Plant Physiol. (1998) 118: 1181-1190
Abscisic Acid-Dependent and -Independent Expression of the Carrot
Late-Embryogenesis-Abundant-Class Gene Dc3 in Transgenic
Tobacco Seedlings1
Najeeb U. Siddiqui2,
Hwa-Jee Chung3,
Terry
L. Thomas, and
Malcolm C. Drew*
Department of Horticultural Sciences (N.U.S., M.C.D.), and
Department of Biology (H.-J.C., T.L.T.), Texas A&M University,
College Station, Texas 77843
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ABSTRACT |
We
studied the expression of three promoter 5
deletion constructs (
218,
599, and 1312) of the LEA (late
embryogenesis abundant)-class gene
Dc3 fused to 
-glucuronidase (GUS), where each
construct value refers to the number of base pairs upstream of the
transcription start site at which the deletion occurred. The
Dc3 gene is noted for its induction by abscisic acid
(ABA), but its response to other plant hormones and various
environmental stresses has not been reported previously for vegetative
cells. Fourteen-day-old transgenic tobacco (Nicotiana
tabacum L.) seedlings were exposed to dehydration, hypoxia,
salinity, exogenous ethylene, or exogenous methyl jasmonate (MeJa). GUS
activity was quantified fluorimetrically and expression was observed by
histochemical staining of the seedlings. An increase in GUS activity
was observed in plants with constructs 599 and 1312 in response to
dehydration and salinity within 6 h of stress, and at 12 h in
response to hypoxia. No increase in endogenous ABA was found in any of
the three lines, even after 72 h of hypoxia. An ABA-independent
increase in GUS activity was observed when endogenous ABA biosynthesis was blocked by fluridone and plants were exposed to 5 µL
L
1 ethylene in air or 100 µM MeJa.
Virtually no expression was observed in construct 218 in response to
dehydration, salinity, or MeJa, but there was a moderate response to
ethylene and hypoxia. This suggests that the region between 218 and
599 is necessary for ABA (dehydration and salinity)- and
MeJa-dependent expression, whereas ethylene-mediated expression does
not require this region of the promoter.
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INTRODUCTION |
LEA (late embryogenesis
abundant) proteins accumulate in the seeds of many higher
plants and are probably universal in occurrence in plant seeds. LEA
proteins were first identified and characterized in cotton as a set of
proteins that are highly accumulated in the embryos at the late stage
of seed development (Dure et al., 1981
). Subsequently, more than 100 LEA genes/cDNAs or their homologs have been identified from
both monocots and dicots. In many cases, the timing of LEA
mRNA and protein accumulation is correlated with the seed-desiccation
process and associated with elevated in vivo ABA levels. The products
of these genes are thought to function in protecting cells from
dehydration (Baker et al., 1988; Dure et al., 1989), possibly by ion
sequestration in dehydrated cells (Dure, 1993). The high abundance of
LEA proteins in desiccation-tolerant seed embryos (Roberts et al.,
1993), water-stress inducibility of specific LEA genes in
vegetative tissues (Piatowski et al., 1990), and particular structural
features (Baker et al., 1988; Dure, 1993) support this role. The
Dc3 promoter fused to GUS can drive GUS expression in leaves
of mature, transgenic plants in response to desiccation stress and/or
treatment with ABA (Vivekananda et al., 1992).
ABA is involved in the signal transduction pathway regulating several
genes that are expressed at specific developmental stages or as a
result of an environmental stress. ABA accumulates in vegetative cells
in response to water deficit, salinity, cold temperature, and light
variation, and it is thought to act as a signal for the initiation of
acclimation to these stresses (Marcotte et al., 1992; Chandler and
Robertson, 1994; Weatherwax et al., 1996). Constitutive expression of
the barley LEA gene HVA1 conferred increased
tolerance to water deficit and salinity in second-generation transgenic
rice plants (Xu et al., 1996). The extent of stress tolerance was
correlated with the level of HVA1 protein accumulation. In rice more
salt-tolerant varieties showed higher concentrations in their roots of
dehydrins and group 3 LEA proteins compared with salt-sensitive
varieties (Moons et al., 1995).
Dc3 is a carrot (Daucus carota) gene encoding an
mRNA that is expressed at high levels in developing embryos (Seffens et
al., 1990). Dc3 belongs to a small gene family (Wilde et
al., 1988) encoding Dc3 and Dc3-like mRNAs, and
is closely homologous to the D-7 LEA family of cotton (Dure
et al., 1989). In transgenic tobacco (Nicotiana tabacum L.)
containing a GUS reporter gene fused to the full-length 5 promoter
element of Dc3, there was expression in seedlings in
response to desiccation or ABA (Seffens et al.,
1990). Also, Dc3 is expressed in
developing transgenic tobacco seeds, and detailed evidence has been
obtained for the differential response of different promoter deletion
constructs in response to desiccation and exogenous ABA during
germination (Chung, 1996). The Dc3 promoter can be
divided into two regulatory regions. The PPR from 117 to +27 is the
minimal sequence for seed-specific expression and contains the
cis-acting elements that are responsible for the regulation
of Dc3 expression in seeds (Thomas, 1993; Kim et al.,
1997). The second region, the DPR from 1457 to 118, contains
several repeats of additional cis-regulatory regions, namely
the TCGT motifs (Chung, 1996). The TCGT motif, interacting with the
PPR, is required for ABA-induced expression in vegetative cells in
transient expression assays. It is proposed that bZIP proteins bind to
the PPRs and DPRs and regulate gene expression in response to
developmental and environmental cues (Kim et al., 1997). Several
studies have shown that bZIP proteins are induced by a variety of
stresses, including: hypoxia (de Vetten and Ferl, 1995), low light
(Feldbrugge et al., 1994; Mikami et al., 1995), low temperature (Kusano
et al., 1995), and ABA (Lu et al., 1996). Thus, a bipartite structure
for the Dc3 promoter is proposed for seed-specific
expression, as well as for ABA-inducible expression in vegetative
tissues. Deletion of the DPR eliminates all expression in vegetative
tissues, allowing only seed-specific expression (Chung, 1996).
The aim of the present work was to study the response of the
Dc3 promoter in vegetative cells to several environmental
stresses that may involve mediation by ABA and other hormones. Although the sensitivity of the Dc3 promoter in transgenic plants to
dehydration and/or ABA is well established, it is not known whether
other environmental factors or hormonal signals are able to modulate the activity of the gene. Therefore, the sensitivity of
Dc3-driven GUS expression was tested with ethylene and MeJa,
and with the environmental stresses hypoxia and salinity. By examining
the response of the Dc3 promoter to various 5 upstream
deletions, we hoped to identify regions that are essential for
differential expression.
Plants encounter hypoxia or O2 shortage with
excess soil water (Drew, 1997) and the ethylene-biosynthetic rate is
then increased (Jackson et al., 1985; Atwell et al., 1988; He et al.,
1996). Endogenous ABA levels may also increase under hypoxic conditions (Smit et al., 1990; Neuman and Smit, 1991). Ethylene biosynthesis is
increased in response to stimuli such as wounding, pathogen attack,
hypoxia, and water deficit (for review, see Abeles et al., 1992; Morgan
and Drew, 1997). In leaves of plants growing under high-salt stress,
ABA accumulation may assist in the acclimation to salinity (Zeevaart
and Creelman, 1988).
JA and its methyl ester MeJa are plant growth substances that modulate
plant development and defense mechanisms (Creelman and Mullet, 1997).
JA and MeJa influence many physiological and developmental processes
that are affected by ABA (Hildmann et al., 1992; Melan et al., 1993;
Sembdner and Parthier, 1993; Lehmann et al., 1995). ABA and JA not only
exert similar physiological effects, but they also share common actions
at the level of gene expression by also inducing similar, or even the
same, polypeptides, e.g. Pin II, the proteinase
inhibitor of potato (Farmer and
Ryan, 1992); VspB, the vegetative storage
proteins of soybean (Mason et al., 1993); or the seed
storage proteins of Brassica napus (Wilen et al., 1991).
Jasmonates can accumulate in plants in response to water deficit and
wounding (Creelman et al., 1992; Creelman and Mullet, 1995). Promoters
of both the Pin II and VspB genes contain similar
DNA domains called G-boxes, which are identical to the G-box in
ABA-responsive promoters, including Dc3. One aim of our
study was to determine whether exogenous MeJa can induce Dc3
when the synthesis of ABA is blocked.
Regulation of the Dc3 promoter in response to
dehydration and exogenous ABA has been studied previously using GUS
fusions in vegetative tissues of transgenic plants (Vivekananda, et
al., 1992). Plants subjected to mild dehydration showed a slight
decrease in leaf water potential, with a dramatic increase in the
levels of GUS activity. However, this earlier study was with the
full-length promoter construct. It focused only on leaves of mature
plants and did not examine the first signs of expression in roots and shoots. The specific objectives of the present research were: (a) to
study the tissue-specific expression of Dc3 in response to
three major environmental stresses that might involve ABA-mediated responses, namely dehydration, salinity, and hypoxia; (b) to determine the responses of various promoter deletion lines to these stresses; and
(c) to examine the possibility of ABA-independent expression of
Dc3.
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MATERIALS AND METHODS |
Plant Material
Transgenic tobacco (Nicotiana tabacum L. cv
Xanthi) seeds containing the promoter region of the gene for
carrot (Daucus carota), Dc3, fused to a GUS
reporter gene were used. Three different 5 promoter deletion
constructs were chosen, 218, 599, and 1312, where each construct
value represents the number of base pairs upstream of the transcription
start site at which the deletion occurred. The full-length
Dc3 promoter is 1.5 kb long. Thus, the 218 construct is
the shortest promoter sequence and the 1312 construct is the longest.
For convenience, in the remainder of the text the promoter deletion
constructs will be referred to as 218, 599, and 1312. Details
concerning tobacco transformation and construction of these deletion
lines are given by Chung (1996).
Agrobacterium tumefaciens strain LBA4404 was transformed
with the pBI101.1 cassette containing the promoter deletions and chimeric constructs via electroporation. Leaf discs of tobacco were
inoculated for 10 min with A. tumefaciens grown to
saturation in M9 medium (Maniatis et al., 1982). The inoculated
leaf discs were then incubated for 2 d on Murashige and Skoog
medium (1× Murashige and Skoog salts with 3% Suc, 1 µg/mL
pyridoxine-HCl, 1 µg/mL nicotinic acid, 100 µg/mL
myo-inositol, and 0.25% Gel-Rite) at 25°C with 16 h
of light and 8 h of darkness. The inoculated leaf discs were
transferred to a shoot-inducing medium (Murashige and Skoog medium,
0.01 µg/mL NAA, and 2.0 µg/mL 6-BA) containing 100 µg/mL
kanamycin and 200 µg/mL carbenicillin. Young shoots were transferred
to a growth medium (Murashige and Skoog medium containing 100 µg/mL
kanamycin and 200 µg/mL carbenicillin). Regenerated plants were
transferred to soil and grown under optimal conditions. The seed used
in this study was from plants that were representative of between 8 and
10 independent transformation events for each construct. Both
histochemical and fluorimetric tests were made on embryos and on
seedling leaves (Chung, 1996). Thus, the lines used in the
present work gave GUS expression under a variety of environmental
conditions that was close to the median for each group of independent
lines. These particular constructs were chosen because in transgenic
tobacco embryos the sequence and properties of 1312 were very close
to those of the full-length promoter; 599 gave maximal response to
ABA, whereas 218 was relatively insensitive to ABA.
Seeds were surface sterilized with 5% (v/v) bleach and washed with
sterile distilled water several times. Seedlings were grown hydroponically on an inorganic nutrient solution containing (in mM): 0.1 KNO3, 0.4 Ca(NO3)2, 0.1 NH4H2PO4/(NH4)2PO4
(pH 5.0), 0.05 MgSO4, and 0.05 Fe-EDTA, together
with micronutrients (Rodriguez et al., 1997). To reduce damage and
stress to plants during transplanting, a hydroponic system was chosen
for growing plants. In early experiments, in which plants were grown on
Murashige and Skoog medium before transfer to hydroponics, they had
significantly higher expression of GUS because of the physical damage
to the plants (data not shown). Kanamycin (5 µg
mL1) was used for selection. This antibiotic
concentration was chosen after a separate experiment in which plants
were grown in the presence of kanamycin concentrations ranging from 5 to 100 µg mL1. At 5 µg
mL1, there was clearly no visible damage to the
kanamycin-resistant transgenic seedlings. A higher concentration
resulted in late germination, restricted growth of roots, smaller
cotyledons, delayed emergence of true leaves, and yellowing of leaves
after 10 d (data not shown). Hydroponic containers were made from
Magenta (Chicago, IL) boxes by mounting stainless-steel mesh over the
top and laying a piece of cheesecloth to hold the seeds and provide a
moist substratum for the germinating seeds. Plants were grown in a
controlled-environment growth chamber at 25°C in 16 h of light
and 8 h of darkness. Seeds were germinated in contact with
nutrient solution containing kanamycin for 6 d and resistant
seedlings were then transferred to kanamycin-free nutrient solution.
Stress treatments started 14 d after germination.
Stress and Hormone Treatments
Fourteen-day-old plants were subjected to dehydration by removing
water from the hydroponic container and then very gently blotting the
plants with filter paper to remove excess water. Seedlings were then
transferred to a Styrofoam box and covered with a lid to maintain a
near-constant RH level inside of the box for uniform dehydration. The
RH was monitored using an instant digital hygrometer (Fisher) at
regular intervals during the stress period, and was maintained at 80%
to 85% by adjusting the lid of the Styrofoam box. Control plants were
grown in hydroponic containers during the stress period. Samples were
taken every 6 h (starting at 0 h) for up to 24 h.
For salinity treatment, 14-d-old seedlings were transferred to nutrient
solution containing 100 mM NaCl. Control plants continued to grow on regular nutrient solution with no NaCl. Samples were taken
every 6 h for up to 24 h.
For hypoxia treatment, at 14 d after germination hydroponic
containers were transferred to 4-L sealed plastic boxes. To induce hypoxia, air (20.6% [v/v] O2) and
prepurified N2 gas, both from pressurized
cylinders and regulated by electronic mass flow meters, were mixed to
give an O2 concentration of 4% (v/v). This gas
mixture was passed into the nutrient solution at a flow rate of 100 mL min1 per container. The top of the plastic
container was sealed except for a tube to vent the gas mixture to
ensure that the tops of the plants were also hypoxic. Control plants
were transferred to similar containers but received normal air.
For exogenous ethylene and MeJa treatments, plants were grown as
described above and were then subjected to fluridone to block ABA
biosynthesis. Fluridone solution was prepared by dissolving the solid
in 100 µL of ethanol and then adding it to vigorously stirred water.
The final concentration of ethanol in the nutrient solution was 100 µL L1. Fluridone precipitates rapidly if it
is added to unstirred water or if the solution is more than 3 to 4 d old. Fresh fluridone solution was prepared for each experiment. To
determine the effect of fluridone exposure on growth, plants in a
preliminary experiment were subjected to 100 µM fluridone
for 1, 2, 3, and 4 d, and the morphological changes and GUS
expression in seedlings were observed. No noticeable damage to
seedlings was seen after 2 d of fluridone treatment, and no GUS
activity was detected even after 24 h of dehydration. However, we
were cautious about continuous exposure to fluridone because it can be
lethal to plants if it is present for too long in the growing medium.
These results suggested that 2 d of treatment might be enough to
block endogenous ABA biosynthesis without any significant damage to the
seedlings.
In all subsequent experiments involving fluridone, 14-d-old seedlings
were transferred to nutrient solution containing 100 µM
fluridone for 48 h before being transferred back to the regular nutrient solution. Hydroponic containers were transferred to sealed plastic boxes. Ethylene was continuously bubbled through the nutrient solution. Ethylene (10 mL min1 of 100 µL
L1 [v/v]) and air (190 mL
min1), both from pressurized cylinders and
regulated by electronic mass flow meters, were mixed to give a final
ethylene concentration of 200 nmol L1,
equivalent to 5 µL L1 (v/v). Control plants,
initially grown on fluridone, were also transferred to the plastic
containers but received no ethylene. Samples of plant tissue were taken
at intervals for histochemical staining and GUS-activity analysis.
For exogenous MeJa treatment, 100 µM MeJa was prepared by
dissolving 95% MeJa (Aldrich) in 200 µL of ethanol before adding it
to the nutrient solution. The final concentration of ethanol in the
nutrient solution was 100 µL L1. A separate
aliquot of MeJa (100 µM final concentration) was mixed in
water instead of the nutrient solution. This solution was sprayed on
the leaves at the start of the stress period. Control plants were grown
on nutrient solution containing the same amount of ethanol used to
dissolve MeJa and were also sprayed with the same concentration of
ethanol mixed in water.
GUS Analysis
GUS activity was determined according to the method of Jefferson
et al. (1987). For the fluorimetric assay, roots or shoots of stressed
or control seedlings were homogenized in GUS extraction buffer (50 mM NaPO4 [pH 7.0], 10 mM EDTA, 0.1% sarkosyl, 0.1% Triton X-100, and 10 mM -mercaptoethanol). The homogenate was centrifuged in
a microcentrifuge for 5 min, and the supernatant was used for the GUS
assay. For fluorimetric reactions, 100 µL of supernatant was used.
Duplicate reactions were started by adding 100 µL of extraction
buffer containing 2 mM 4-methylumbelliferyl -D-glucuronide (Sigma) and incubating at 37°C for 60 min. One reaction was terminated at time 0 and the second at 60 min,
with the addition of 800 µL of 0.2 M
Na2CO3 to each reaction.
The 4-methylumbelliferyl -D-glucuronide fluorescence
product was then measured on a fluorimeter (model no. TKO 100, Hoefer,
San Francisco, CA) (excitation wavelength = 365 nm, photodetector
wavelength = 460 nm). The protein content of the samples was
determined by the procedure of Bradford (1976). For histochemical
GUS analysis, whole seedlings were immersed in the GUS reaction buffer
(2 mM
5-bromo-4-chloro-3-indolyl--D-glucuronic acid,
1% dimethylformamide, 1× KFeCN, 1 mM EDTA, and 50 mM NaPO4 [pH 7.0]). Tissue was
incubated in the dark at 37°C for 18 to 24 h. The reaction was
stopped by washing the seedlings several times with 50 mM
phosphate buffer without
5-bromo-4-chloro-3-indolyl--D-glucuronic acid. For
better visualization of the stained tissue, seedlings were cleared with
an ethanol series and stored in 80% glycerol. Mounted seedlings were
photographed with Kodak Ektachrome 60 ASA tungsten film.
Endogenous ABA Measurements
Approximately 25 seedlings per sample were cut to separate roots
and shoots and were immediately frozen in liquid
N2. Three replicates of each sample were taken
for a statistical comparison. Samples were lyophilized overnight before
measuring dry weights. The extraction and determination (HPLC-GC) were
done as described by Creelman et al. (1990). An internal standard of
[3H](±)-ABA was added for the determination of
ABA recovery.
| |
RESULTS |
Strong Expression of the 1312 and 599 Constructs in Response to
Dehydration
We studied the effects of dehydration on Dc3-driven GUS
expression in roots and shoots of 14-d-old seedlings. The exact method used to bring about dehydration is critical in studying responses. The
method frequently used to dehydrate plants or detached leaves is to
expose them to laboratory air at an RH level of around 50% to 55%.
This RH level brings about rapid desiccation, and damaged seedlings may
be unable to acclimate or show expression of potentially responsive
genes. To avoid this, we subjected seedlings to 80% to 85% RH, in
which seedlings survived, even after 24 h of dehydration, in
contrast to seedlings exposed to laboratory RH, which did not survive
(data not shown). A 30-fold increase in GUS activity in roots and a
40-fold increase in GUS activity in shoots was observed after
24 h of dehydration in transgenic seedlings carrying the 1312
and 599 constructs (Fig. 1). The first
sign of Dc3-GUS expression was observed at 6 h in root
tips, vascular tissues, and hypocotyls. A low level of GUS expression
was also observed in leaves of seedlings carrying the 1312 and 599
constructs at 6 h. A minimal increase in expression was observed
in the tissue of the transgenic seedlings carrying the 218 construct.
Figure 2, A to C, shows the lack of GUS
expression in all tissues of the controls. By contrast, GUS expression
in the 1312 and 599 constructs was uniformly increased throughout
the seedlings at 24 h, with a concentration of activity in the
densely packed cells of the root tip (Fig. 2, D-F). The relative
responses of the different deletion constructs of the Dc3
promoter to dehydration, as determined by both fluorimetric and
histochemical assays, are summarized in Table
I.
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| Figure 1.
Effects of dehydration on GUS activity in roots
and shoots of transgenic tobacco seedlings containing promoter deletion
constructs of the carrot gene Dc3 fused to GUS. Stress
started at 0 h. In this and subsequent figures, bars indicate
SE (n = 3).
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| Figure 2.
Histochemically stained transgenic tobacco
seedlings containing promoter deletion constructs of Dc3
fused to GUS. Line 1312 contained the longest promoter sequence and
218 contained the shortest. A to C, Control seedlings; D to F,
24 h of dehydration; G to I, 24 h of exposure to 100 mM NaCl; J to L, 24 h of hypoxia; M to O, 24 h of
exposure to 5 µL L1 exogenous ethylene; and P to R,
24 h of 100 µM exogenous MeJa treatment.
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Table I.
Comparison of Dc3-driven GUS activity in roots and
shoots of transgenic tobacco seedlings in response to environmental
stresses and hormone treatments
Activity is shown as strong (+++), medium (++), mild (+), or no
activity ().
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The possibility of ABA-independent expression during dehydration was
tested with seedlings previously exposed to fluridone. Seedlings
containing the 599 deletion construct showed no GUS expression when
subjected to dehydration for 24 h, whereas control seedlings
(those not treated with fluridone and subjected to similar dehydration)
showed an increase in GUS activity (Table
II). This test confirmed that fluridone
effectively blocked ABA biosynthesis, and proved that fluridone itself
did not induce Dc3 expression.
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Table II.
Dc3-driven GUS expression in tobacco seedlings
exposed to dehydration, in the presence (+) or absence (0) of
fluridone
Values are means from three independent experiments with the 599
line; SE values are in parentheses. Zero time corresponds
to the end of the pretreatment with fluridone, immediately before the
start of dehydration.
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Dc3 Promoter Responds to Salt Stress
Expression of the three Dc3 promoter lines was
studied with seedlings exposed to 100 mM NaCl. We decided
to study the response of the Dc3 promoter to salt stress
because endogenous ABA increases in plants exposed to salinity (for
review, see Zeevaart and Creelman, 1988), and exogenous ABA or
dehydration induces Dc3 expression. In a preliminary
experiment, 50 mM NaCl caused very little change in GUS
activity compared with the controls (data not shown). In later
experiments, 14-d-old seedlings were exposed to 100 mM NaCl
for 24 h. Salt was added to the nutrient solution, and plants
continued to grow under ambient conditions of light, temperature, and
photoperiod. NaCl (100 mM) caused the leaves to wilt within
2 h of exposure, a characteristic response of plants to salt-shock
conditions, but the leaves regained turgor after 6 to 8 h. A
5-fold increase in GUS activity was observed in roots of the 599 and
1312 lines at 6 h, and the level of GUS activity increased to
20- to 25-fold over that of the controls at 24 h of salt stress
(Fig. 3). No appreciable increase in GUS activity was observed in roots of the 218 line (Fig. 3). This response of the 218 line was similar to its response under
dehydration stress, presumably because ABA mediates expression driven
by either salt stress or dehydration. A 5-fold increase in GUS activity was also observed in shoots of the 599 and 1312 lines at 6 h, and up to a 30-fold increase was observed at 24 h of salt stress (Fig. 3). The 218 line showed only very small increases in GUS activity in shoots even after 24 h of salt stress (Fig. 3; Table I), which was similar to the response to dehydration. From
histochemical tests (Fig. 2, G-I), GUS expression could be seen
throughout roots and leaves, particularly in cotyledons and root tips
of the 599 and 1312 lines, whereas there was no expression in any
tissues in the 218 line.
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| Figure 3.
Effects of 100 mM NaCl on GUS activity
in roots and shoots of transgenic tobacco seedlings containing promoter
deletion constructs of Dc3. Stress started at 0 h.
Control values in this experiment were <2 pmol mg1
protein min1.
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ABA-Independent Expression under Hypoxia
To investigate whether there was sufficient ABA released to induce
Dc3 under hypoxic conditions and to study the tissue
specificity and appearance of first expression, we subjected 14-d-old
seedlings to hypoxia by supplying roots with 4%
O2. ABA concentration did not change in hypoxic
roots and shoots sampled at regular intervals over 72 h, with
initial (prestress) values (nmol/g fresh weight) of 0.205 (roots) and
0.303 (shoots), and final values of 0.199 (roots) and 0.326 (shoots) at
72 h. Despite the absence of a change in ABA concentration, an
increase in GUS activity was observed in all three lines, although
218 showed the least response (Fig. 4;
Table I). Increases in GUS activity of 12-, 15-, and 10-fold were
observed in roots of the 1312, 599, and 218 lines, respectively, at 24 h of hypoxia (Fig. 4). Increases of 12-, 15-, and 17-fold were observed in shoots of the 1312, 599, and 218 lines,
respectively, at 24 h of hypoxia (Fig. 4). It is notable that
there was a delay in GUS expression with hypoxia relative to the more
rapid increase with dehydration or salt shock (Figs. 1 and 3). The
weaker response of the 218 line is apparent in Figure 2, J-L, where
the densely staining cotyledons and leaves that are apparent in lines
1312 and 599 are not seen.
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| Figure 4.
Effects of hypoxia on GUS activity in roots
and shoots of transgenic tobacco seedlings containing promoter deletion
mutant constructs of Dc3. Stress started at 0 h.
Control values in this experiment were <1.5 pmol mg1
protein min1.
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To test further whether Dc3 expression might be driven in an
ABA-independent manner under hypoxia, seedlings were exposed to
fluridone for 48 h and then transferred to nutrient solution lacking fluridone and subjected to 4% O2. The
increase in GUS activity under hypoxia (after fluridone treatment) was
similar to the GUS activity levels observed in the earlier experiment without fluridone treatment (data not presented). This suggests that
Dc3 expression during hypoxia could be triggered by a signal other than ABA.
Expression of Dc3 Induced by Exogenous Ethylene
When 14-d-old seedlings were subjected to 5 µL
L1 ethylene in air after a 48-h fluridone
treatment, GUS activity showed an increase in the roots and shoots of
all three lines, but the induction of GUS was delayed, as it was under
hypoxia. A slight increase in GUS activity was seen at 6 h. The
GUS activity in roots at 12 h of stress increased 5-fold in the
218 line and approximately 15-fold in the 599 and 1312 lines,
which increased to 22- and 30-fold, respectively, at 24 h (Fig.
5; Table I). A similar pattern was
observed in shoots but the final expression at 24 h was slightly more than for the roots (Fig. 5). Histochemical staining showed an
increase in GUS expression in all three lines (Fig. 2, M-O), although
the weaker response of line 218 to ethylene, particularly the absence
of intense expression in cotyledons and leaves, was apparent. Overall,
the results with ethylene matched closely the results obtained under
hypoxic conditions.
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| Figure 5.
Effects of exogenous ethylene on GUS
activity in roots and shoots of transgenic tobacco seedlings containing
promoter deletion constructs of Dc3. Stress started at
0 h, and seedlings had been exposed to fluridone. The
concentration of ethylene in air was 5 µL L1. Control
values in this experiment were <1.5 pmol mg1 protein
min1.
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Expression of Dc3 by MeJa in the Absence of ABA
JA is primarily involved in wounding response, senescence,
pathogen attack, and fruit removal (Creelman and Mullet, 1997), but it
is also accumulated under water deficit. Several studies have shown an
involvement of JA in ABA-mediated events. ABA and JA levels increase in
response to water deficit, and some JA-inducible genes are also
responsive to ABA, although it is still unknown whether they share
common signal transduction pathways. To investigate whether MeJa can
induce Dc3, we subjected 14-d-old seedlings to 100 µM MeJa. To isolate the effects of MeJa, seedlings were pretreated with 100 µM fluridone to block ABA synthesis.
The 1312 and 599 lines showed a 3-fold increase in Dc3
expression in roots and a 5-fold increase in GUS activity in shoots
after 6 h of exposure to MeJa. A 10-fold increase in GUS activity
was observed in roots, and 20- and 15-fold increases were seen in
shoots of 1312 and 599 mutants, respectively, after 24 h (Fig.
6). The increase in GUS activity was very
small in roots or shoots of the 218 line compared with the controls
even after 24 h of exposure to MeJa (Fig. 6; Table I).
Histochemical staining of seedlings reflected the same pattern of
expression (Fig. 2, P-R). Hypocotyls and cotyledons had a high
expression of GUS. Irregular patches of GUS expression were also
observed on the first true leaves. This pattern of expression among the
three deletion lines resembles that found with dehydration. This
suggests that jasmonates cause a similar but less-pronounced induction
of Dc3, as does ABA.
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| Figure 6.
Effects of 100 µM MeJa on GUS
activity in roots and shoots of transgenic tobacco seedlings containing
promoter deletion constructs of Dc3. Stress started at
0 h, and seedlings had been exposed to fluridone. Control values
in this experiment were <2.5 pmol mg1 protein
min1.
|
|
| |
DISCUSSION |
This study shows the sensitivity of three promoter deletion
lines of Dc3 to three major plant-stress hormones, ABA,
ethylene, and JA, and the response of these Dc3 promoter
deletion lines to three environmental stresses, dehydration, salinity,
and hypoxia. Induction of GUS activity in vegetative cells by
dehydration presumably was associated with water-deficit-induced
increases in endogenous ABA in tobacco seedlings (Vivekananda et al.,
1992). The Dc3 promoter has a bipartite structure, and
interaction of upstream sequences (TCGT motifs) between 449 and 351
and the PPR from 117 and +27 is necessary for the ABA-driven
expression in nonembryonic tissues (Chung, 1996). Only a very small
increase in GUS activity was observed in the 218 line even after
24 h of dehydration, indicating that deletion of the upstream
region prevented ABA-inducible expression in vegetative cells. GUS
expression in the 1312 and 599 lines was observed in root tips and
leaves within 6 h of water-deficit stress, followed by a more
uniform expression in all vegetative tissues within 24 h. This
indicates that the Dc3 promoter responds to ABA at the
whole-plant level and expression is not localized to a specific tissue.
GUS expression in leaves within 6 h of water deficit, before
wilting was seen, could imply that the Dc3 promoter is
sufficiently sensitive to ABA that it responds to presumed increases in
endogenous ABA that occur before loss of cell turgor. Induction of
Dc3 during dehydration is necessarily dependent on ABA
signaling, as the experiments with fluridone showed. It is interesting
that with the 218 construct, very small but possibly significant
increases above the control levels of GUS activity occurred with
dehydration, salinity, and exposure to MeJa (Figs. 1, 3, and 6). The
possibility cannot be ruled out that within the 218-bp promoter
sequence there are sequences that confer very weak but detectable
levels of response to ABA or other signals.
Salinity stress is associated with increases in endogenous ABA
(Zeevaart and Creelman, 1988; Moons et al., 1997). With 100 mM NaCl, there was a marked increase in
Dc3-driven GUS activity that followed the same trend that
was seen with dehydration, perhaps because the endogenous ABA levels
increased similarly with dehydration stress. Moons et al. (1995) found
expression of a group 3 LEA gene under salt stress in
different cultivars of rice, with a simultaneous increase in endogenous
levels of ABA and jasmonates. There was slightly greater GUS expression
in tobacco leaves than in roots, as would be expected if there was a
more rapid accumulation of ABA levels in leaves during salt shock.
In our study we also looked at Dc3 expression under
root hypoxia. To explore the possibility of increased ABA levels, we
measured the endogenous ABA levels in hypoxic roots and shoots.
Although other studies have reported an increase in ABA level in
hypoxic tissues (Zhang and Davies, 1987), we did not detect any
increase even at 72 h of hypoxia. Hypoxia caused an increase in
GUS expression within 12 h of stress imposition (Fig. 4).
Transcription of LEA genes has been reported to be inducible
by ABA-dependent and -independent signals (Chandler and Robertson,
1994; Shinozaki and Yamaguchi-Shinozaki, 1997). We observed expression
in the 218 line, which did not respond to dehydration/ABA, salt
shock, or exogenous MeJa. This raises the possibility of yet another
signal involved in Dc3 expression. Ethylene may be working
as the signal for hypoxia. Numerous studies have shown an increase in
endogenous ethylene in hypoxic plants (Atwell et al., 1988; He et al.,
1996; Drew, 1997). The endogenous ethylene levels in hypoxic tissues of
tobacco seedlings were not determined, but we observed a very similar
GUS expression in all three constructs in the presence of exogenous
ethylene. This suggests that ethylene could be the signal for
Dc3 expression in hypoxic tissues. ABA does not seem to be
essential for the induction of Dc3 in hypoxic tissues
because there was no increase in ABA concentration in roots or shoots,
but there was GUS expression in hypoxic seedlings pretreated with
fluridone. Fluridone effectively blocks synthesis of ABA in seedlings
(Saab et al., 1990; Sharp et al., 1994) and inhibits expression of
ABA-responsive genes (Lang and Palva, 1992). Although fluridone
was removed from the root environment during the stress period in the
present experiments, pretreatment with fluridone was clearly effective
in inhibiting Dc3-driven GUS expression during dehydration.
It could be argued that fluridone might be washed out of plants with
their roots immersed in nutrient solution, as in the hypoxia or
ethylene treatments, but that seems unlikely because the compound binds
tightly to surfaces and could not be washed out of the shoots, which
were exposed to the air.
Ethylene- and hypoxia-induced GUS expression in the 218 line
suggests that the region between 599 and 218 is not absolutely necessary for ABA-independent expression, but is necessary for ABA-dependent expression. The transcription factors associated with
ABA-independent expression in the Dc3 promoter are
unknown. The dehydration-response element in a number of promoters
is recognized as responding to some aspect of dehydration and not to
ABA (Bray, 1997; Shinozaki and Yamaguchi-Shinozaki, 1997). However, the
failure of dehydration to induce expression in fluridone-treated roots and shoots suggests that the dehydration-response element may not play
a role in Dc3.
Induction of Dc3 expression by MeJa adds
Dc3 to the growing list of ABA-inducible genes that are also
sensitive to JA (Wilen et al., 1991; Hildmann et al., 1992; Lehmann et
al., 1995). Several reports have shown that ABA and JA exert similar
physiological effects and induce similar and/or related genes. However,
it is not known whether MeJa-induced expression involves activation of
transcription factors similar to those involved in ABA-dependent expression, or activation of a new set of transcription factors specific to MeJa. It is interesting that MeJa, like ABA, could not
induce expression in the 218 line. This implies that the region
between 599 and 218 is also necessary for MeJa-induced expression.
Therefore, our results point to a similar mode of action of ABA and
MeJa at the level of the Dc3 gene promoter. This contrasts
with rice, in which a group 3 LEA protein that is induced by ABA was
not induced by JA (Moons et al., 1997). Furthermore, ABA and JA exerted
antagonistic effects with respect to induction of some
salinity-inducible genes in rice seedlings (Moons et al., 1997).
Clearly, ABA and jasmonates cannot be regarded as exerting overlapping
influences on all genes.
In summary, GUS expression driven by the Dc3 promoter
in transgenic tobacco seedlings is inducible not only by ABA, but also by salt shock, hypoxia, ethylene, and MeJa. Induction is virtually arrested in the 218 deletion mutants in response to dehydration, salt
shock, and exogenous MeJa treatments, implying that the promoter region
between 599 and 218 is absolutely necessary for ABA- and
MeJa-dependent expression, but not for ethylene-mediated expression. This work shows that Dc3, an ABA-responsive,
embryogenesis-specific gene, can be modulated by many different stress
signals. The potential significance of these observations is that
subtle differences in the pattern of response to different
environmental/hormonal factors may result from various combinatorial
interactions between cis- and trans-acting
factors in the Dc3 promoter.
| |
FOOTNOTES |
1
This work was supported by the U.S.
Department of Agriculture National Research Initiative Competitive
Grants Program (no. 9437304 to T.L.T.).
2
Present address: Department of Botany,
University of Toronto, Toronto, Ontario, Canada M5S 3B2.
3
Present address: Department of Horticultural
Science, University of Florida, Gainesville, FL 32611.
*
Corresponding author; e-mail mcdrew{at}tamu.edu; fax
1-409-845-0627.
Received June 17, 1998;
accepted August 27, 1998.
| |
ABBREVIATIONS |
Abbreviations:
DPR, distal promoter region.
JA, jasmonic acid.
MeJa, methyl jasmonate.
PPR, proximal promoter region.
| |
ACKNOWLEDGMENTS |
We thank Dr. Robert Creelman for his assistance in HPLC-GC work
on ABA. We also thank Dr. Keerti Rathore for his help with photography.
| |
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