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Plant Physiol. (1999) 119: 599-608
Mutations Affecting Induction of Glycolytic and Fermentative
Genes during Germination and Environmental
Stresses in
Arabidopsis1
Terry R. Conley,
Hsiao-Ping Peng, and
Ming-Che Shih*
Department of Biological Sciences, 204 Chemistry Building,
University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
Expression
of the alcohol dehydrogenase gene (ADH) of Arabidopsis
is known to be induced by environmental stresses and
regulated developmentally. We used a negative-selection approach to
isolate mutants that were defective in regulating the expression of the ADH gene during seed germination; we then characterized
three recessive mutants, aar1-1,
aar1-2, and aar2-1, which belong to two
complementation groups. In addition to their defects during seed
germination, mutations in the AAR1 and
AAR2 genes also affected anoxic and hypoxic induction of
ADH and other glycolytic genes in mature plants. The
aar1 and aar2 mutants were also defective in responding to cold and osmotic stress. The two allelic mutants aar1-1and aar1-2 exhibited different
phenotypes under cold and osmotic stresses. Based on our results we
propose that these mutants are defective in a late step of the
signaling pathways that lead to increased expression of the
ADH gene and glycolytic genes.
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INTRODUCTION |
Exposure to oxygen deprivation (anoxia) and decreased oxygen
availability (hypoxia) due to flooding are common plant environmental stresses. After a change from aerobic to anoxic conditions, carbon metabolism of plant cells switches from aerobic respiration to fermentation (Drew, 1990 ). Biochemical studies have shown that oxygen
deficiency in maize roots induces synthesis of approximately 20 anaerobic polypeptides (for review, see Sachs et al., 1980, 1996),
several of which have been identified as glycolytic and fermentative
enzymes, including ADH and GAPDH (Freeling and Bennett, 1985; Ricard et
al., 1989; Russell and Sachs, 1989; Xie and Wu, 1989; Yang et al.,
1993). Several cis-acting elements and
trans-acting factors involved in anaerobic induction of the
ADH genes in maize and Arabidopsis have been identified
(Ferl and Laughner, 1989; Dolferus et al., 1994; Kyozuka et al., 1994;
Hoeren et al., 1998). Recent reports indicate that
Ca2+ may play an important role in the induction
of ADH gene expression during anoxia in maize (Subbaiah et
al., 1994a, 1994b; Sedbrook et al., 1996).
Most crop plants, including barley, maize, sorghum, and wheat, can
tolerate only very transient flooding (Kennedy et al., 1992). In
contrast, rice plants can survive much longer under anaerobic
conditions. Formation of aerenchyma, characterized by continuous gas
spaces in roots and shoots (Drew et al., 1979; Justin and Armstrong,
1987), has been proposed to correlate with tolerance to flooding
(Justin and Armstrong, 1987). Campbell and Drew (1983) have shown that
lysogenic aerenchyma formation occurs in the root cortex of maize
during hypoxia. A recent report indicates that an ethylene signal is
required for cell death during aerenchyma formation induced by hypoxia
(He et al., 1996). These results, in conjunction with reports that
Ca2+ is also involved in the induction of the
ADH gene in the early stage of anoxia (Subbaiah et al.,
1994a, 1994b; Sedbrook et al., 1996), suggest that multiple signaling
events may be involved.
In addition to hypoxia or anoxia, other environmental stresses,
including cold and dehydration, also induce the expression of the
ADH gene in Arabidopsis (Dolferus et al., 1994). Recent genetic evidence suggests that both an ABA-dependent and an
ABA-independent pathway are involved in mediating the response to cold
and osmotic stress (Ishitani et al., 1997; Zhu et al., 1997). The
molecular relationship, if any, between ADH induction by
hypoxia or anoxia and response to cold and osmotic stress is
essentially unknown.
We are taking advantage of the amenability of Arabidopsis to genetic
manipulation to dissect these complex signaling pathways. It has been
shown that mRNA levels for both ADH and GAPC
genes increase during anoxia (Chang and Meyerowitz, 1986; Yang et al., 1993). Analysis of transgenic plants containing ADH- or
GAPC-promoter::GUS fusion genes show
that transcriptional control is involved in anaerobic induction of both
genes in Arabidopsis (Yang et al., 1993; Dolferus et al., 1994). We
have used a genetic scheme that allowed us to identify regulatory
mutants that were defective in the induction of the ADH gene
during seed germination. Characterization of some of these mutants
revealed that they were also defective in other stress-induced signal
transduction pathways that lead to the activation of several glycolytic
genes.
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MATERIALS AND METHODS |
Generation of an
ADH-Promoter::GUS Reporter Gene
Construct in the Binary Vector pBI101
The genomic clone pAtAdh-7 served as the source of a 2.4-kb
HindIII-SmaI fragment, which included
approximately 1 kb of sequence upstream from the transcription start
site of the ADH gene of Arabidopsis (Chang and Meyerowitz,
1986). The 2.4-kb HindIII-SmaI fragment was
subcloned into pBS (plasmid
BlueScript, Stratagene), generating pAdh. This
plasmid, along with two oligonucleotide primers, was used in PCR to
generate an approximately 1-kb ADH-promoter fragment. The
upstream primer was complementary to the T7 RNA polymerase-promoter
site in the pBS sequences flanking the multilinker region, whereas the
downstream primer encompassed the ADH translation initiation
codon. The downstream primer was designed to engineer a new
HincII site by changing the original
5 -GTTGATAATG-3 sequence (the translation
initiation codon is underlined) to
5-GTTGACCATG-3 (the new HincII site
is designated by underlining; modified bases are designated in bold).
The HincII site served to facilitate subsequent
manipulations. The 1-kb PCR product was subcloned into pBS, generating
pAdh(Pro). Digesting pAdh(Pro) with XbaI and
HincII produced a 1072-bp fragment that was subcloned
directionally into the XbaI and SmaI sites of the
multilinker region of the binary vector pBI101 (Jefferson et al.,
1987). Restriction mapping was used to confirm the presence and the
correct orientation of the ADH-promoter insert in the
resulting plasmid clone pBI101/Adh-Gus.
Generation of Transgenic Arabidopsis Plants
Root transformation was used to generate transgenic Arabidopsis
plants, essentially as described by Valvekens et al. (1988). Rooted
transformed plantlets were transferred to Jiffy Mix Plus soil mix
(Hummert International, St. Louis, MO). Plants were grown in
environmental chambers (Percivall, Boone, IA) at 20°C under a mixture
of incandescent and fluorescent lamps (fluence rate, 100 µE
m 2s1) under long-day
conditions (16-h light/8-h dark).
The four independent transformants that were obtained were propagated
to maturity for subsequent analysis. All results reported here are
based on ADH::GUS expression in
F4 progeny of the line designated AG2. The
F1 progeny of the primary transformant line of AG2 had an approximate 3:1 segregation of
kanamycin-resistant/kanamycin-sensitive progeny. Kanamycin-resistant
F1 plants were analyzed through the F2 and F3 generations to
identify homozygous lines. The presence of a single copy of the
ADH::GUS construct in line AG2 was confirmed by
genomic Southern analysis.
Chemical Mutagenesis
The AG2 line of the transgenic ADH::GUS was
mutagenized. Three 0.5-g aliquots of seeds were placed separately into
sterile 50-mL centrifuge tubes and surface-sterilized by soaking in
1.5% (v/v) NaOCl with 0.04% (w/v) sodium dodecyl sulfate for 30 min, followed by five sterile water rinses. Seeds were then soaked in 0.25%
(v/v) aqueous ethyl methanesulfonic acid for 10 to 12 h
(Somerville and Ogren, 1982) and then rinsed several times with sterile
water. The seeds were planted on Jiffy Mix Plus soil mix and grown to
maturity. Approximately 4000 plants from each of the three 0.5-g
aliquots of mutagenized seeds were grown to maturity. The
M2 seeds from each of the resulting populations
of seeds were collected, dried, and stored in a sealed container at
4°C.
Allyl Alcohol Selection
Approximately 50,000 seeds from each of the three mutagenized
populations were surface-sterilized as described above. The seeds were
then soaked in sterile water at room temperature for 6 h. The
imbibed seeds were next exposed to 45 mM (aqueous) allyl alcohol for 2 h at room temperature (Jacobs et al., 1988),
followed by five rinses with sterile water and treatment overnight with 15 µM GA (aqueous) at 4°C. Each aliquot of seeds was
subdivided and transferred into ten 250-mL flasks, each containing
liquid germination medium (Valvekens et al., 1988). The flasks were
incubated under fluorescent lights in an environmental chamber on a
shaker operated at 150 rpm. Green seedlings that appeared after several days were transferred to Petri dishes containing germination medium solidified with 0.8% agar.
Growth Conditions and Stress Treatment
Seeds of line AG2 and aar mutants were
surface-sterilized and treated with 15 µM GA at
4°C overnight. Seeds were sown onto plates with Murashige and Skoog
medium containing 1% Suc and 0.8% agar and grown at 20°C with 16-h
light/8-h dark cycles. After 1 week, seedlings were transferred to
fresh Murashige and Skoog plates in a vertical position under the same
conditions for an additional 3 weeks. For anoxic treatments, plants
were submerged in liquid Murashige and Skoog medium through which
nitrogen gas (>99% purity) was bubbled to purge O2. For
hypoxic treatments, similar conditions were used except that pure
N2 gas was replaced with gas containing 4.5% to 5%
O2 and balanced with N2.
We used a modification of the procedures described by Dolferus et al.
(1994) for osmotic-stress experiments. Arabidopsis plants were
submerged in liquid Murashige and Skoog medium containing 0.6 M mannitol through which air (18%
O2) was bubbled for 24 h at 20°C. For cold
treatment, plants were submerged in aerated liquid Murashige and Skoog
medium at 4°C for 24 h.
GUS and ADH Enzymatic Assays
Histochemical staining and enzyme activity assays for GUS were
performed essentially as described by Jefferson et al. (1987). Fluorescence of the 4-methylumbelliferyl product was quantified using a
minifluorometer (model TKO-100, Hoefer Scientific Instruments, San Francisco, CA).
ADH enzyme activity assay was performed according to the procedures
described by Freeling (1973) and modified by Xie and Wu (1989). The
assay uses ethanol as the substrate and measures the production of NADH
in a spectrophotometer (model DU 64, Beckman). One unit of ADH enzyme
is defined as an increase in A340 of 0.01 per min (Xie and Wu, 1989).
RNA Isolation and RT-PCR Reactions
Total RNA was isolated by an acidic phenol protocol adapted from
the procedures described in Chomczynski and Sacchi (1987). Prior to
RT-PCR reactions, RNA samples were treated with DNase I twice to
deplete contaminating genomic DNA. First-strand cDNA synthesis was
performed in a 50-µL reaction mixture containing 2 µg of RNA, 1 µg of random hexamer, 40 µM each of the four
deoxynucleotides, 5 µL of 10× buffer, 200 units of Moloney murine
leukemia virus RT, and 20 units of ribonuclease inhibitor. The reaction
mixture was incubated at 42°C for 60 min and stopped by heating at
65°C for 10 min.
PCR was performed in a DNA Thermal Cycler (Perkin Elmer/Cetus)
programmed for 28 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min
at 72°C. To find the amounts of cDNA suitable for linear amplification, we used 1, 2, 4, 6, 8, and 10 µL of RT reaction mixture from the hypoxic-treated AG2 line. For all of the genes shown
in Table I, there were linear increases
of PCR products in reactions with 1, 2, and 4 µL of cDNA (data not
shown). Therefore, we used 1 or 2 µL of the RT reaction mixture for
subsequent PCR and analyzed the products using agarose-gel
electrophoresis. Sequencing the products from the reactions of the AG2
line confirmed the identities of the PCR products for each primer set .
We quantified the PCR products by analyzing the digitized images of
agarose gels using the ImageQuant software program, version 1.1 (Molecular Dynamics, Sunnyvale, CA). Nucleotide sequences of the primer
pairs for amplification of each gene and the sizes of the corresponding
products appear in Table I. The accession numbers (in parentheses) for
the genes on which the primer sequences were based are as follows:
ADH (M12196), GAPC (M64116), PDC1
(U71121), and PDC2 (U71122).
Chemical and Enzyme Suppliers
We purchased synthetic plant hormones (benzylaminopurine,
GA3, and kinetin), antibiotics (kanamycin, rifampicin, and
vancomycin), and other chemicals from Sigma, and
5-bromo-4-chloro-3-indolyl- -D-glucuronic acid
cyclohexylammonium salt from Gold Biotechnology (St. Louis, MO).
Restriction and modification enzymes were from New England BioLabs and
Promega, and the sequenase and radiochemicals used in DNA sequencing
were from Amersham.
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RESULTS |
Isolation of Mutants Affecting ADH Gene Expression
The genetic scheme we adapted uses the ADH enzyme as a selectable
marker. ADH normally functions in alcoholic fermentation, catalyzing
the reduction of acetaldehyde to ethanol. However, ADH also uses allyl
alcohol (2-propen-1-ol) as a substrate, forming acrolein (2-propenal),
which is highly toxic to plant cells. Therefore, in the presence of
allyl alcohol, wild-type plants with normal levels of functional ADH
enzyme will die, whereas plants that lack ADH will survive (Freeling
and Bennett, 1985; Jacob et al., 1988). This property can be exploited
to isolate mutants that are defective in regulating the expression of
the ADH gene.
Several transgenic Arabidopsis lines that contain one copy of
ADH::GUS were constructed. We chose one of these
lines, AG2, in which the expression pattern of the
ADH::GUS transgene reflected the expression
pattern of the endogenous ADH gene (Chang and Meyerowitz, 1986; Dolferus et al., 1994), for ethyl methanesulfonic acid
mutagenesis. To screen for mutants affecting ADH gene
expression, 150,000 M2 seeds were treated with
allyl alcohol (for details, see ``Materials and Methods''). A total
of 40 seeds germinated and produced green seedlings. These we
designated as aar (allyl alcohol
resistant) mutants, which we further divided into two
classes. The first, with mutations to the endogenous ADH
gene, were to have normal ADH::GUS expression. The
second, with mutations in genes involved in the signaling pathway(s)
leading to the induction of ADH, were to have reduced levels
of expression of both the endogenous ADH gene and the
GUS reporter gene. To distinguish between these two classes,
we stained imbibed seeds of aar mutants for GUS activity.
Thirteen of the 40 aar mutants had pronounced GUS staining
after 6 h or less of incubation, similar to the parental AG2
plants, indicating that the ADH::GUS gene was
expressed. In contrast, the remaining 27 aar plants
exhibited no detectable GUS staining even after 24 h of incubation
(data not shown). We characterized three of these mutants,
aar7, aar10, and aar17, which are
described below.
Table II showed that
F1 progeny from crosses between line AG2 and each
of the three aar mutants were sensitive to allyl alcohol treatment. In addition, allyl alcohol-sensitive and -resistant plants
in the F2 progeny from all three crosses showed a
3:1 ratio. These results indicated that each aar mutant
contains a monogenic and recessive mutation. Pair-wise crosses among
these mutants showed that aar7 and aar17 belonged
to the same complementation group, whereas aar10 constituted
a second group (Table III). Therefore, aar7 and aar17 were renamed aar1-1
and aar1-2, respectively, whereas aar10 was
renamed aar2-1.
Effects of Mutations in AAR Genes during Seed
Germination
To quantify the effects of mutations on the expression of the
ADH::GUS transgene during seed germination, we
compared the levels of GUS activity of the wild-type AG2 line and the
aar mutants (Fig. 1A). In AG2
GUS activity was highest in imbibing seeds (Fig. 1A, d 0) and 1-d-old
germinating seedlings. The activity decreased 50% by 5 d, and had
decreased to a steady-state level by 15 d, which was less than
10% of the 1-d level. In contrast, GUS activity was much lower in the
imbibing seeds and the germinating seedlings in all three
aar mutants. In 1-d-old germinating seedlings, GUS activity
in line AG2 was 8- to 10-fold higher than in the aar mutants. In 5-d-old seedlings, GUS activity in line AG2 was 2- to
3-fold higher than in the mutant plants. In contrast, at later developmental stages GUS activity was very similar in line AG2 and in
all of the mutants (Fig. 1A, d 15).
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| Figure 1.
GUS and ADH activities of AG2 and of the
aar mutants during seed germination. Seedlings of AG2
and aar1-1, arr1-2, and
aar2-1 at different developmental stages were harvested
and assayed for GUS and ADH activities as described in ``Materials and Methods''. A, GUS activity is expressed as pmol 4-methylumbelliferone
min1 mg1 protein. B, One unit of ADH enzyme
is defined as an increase in A340 of 0.01 per min. The data presented are the averages of three independent
treatments. Plants grown at different times were used for replicate
treatments. Bar graphs at each time point (from left to right)
represent activities for AG2, aar1-1,
aar1-2, and aar2-1. Bars indicate
SD.
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We also measured ADH activity in germinating seedlings of AG2 and
aar mutants. Figure 1B shows that the pattern of ADH
activity level in AG2 was similar to that of GUS, except that ADH
activity decreased more rapidly, reaching an undetectable level in
10-d-old seedlings. However, as in the case for GUS, there was much
lower ADH activity in imbibing seeds and germinating seedlings than in
any of the three aar mutants. These results showed that
mutations in the AAR1 and AAR2 genes affected the
expression of both the ADH::GUS transgene and the
endogenous ADH gene during germination.
Anoxic and Hypoxic Responses in Mature Plants
To determine whether mutations in the AAR1 and
AAR2 genes affected regulation of the
ADH::GUS transgene in mature plants in response to
anoxia and hypoxia, 4-week-old plants from line AG2 and from the
aar1-1, aar1-2, and aar2-1 mutants
were subjected to hypoxic or anoxic treatments for 8 h and
harvested for the assay of GUS activity. The data showed that after
8 h, there were 10- and 15-fold increases in GUS activity,
respectively, in anoxic- and hypoxic-treated AG2 plants (Fig.
2). In contrast, there was no increase in
GUS activity in either anoxic- or hypoxic-treated plants in all three
aar mutants (Fig. 2).
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| Figure 2.
Anoxic and hypoxic induction of GUS activity in
mature plants. Twenty-day-old AG2 and aar1-1,
aar1-2, and aar2-1 plants were
subjected to anoxic or hypoxic treatment and assayed for GUS activity
as described in ``Materials and Methods''. The data presented are the
averages of three independent treatments. Bars indicate
SD.
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To examine the expression pattern of the endogenous ADH
gene, we subjected the 4-week-old AG2 plants and the aar
mutants to 8 h of anoxic or hypoxic treatments. Total RNA was
isolated and analyzed by RT-PCR. The nuclear gene ATP
that encodes the -subunit of mitochondrial ATP synthase, the
expression of which was not affected by either hypoxic or anoxic
treatment (T.R. Conley and M.-C. Shih, unpublished data), was used as
an internal standard. The results from one set of representative RT-PCR
reactions are illustrated in Figure 3.
The data show that under normoxic conditions, there was little
ADH mRNA in either line AG2 or the three aar mutants (Fig. 3A, lanes N). Levels of ADH mRNA were greatly
induced in anoxic- or hypoxic-treated AG2 plants (Fig. 3A, lanes N and H). Quantification of the RT-PCR products indicated that anoxic treatment resulted in a 25-fold increase in ADH mRNA level
in AG2, whereas hypoxic treatment resulted in a 50-fold induction (Fig.
3B). In contrast, there was no increase in the ADH mRNA level in either anoxic- or hypoxic-treated plants in any of the three
aar mutants. These results demonstrate that mutations in AAR1 and AAR2 genes affected anoxic and hypoxic
induction of expression of both the endogenous ADH gene and
the ADH::GUS transgene in mature plants.
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| Figure 3.
RT-PCR analysis of anoxic and hypoxic induction of
ADH. Total RNA (1 µg) from anoxic- or hypoxic-treated
plants was used in a 100-µL reaction for first-strand cDNA synthesis.
One-tenth of the synthesized cDNA from each treatment was then used in
subsequent PCR reactions. A, RT-PCR products for ADH
were analyzed by agarose-gel electrophoresis. The sizes of PCR products
for each gene are 525 bp for ADH and 350 bp for
ATP. The notations on top of each lane represent
treatment conditions of normoxia (N), anoxia (A), and hypoxia (H). B,
Digitized images of the ADH bands in A were quantified
and normalized to the ATP band in each lane. The
normalized mRNA levels from normoxic-treated plants (solid bars) of AG2
and each aar mutant were used as the reference levels to
calculate magnitudes of induction. Checked and striped bars correspond
to anoxic- and hypoxic-treated samples, respectively. The data
presented are the averages of three independent treatments. Bars
indicate SD.
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In addition to ADH, PDC was also required to catalyze the conversion of
pyruvate to ethanol during alcoholic fermentation. Therefore, we
examined whether the expression of PDC1 and PDC2 was affected in the aar1 and aar2 mutants. The
data show that there was a low level of PDC1 mRNA present in
AG2 plants grown under normoxic conditions (Fig.
4A). However, the PDC1 mRNA
level increased by greater than 20- and 40-fold, respectively, in
anoxic- and hypoxic-treated AG2 plants (Fig. 4, top panel). In
contrast, in the three aar mutants, PDC1 mRNA
levels were not induced by either anoxic (lanes A) or hypoxic (lanes H)
treatments. Figure 6 also shows that there was a low level of
PDC2 mRNA present in AG2 and aar mutant plants
grown under normoxic conditions (bottom panels). However, unlike
PDC1, levels of PDC2 mRNA were not induced in
either anoxic- or hypoxic-treated AG2 plants or in any of the three
aar mutants. These results showed that only the expression of PDC1, not that of PDC2, was inducible by
anoxic or hypoxic treatment in Arabidopsis, and that mutations in
AAR1 and AAR2 genes could affect PDC1
induction.
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| Figure 4.
RT-PCR analysis of anoxic and hypoxic induction of
PDC1 and PDC2. Analysis of RT-PCR
products (A) and quantification of levels of induction (B) of
PDC1 and PDC2 were performed as described
in Figure 3. The symbols used are as described in the Figure 3 legend.
The sizes of PCR products are 682 bp for PDC1 and 740 bp
for PDC2.
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| Figure 6.
Effects of cold and mannitol on ADH
and PDC1 expression. A, RNA samples from AG2 and
aar mutants grown under normal conditions (lanes N),
cold treatment (lanes C), and mannitol treatment (lanes M) were
subjected to RT-PCR analysis. B, Quantification of cold and mannitol
induction of ADH and PDC1 was performed
as described for Figure 3B. Solid bars correspond to samples from
control experiments, whereas checked and striped bars correspond to
samples from cold and mannitol treatments, respectively. The data
presented are the averages of three independent treatments. Bars
indicate SD.
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The ADH and PDC genes belong to a class of genes
that function exclusively in alcoholic fermentation. The data presented
here show that these genes were expressed at extremely low levels under normoxic conditions, but could be greatly induced by anoxic and hypoxic
treatments. Russell and Sachs (1989) and Yang et al. (1993) have shown
that glycolytic genes whose functions are required for both glycolysis
and fermentation were expressed at high levels under normoxia, but
could be induced further by anoxia or hypoxia. We chose to examine the
expression pattern of the GAPC gene, which encodes cytosolic
GAPDH, to determine whether its expression was affected in
aar mutants. Figure 5A shows
that there was a detectable mRNA level of GAPC in AG2 plants
grown under normoxic conditions (lanes N) and that the expression of
GAPC was induced by both anoxic and hypoxic treatments
(lanes A and H). Quantification of the RT-PCR products indicated that
the anoxic treatment resulted in a 3-fold increase and the hypoxic
treatment resulted in a 5-fold increase in the GAPC mRNA
level in AG2 plants (Fig. 5B). Figure 5 also shows that there were
detectable and similar-to-wild-type GAPC mRNA levels in the
three aar mutants grown under normoxic conditions. In
contrast to the wild-type AG2 line, the expression level of
GAPC remained unchanged after the anoxic or hypoxic
treatments in all three mutants. Therefore, mutations in
AAR1 or AAR2 genes affect anoxic and hypoxic
induction of the GAPC gene, as well as induction of
ADH and PDC1 genes.
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| Figure 5.
Effect of aar mutations on anoxic
and hypoxic induction of GAPC. A, Levels of
GAPC mRNA from normoxic-treated (lanes N),
anoxic-treated (lanes A), and hypoxic-treated (lanes H) AG2 plants and
aar mutants were analyzed by relative RT-PCR. The sizes
of the PCR products are 670 bp for GAPC and 350 bp for
ATP. B, Quantification of levels of induction of
GAPC by anoxia and hypoxia in AG2 and aar
mutants were calculated as described for Figure 3B.
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Allelic-Specific Effects of aar1 Mutants under Cold and
Osmotic Stress
The expression of the ADH gene is also affected by
other environmental conditions, such as cold and osmotic stress, in
several plant species (Xie and Wu, 1989; Dolferus et al., 1994). Our
RT-PCR analysis showed that in AG2 plants, levels of ADH and
PDC1 mRNA were greatly increased by cold or mannitol
treatment (Fig. 6). However, the mRNA
levels for PDC2 and GAPC were not affected by either treatment (data not shown). Therefore, we examined whether the
induction of ADH and PDC1 by cold or mannitol
treatment was affected in the aar mutants. In AG2, following
24 h at 4°C, there were 35-fold increases in mRNA levels
measured for both ADH and PDC1, whereas exposure
to 0.6 M mannitol resulted in approximately 30-fold increases in ADH and PDC1 mRNA levels
(Fig. 6, A [lanes C and M] and B). However, the data presented in
Figure 6 also show that the three mutants exhibited different
allele-specific effects on induction of ADH and
PDC1 genes by cold and mannitol treatment. In
aar1-1 mannitol treatment resulted in a 15-fold induction
of ADH (Fig. 6, A [top panel] and B). In contrast, the induction of ADH by cold treatment (Fig. 6A, lanes C [top
panel]) and the induction of PDC1 by cold and mannitol
treatments (Fig. 6A, lanes C and M [bottom panel]) were abolished
completely in aar1-1. In aar1-2 the induction
of PDC1 by mannitol treatment and the induction of
ADH by cold and mannitol treatment were abolished, whereas
there was a 30-fold induction of the PDC1 gene by mannitol treatment. In aar2-1 the induction of both ADH
and PDC1 by cold or mannitol treatment was abolished
completely.
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DISCUSSION |
We have identified and characterized trans-regulatory
mutations that affect the expression of the ADH gene in
Arabidopsis. The genetic approach used here takes advantage of the fact
that ADH catalyzes the conversion of allyl alcohol into a toxic
compound (acrolein) that is lethal to wild-type plants. In addition, we have used a transgenic line bearing an ADH::GUS
construct as the starting point for mutagenesis. The GUS
marker gene allowed us to distinguish between the two classes of
mutants that were predicted to be isolated: mutations affecting the
promoter or coding region of the endogenous ADH gene, and
mutations affecting any step in the signal transduction pathway leading
to the activation of the ADH gene. In our screening, about
one-third of the putative mutants were potentially members of the first
group, whereas the remaining two-thirds potentially belong to the
latter group of mutants. Characterization of three of these regulatory
mutants has provided us with information for understanding the
molecular mechanism of induction of ADH and PDC1
genes by three different environmental stresses. Li et al. (1995) used
a similar screening scheme successfully to isolate
trans-regulatory mutants of the CAB gene in
Arabidopsis.
Expression of ADH in Arabidopsis is known to be induced by
several environmental stresses and to be regulated developmentally (Dolferus et al., 1994). The selection scheme we have adapted allowed
us to identify mutants that were defective in regulating the expression
of the ADH gene in germinating seedlings. Three such
mutants, aar1-1, aar1-2, and
aar2-1, were also defective in regulating the anoxic and
hypoxic induction of the ADH gene in mature plants. These
results can be interpreted in two ways. First, the regulatory pathways
controlling the expression of ADH during germination and
anoxia/hypoxia are identical. Second, two signaling pathways share some
common steps and the aar mutants characterized here affect
these steps. If the second hypothesis is correct, it is conceivable
that some of the other aar mutants identified in these
screens may affect ADH expression during germination, but
exhibit normal anoxic or hypoxic induction of ADH.
Experiments are under way to further characterize the other
aar mutants that we have isolated.
In addition to the ADH gene, glycolytic and other
fermentative genes are induced by anoxia and hypoxia (Yang et al.,
1993; Sachs et al., 1996; Rivoal et al., 1997). We have also examined the effects of the aar mutations on the expression of other
glycolytic genes. Among these genes, GAPC gene products are
required for both aerobic respiration and anaerobic fermentation,
whereas ADH and PDC1 gene products are required
only for alcoholic fermentation. The expression patterns of these genes
reflect their physiological roles. ADH and PDC1
genes are expressed at extremely low levels in plant cells under normal
growth conditions and their expression is strongly induced by hypoxia
or anoxia. In contrast, the GAPC gene is expressed
constitutively at a fairly high level under normal growth conditions,
but its expression can be induced further by hypoxia or anoxia. This
raises the interesting question of whether similar regulatory elements
are involved in the activation of these two different classes of genes.
Our data show that mutations in AAR1 and AAR2
genes affected induction of both classes of glycolytic genes by anoxia
and hypoxia. These results suggested that similar regulatory elements
mediated the induction of these two different classes of genes in
response to anoxic and hypoxic stresses.
Cold and osmotic stress also induce the expression of ADH in
several plant species (Xie and Wu, 1989; Dolferus et al., 1994). Our
results show that both ADH and PDC1 genes could
be induced by cold and mannitol treatment in Arabidopsis. Recent
genetic and molecular studies suggested that both ABA-dependent and
-independent pathways interact and converge to activate the expression
of downstream genes (Ishitani et al., 1997, 1998; Zhu et al., 1998).
Our results show that the aar2-1 mutant was defective in
the induction of ADH and PDC1 by anoxic/hypoxic,
cold, and osmotic stresses (Fig. 6). This suggests that the three
stress-induced signaling pathways shared some common intermediate steps
and that aar2-1 was defective in one of these steps.
However, our results also show that the induction of
ADH and PDC1 by these three stresses was affected differently in the two allelic mutants, aar1-1 and
aar1-2. There, the induction of ADH and
PDC1 by the three stresses was abolished, with two
exceptions: in aar1-1 there was a 15-fold induction of ADH in response to the mannitol treatment, which was about
50% of the induction level of the wild-type AG2 line (Fig. 6); second, the induction of PDC1 by cold treatment was unaffected in
aar1-2.
A report by de Bruxelles et al. (1996) indicated that although
induction of the ADH gene by osmotic and cold stresses
required some common cis-acting promoter elements,
additional, but distinct, regulatory elements were required for each
pathway. Their results also showed that regulatory elements different
from those involved in cold and osmotic responses were required for
induction of ADH by anoxia and hypoxia. This implies
that transcription complexes for the ADH gene under cold,
osmotic, and low-oxygen stresses may share some common factors, but
probably also embody some discrete factors. If this hypothesis is true,
one can propose that the AAR1 gene may encode a
transcription factor shared by these complexes, and that mutations in
aar1-1 and aar1-2 have different effects on the
interaction between the AAR1 factor and factors that are specific to
each stress condition.
Current information suggests complex patterns of interactions
among the signal transduction pathways elicited by cold, osmotic, and
low-oxygen stresses in plants (de Bruxelles et al., 1996; Stockinger et
al., 1997; Zhu et al., 1997). Ishitani et al. (1997) have isolated
mutants that affect cold- and osmotic-stress signaling pathways by a high-throughput screening approach. Some of these mutants
are specifically defective in cold- or osmotic-stress-induced signaling
pathways (Ishitani et al., 1997; Zhu et al., 1998). It is likely that
some of the aar mutants we have isolated are specifically defective in the signaling pathway that corresponds to decreased oxygen. Combining studies of these different classes of mutants should greatly facilitate the elucidation of these signaling pathways.
| |
FOOTNOTES |
1
This work was supported by the U.S. Department
of Agriculture National Research Initiative Competitive Grants Program
(grant nos. 9401454 and 9700603 to M.-C.S.).
*
Corresponding author; e-mail mcshih{at}blue.weeg.uiowa.edu; fax
1-319-335-3620.
Received September 8, 1998;
accepted October 28, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ADH, alcohol dehydrogenase.
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
PDC, pyruvate decarboxylase.
RT, reverse transcriptase.
| |
ACKNOWLEDGMENTS |
We thank Drs. Joseph Frankel and Andy Wang for comments
on the manuscript. We also thank the Arabidopsis Biological Resource Center at the Ohio State University, Columbus, for providing us with
expressed sequence tag clones.
| |
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Top
Abstract
Introduction