to salt stress and drought.
Plant Cell
2:
19-27
[Abstract/Free Full Text]
Deutch CE,
Winicov I
(1995)
Post-transcriptional regulation of a salt-inducible alfalfa gene encoding a putative chimeric proline-rich cell wall protein.
Plant Mol Biol
27:
411-418
[CrossRef][ISI][Medline]
Foolad MR,
Jones RA
(1993)
Mapping salt-tolerance genes in tomato (Lycopersicon esculentum) using trait-based marker analysis.
Theor Appl Genet
87:
184-192
Grotewold E,
Chamberlin M,
Snook M,
Siame B,
Butler L,
Swenson J,
Maddock S,
St. Clair G,
Bowen B
(1998)
Engineering secondary metabolism in maize cells by ectopic expression of transcription factors.
Plant Cell
10:
721-740
[Abstract/Free Full Text]
Guiltinan MJ,
Marcotte WR,
Quatrano RS
(1990)
A plant leucine zipper protein recognizes an abscisic acid response element.
Science
250:
267-270
[Abstract/Free Full Text]
Gyorgyey J,
Gartner A,
Nemeth K,
Magyar Z,
Hirt H,
Heberle-Bors E,
Dudits D
(1991)
Alfalfa heat shock genes are differentially expressed during somatic embryogenesis.
Plant Mol Biol
16:
999-1007
[CrossRef][ISI][Medline]
Hoagland DT,
Arnon DI
(1938)
The water culture method for growing plants without soil.
Calif Agric Exp Stn Circ
347:
1-39
Hoekema A,
Hirsch PR,
Hooykaas PJJ,
Schilperoort RA
(1983)
A binary plant vector strategy based on separation of the vir- and T-region of A. tumefaciens Ti plasmid.
Nature
303:
179-180
[CrossRef]
Ingram J,
Bartels D
(1996)
The molecular basis of dehydration tolerance in plants.
Annu Rev Plant Physiol Plant Mol Biol
47:
377-403
[CrossRef][ISI][Medline]
Jaglo-Ottosen KR,
Gilmour SJ,
Zarka DG,
Schabenberger O,
Thomashow MF
(1998)
Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance.
Science
280:
104-106
[Abstract/Free Full Text]
Kakidani H,
Ptashne M
(1988)
Gal4 activates gene expression in mammalian cells.
Cell
52:
161-167
[CrossRef][ISI][Medline]
Kishor PBK,
Hong Z,
Miao G-H,
Hu C-A,
Verma SPS
(1995)
Over-expression of 
1-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants.
Plant Physiol
108:
1387-1394
[Abstract]
Michel D,
Salamini F,
Bartels D,
Dale P,
Baga M,
Szalay A
(1993)
Analysis of a desiccation and ABA-responsive promoter isolated from the resurrection plant Craterostigma plantagineum.
Plant J
4:
29-40
[CrossRef][ISI][Medline]
Pilon-Smits EAH,
Ebskamp MJM,
Paul MJ,
Jeuken MJW,
Weisbeek PJ,
Smeekens SCM
(1995)
Improved performance of transgenic fructan-accumulating tobacco under drought stress.
Plant Physiol
107:
125-130
[Abstract]
Pla M,
Gomez J,
Goday A,
Pages M
(1991)
Regulation of the abscisic acid-responsive gene rab28 in maize viviparous mutants.
Mol Gen Genet
230:
394-400
[Medline]
Raghothama G,
Liu D,
Nelson DE,
Hasegawa PM,
Bressan RA
(1993)
Analysis of an osmotically regulated pathogenesis-related osmotin gene promoter.
Plant Mol Biol
23:
1117-1128
[CrossRef][Medline]
Sambrook J,
Fritsch EF,
Maniatis T
(1989)
Molecular Cloning: A Laboratory Manual, Ed 2.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Schenk RU,
Hildebrandt AC
(1972)
Medium and techniques for induction of growth of monocotyledonous and dicotyledonous plant cell cultures.
Can J Bot
50:
199-204
[ISI]
Shen Q,
Zhang P,
Ho T-HD
(1996)
Modular nature of abscisic acid (ABA) response complexes: composite promoter units that are necessary and sufficient for ABA induction of gene expression in barley.
Plant Cell
8:
1107-1119
[Abstract]
Shinozaki K,
Yamaguchi-Shinozaki K
(1997)
Gene expression and signal transduction in water-stress response.
Plant Physiol
115:
327-334
[CrossRef][ISI][Medline]
Stockinger EJ,
Gilmour SJ,
Thomashow MF
(1997)
Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit.
Proc Natl Acad Sci USA
94:
1035-1040
[Abstract/Free Full Text]
Straub PF,
Shen Q,
Ho T-hD
(1994)
Structure and promoter analysis of an ABA- and stress-regulated barley gene HVA1.
Plant Mol Biol
26:
617-630
[CrossRef][ISI][Medline]
Tamagnone L,
Merida A,
Parr A,
Mackay S,
Culianez-Macia FA,
Roberts K,
Martin C
(1998)
The AmMYB308 and AmMYB330 transcription factors from Antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco.
Plant Cell
10:
135-154
[Abstract/Free Full Text]
Tarczynski MC,
Jensen RG,
Bohnert HJ
(1993)
Stress protection of transgenic tobacco by production of the osmolyte mannitol.
Science
259:
508-510
[Abstract/Free Full Text]
Urao T,
Yamaguchi-Shinozaki K,
Urao S,
Shinozaki K
(1993)
An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence.
Plant Cell
5:
1529-1539
[Abstract]
Winicov I
(1991)
Characterization of salt tolerant alfalfa (Medicago sativa L.) plants regenerated from salt tolerant cell lines.
Plant Cell Rep
10:
561-564
Winicov I
(1993)
cDNA encoding putative zinc finger motifs from salt-tolerant alfalfa (Medicago sativa L.) cells.
Plant Physiol
102:
681-682
[Medline]
Winicov I
(1996)
Characterization of rice (Oryza sativa L.) plants regenerated from salt-tolerant cell lines.
Plant Sci
113:
105-111
[CrossRef]
Winicov I
(1998)
New molecular approaches to improving salt tolerance in crop plants.
Ann Bot
82:
703-710
[Abstract/Free Full Text]
Winicov I,
Bastola DR
(1997)
Salt tolerance in crop plants: new approaches through tissue culture and gene regulation.
Acta Physiol Plant
19:
435-449
Winicov I,
Button JD
(1991)
Induction of photosynthesis gene transcripts by sodium chloride in a salt tolerant alfalfa cell line.
Planta
183:
478-483
Winicov I,
Deutch CE
(1994)
Characterization of a cDNA clone from salt-tolerant alfalfa cells that identifies salt inducible root specific transcripts.
J Plant Physiol
144:
222-228
Winicov I,
Krishnan M
(1996)
Transcriptional and post-transcriptional activation of genes in salt-tolerant alfalfa cells.
Planta
200:
397-404
Winicov I,
Waterborg JH,
Harrington RE,
McCoy TJ
(1989)
Messenger RNA induction in cellular salt tolerance of alfalfa (Medicago sativa).
Plant Cell Rep
8:
6-11
Xu D,
Duan X,
Wang B,
Hong B,
Ho T-HD,
Wu R
(1996)
Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice.
Plant Physiol
110:
249-257
[Abstract]
Yamaguchi-Shinozaki K,
Shinozaki K
(1994)
A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress.
Plant Cell
6:
251-264
[Abstract]
Top
Abstract
Introduction
