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Plant Physiol. (1999) 121: 189-196
1-Aminocyclopropane-1-Carboxylate Oxidase Activity Limits
Ethylene Biosynthesis in Rumex palustris
during Submergence
Wim H. Vriezen*,
Raymond Hulzink,
Celestina Mariani, and
Laurentius
A.C.J. Voesenek1
Departments of Experimental Botany and Ecology, University of
Nijmegen, Toernooiveld 1, 6525 ED, Nijmegen, The Netherlands
 |
ABSTRACT |
Submergence strongly stimulates
petiole elongation in Rumex palustris, and ethylene
accumulation initiates and maintains this response in submerged
tissues. cDNAs from R. palustris corresponding to a
1-aminocyclopropane-1-carboxylate (ACC) oxidase gene
(RP-ACO1) were isolated from elongating petioles and
used to study the expression of the corresponding gene. An increase in
RP-ACO1 messenger was observed in the petioles and
lamina of elongating leaves 2 h after the start of submergence.
ACC oxidase enzyme activity was measured in homogenates of R. palustris shoots, and a relevant increase was observed within
12 h under water with a maximum after 24 h. We have shown
previously that the ethylene production rate of submerged shoots does
not increase significantly during the first 24 h of submergence
(L.A.C.J. Voesenek, M. Banga, R.H. Thier, C.M. Mudde, F.M. Harren,
G.W.M. Barendse, C.W.P.M. Blom [1993] Plant Physiol 103:
783-791), suggesting that under these conditions ACC oxidase activity
is inhibited in vivo. We found evidence that this inhibition is caused
by a reduction of oxygen levels. We hypothesize that an increased ACC
oxidase enzyme concentration counterbalances the reduced enzyme
activity caused by low oxygen concentration during submergence, thus
sustaining ethylene production under these conditions. Therefore,
ethylene biosynthesis seems to be limited at the level of ACC oxidase
activity rather than by ACC synthase in R. palustris
during submergence.
| |
INTRODUCTION |
The phytohormone ethylene plays an important role in many aspects
of plant growth and development, including fruit ripening and
senescence (Abeles et al., 1992 ). The precursor of ethylene, ACC, is
produced by the conversion of S-adenosyl-Met by ACC synthase (Adams and Yang, 1979). ACC is converted to ethylene, carbon dioxide, and HCN by ACC oxidase in an oxygen-dependent process. The conversion of S-adenosyl-Met to ACC has been considered as the
rate-limiting step in ethylene biosynthesis (Yang and Hoffman, 1984).
However, more recent studies have shown that in some cases ACC oxidase also plays an important role in the regulation of ethylene production, e.g. in melon (Yamamoto et al., 1995; Lasserre et al., 1996) and tomato
(English et al., 1995; Barry et al., 1996). Ethylene synthesis has been
shown to be influenced by feedback regulation (Yang and Hoffman, 1984).
Positive feedback regulation of the ACC synthase and ACC oxidase genes
by ethylene was found in tomato fruits (Rottmann et al., 1991) and
carnation petals (Woodson et al., 1992), while negative feedback of ACC
synthase was found in citrus leaf explants (Sisler et al., 1985) and in
deepwater rice internodes (Bleecker et al., 1987). Thus, biosynthesis
of ethylene could be regulated differently in different processes.
Interestingly, ethylene can promote and inhibit growth depending on the
cell type and plant species. In Arabidopsis it inhibits cell expansion
throughout development in most tissues (Kieber et al., 1993) and
inhibits cell elongation during the formation of the hypocotyl hook in
seedlings, but it stimulates elongation of the hypocotyl and root
through the action of the HLS gene (Lehman et al., 1996) and
enhances elongation of the hypocotyl if nutrient-starved plants are
grown under constant light (Smalle et al., 1997). In general, ethylene
inhibits shoot elongation in most terrestrial plants (Abeles et al.,
1992), whereas it stimulates growth in some aquatic and amphibious
species. This growth stimulation is essential for the survival of these
plants because it enables them to keep their foliage above water during
flooding (Musgrave et al., 1972; Metraux and Kende, 1983; Jackson,
1985; Blom et al., 1994).
One important consequence of flooding is the reduction of gas exchange,
because gases diffuse at a lower rate in water than in air. The oxygen
concentration is 30 times lower in water than in air (Armstrong et al.,
1994), and reduced photosynthesis due to the restricted availability of
carbon dioxide and light causes a further decrease (Setter et al.,
1987; Stünzi and Kende, 1989).
Rumex palustris is a flooding-resistant semiterrestrial
species that responds to flooding with rapid growth stimulation of the
shoot, especially of the petioles of the youngest leaves. This
induction of cell elongation requires ethylene action and is increased
in low oxygen concentrations (Voesenek et al., 1997). In R. palustris, the endogenous ethylene concentration increases from
0.05 to 1 µL L 1 within 1 h of
submergence (Banga et al., 1996) due to physical entrapment. The
ethylene production rate of R. palustris does not increase
during the first 24 h of submergence (Voesenek et al., 1993),
although ACC concentration increases in the shoot (Banga et al., 1996).
Based on these data, we hypothesize that ACC oxidase limits ethylene
production in R. palustris under water.
To understand the role of ACC oxidase in the regulation of ethylene
production during submergence, we studied the accumulation of ACC
oxidase gene transcripts and enzyme activity. We also determined the
influence of reduced oxygen concentrations and elevated ethylene levels
on the gene and enzyme activities. Our results show that both the ACC
oxidase messenger concentration and the ACC oxidase enzyme activity
measured in tissue homogenates increased upon submergence. A low oxygen
concentration proved to have a strong limiting effect on ACC oxidase
enzyme activity. Our results indicate that ethylene production in
R. palustris is limited by ACC oxidase activity due to
hypoxia during submergence.
| |
MATERIALS AND METHODS |
Achenes of Rumex palustris Sm. were collected from
river areas near Millingen, The Netherlands. Germination and growing
conditions were as described by Banga et al. (1996) except for the
plants used to measure the ACC oxidase enzyme activities. All
experiments started at 9 AM. During all
experiments and 24 h before, plants were grown under constant
light (PPFD 100 µmol m2
s1) at 22°C.
Isolation of ACO cDNA Fragments
Total RNA was isolated (van Eldik et al., 1995) from R. palustris shoots that had been submerged for 24 h and
subsequently kept in air for 3 h. Poly(A+)
mRNA was isolated using a mRNA isolation system (PolyATtract, Promega).
A 0.6-µg aliquot of poly(A+) mRNA was dissolved
in 10 µL of diethyl pyrocarbonate-treated water containing 200 ng of
oligo-dT. The mixture was preheated for 10 min at 65°C and then
cooled on ice. First-strand cDNA synthesis was accomplished by
including 200 units of reverse transcriptase (Superscript) in 20 µL
of buffer supplemented with 0.5 mM each dNTP, 20 units of RNasin, and 10 mM DTT. After incubation
for 60 min at 37°C, the reaction was terminated at 90°C for 5 min and then cooled on ice. The mixture was finally incubated for 20 min at
37°C with 1 unit of RNase H. All reaction components were obtained
from GIBCO-BRL except when stated otherwise.
Four microliters of first-strand cDNA served as a template and
was amplified in 100 µL of PCR buffer with 1.25 mM
MgCl2, 100 pmol of each dNTP, 50 pmol of each PCR
primer, and 0.4 unit of thermostable DNA polymerase (Goldstar). Three
minutes at 95°C and 40 cycles of 1 min at 94°C, 1 min at 50°C,
and 1 min at 72°C were performed, followed by 10 min at 72°C in a
thermal cycler (Perkin-Elmer). The sequences of the oligonucleotide
primers corresponding to conserved regions were as follows: upstream,
5 -GCCGAATTCCAAGAGTGTGATGCACAGAGT-3; downstream,
5-GCCAAGCTTCATAGCTTCAAATCTTGGCTC-3(EcoRI
and HinDIII recognition sites are underlined). The 241-bp
PCR product was directly ligated to the pCRII vector using a TA cloning
kit (Invitrogen, Carlsbad, CA).
cDNA Library Screening
We screened approximately 4 × 104
plaques of a R. palustris cDNA library (Vriezen et al.,
1997) with the [ -32P]ATP-labeled ACC oxidase
reverse transcriptase-PCR fragment according to the manufacturer's
protocol (Stratagene). Filters were prehybridized for 1 h and
hybridized overnight at 65°C with a solution containing 5× SSC, 5×
Denhardt's reagent, 0.5% (w/v) SDS, and 100 µg
mL1 of denatured, fragmented salmon-sperm DNA.
Membranes were then washed twice in 2× SSC plus 0.1% (w/v) SDS at
65°C for 15 min each, and twice in 0.2× SSC plus 0.1% (w/v) SDS for
15 min. The blots were exposed to film (X-Omat AR, Kodak) with two
intensifying screens at  80°C for 16 h. The first screening
with the ACC oxidase probe led to 0.25% of positive plaques. Ten
plaques were selected for a second screening, after which seven were
still found to be positive. Based on the nucleotide sequence, we
concluded that these seven cDNAs originated from two different R. palustris ACC oxidase genes, which we designated
RP-ACO1 (accession no. Y10034) and RP-ACO2
(accession no. AF041479). The sequences of the cDNAs were too similar
to distinguish the expression patterns of the corresponding genes by
conventional hybridization techniques, so RP-ACO1 cDNA was
used in all hybridization experiments as a probe to study the
expression of both genes as one.
DNA Manipulations and Sequence Analysis
The pBluescript SK() phagemid containing the positive cDNAs was
excised from the Uni-ZAP XR vector and cloned into Escherichia coli XL1-Blue-MRF cells (ExAssist/SOLR in vivo excision system, Stratagene). The nucleotide sequence of the cDNAs was determined using
a dye terminator cycle-sequencing kit (PRISM Ready Reaction, Applied
Biosystems) with a genetic analyzer (PRISM 310, Applied Biosystems).
Probes for hybridizations were labeled in low-melting-point agarose
with [-32P]dATP by the random-priming method
(Church and Gilbert, 1984).
Experimental Treatments
To determine the ACC oxidase messenger levels in leaf tissue under
water, plants were submerged in an open tank with 25 cm of tap water at
22°C. At several time points after submergence, petioles and lamina
were cut from leaf 4, the youngest fully developed leaf.
The 3% (v/v) oxygen gas treatment was performed as described by
Voesenek et al. (1997). The 5-µL L1 ethylene
treatment was performed in an airtight box (model 1029, Forma
Scientific) with a volume of 670 L. The experiment started with the
release of 3.35 mL of pure ethylene into the box. Samples were taken
after 0, 20, 40, 60 120, 240, and 360 min through an air lock of 48 L. The ethylene concentration was maintained at 5 µL
L1 by releasing 240 µL of ethylene into the
box every time a sample was taken. In all experiments RNA was isolated
from leaf 4, except in the desubmergence experiment, in which complete
shoots were used. The plants used were 26 to 30 d old and were
just starting to develop their fifth leaf. Northern analysis showed
that the expression patterns of the studied genes were comparable in
leaves 3, 4, and 5 of 4-week-old R. palustris plants
(data not shown).
RNA/DNA Isolation and Blot Hybridization Analysis
For RNA gel blots, total RNA was isolated and separated on a 1%
(w/v) agarose gel containing 0.4 M formaldehyde and 0.1 µg mL1 of ethidium bromide. After
electrophoresis, the gel was examined under UV light and photographed
to ensure that equal amounts of RNA were present in each lane. RNA was
transferred to a nylon membrane (Hybond-N, Amersham) using the method
described by the manufacturer. Prehybridization, hybridization, and
washing conditions were the same as described for the library
screening. Hybridization was performed using full-length
RP-ACO1 cDNA as a probe. The blots were exposed to film
(X-Omat AR, Kodak) with two intensifying screens at 80°C for 1 to
2 d. Finally, hybridization with a tobacco ribosomal cDNA (kindly
provided by Dr. K. Weterings, Department of Plant Cell Biology,
University of Nijmegen, The Netherlands) was performed to ensure
equal transfer during the blotting procedure. Genomic DNA was isolated
(van Eldik et al., 1995) and electrophoretically separated on an 0.8%
(w/v) agarose gel. Southern analysis, DNA fixation, and hybridization
were performed on a Hybond-N nylon membrane according to the
manufacturer's directions. The full-length RP-ACO1 cDNA was
used as a probe. Prehybridization, hybridization, and washing
conditions were the same as described for the library screening. All
analyses of messenger concentrations were done at least twice with
different plants in different experiments.
ACC Oxidase Enzyme Assays
To obtain clean plants that were easy to harvest for the ACC
oxidase assays, we grew R. palustris plants hydroponically.
Seeds were spread in a flat plastic tray with a 5-cm-thick layer of glass beads (diameter 5 mm) in a nutrient solution containing 2 mM
Ca(NO3)2, 1.25 mM
K2SO4, 0.5 mM MgSO4, 0.5 mM
KH2PO4, and the
micronutrients 90 µM FeEDTA, 50 µM NaCl, 25 µM
H3BO3, 2 µM MnSO4, 2 µM ZnSO4, 0.5 µM CuSO4, and 0.5 µM
H2MoO4. The seeds and glass
beads were covered with a 1-cm-thick layer of black polyethylene grains
(Lacgtene low-density grains, Elf Atochem, France). The nutrient
solution was well aerated by continuous air bubbling. Germination and
growth conditions were as described by Banga et al. (1996).
The ACC oxidase assay was performed as previously described (Ververdis
and John, 1991; Mekhedov and Kende, 1996) with some modifications. The
shoots of 10 3-week-old R. palustris plants were collected
and frozen immediately in liquid nitrogen. After grinding approximately
0.5 g of fresh tissue in liquid nitrogen, 1.5 mL of extraction
buffer (300 mM Tris-Cl, pH 7.2, 30 mM sodium ascorbate, and 10% [v/v] glycerol)
was added to the ground tissue that was then allowed to thaw to a
slurry. The sample was centrifuged at 15,000g for 10 min at
4°C and the supernatant was used in the assay. The activity assay
contained 1.7 mL of incubation buffer (100 mM
Tris-Cl, pH 7.2, 30 mM sodium ascorbate, and 10%
[v/v] glycerol), 50 µL of 80 mM ACC, 50 µL
of 3 mM FeSO4, 100 µL of 1 M NaHCO3, and 200 µL of
the enzyme extract. This mixture was incubated in the dark at
30°C in a closed 10-mL vial for 1 h. One milliliter of the
headspace was analyzed in a gas chromatograph (model 437A, Chrompack,
Bergen op Zoom, The Netherlands; with a 100-cm Hayesep N column and a
flame-ionization detector). The protein concentration in the extracts
was determined with Bradford reagent according to the manufacturer's
instructions (Bio-Rad). To exclude differences in the recovery of ACC
oxidase activity from tissues isolated before and after submergence,
tissue homogenates of treated and untreated shoots were mixed and the
ACC oxidase enzyme activity was determined. Tissue homogenates with low
ACC oxidase activity did not negatively influence the ACC oxidase activity of homogenates with higher ACC oxidase activities (data not
shown).
Determination of Ethylene Production Rates at Different Oxygen
Concentrations
Aerated, 23-d-old whole plants were moved from the hydroculture
into a 10-mL vial, which was subsequently capped and flushed with
oxygen-free nitrogen gas for 2 min at a rate of 50 mL
min1. The same procedure was performed with
24-h-submerged plants, which were then transferred, still under water,
into the vial. The water was substituted by nitrogen gas and the vials
were additionally flushed with nitrogen for 2 min. Oxygen was injected
into the vials to obtain the intended concentrations. The ethylene
production was determined by GC after incubation of the vials for
1 h at 30°C in the dark. The ethylene production of plants under
these experimental conditions proved to be linear for at least 2.5 h (data not shown).
| |
RESULTS |
Cloning of ACC Oxidase cDNAs from R. palustris
A partial ACC oxidase cDNA clone was generated by RT-PCR on
poly(A+) mRNA from R. palustris
leaves. This cDNA fragment was used to screen the R. palustris cDNA library and was very abundant (one out of 400-600
plaques). Seven of these putative ACC oxidase cDNAs were sequenced and
proved to share high sequence homology (96%), with the main
divergences in the UTRs, and appeared to represent two different genes
(RP-ACO1 and RP-ACO2). The putative RP-ACO proteins have a molecular mass of 35.7 kD (314 amino acids), share 97%
identical residues, and have a pI of 4.9. The deduced amino acid
sequence of RP-ACO1, which was taken to represent both
genes, showed 84% similarity with TOM13, an ACC oxidase from tomato
that is a proven ethylene-forming enzyme (Hamilton et al., 1990).
Furthermore, Southern analysis of R. palustris genomic DNA
showed that RP-ACO1 is a member of a small multigene family
composed of two or three members (Fig.
1), one of which may be
RP-ACO2.
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| Figure 1.
Hybridization of RP-ACO1 cDNA to
digested R. palustris genomic DNA (10 µg per lane).
Molecular length standards are indicated on the right (kb).
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Analysis of RP-ACO1 Messenger Accumulation and Enzyme
Activity in R. palustris under Aerated and Submerged
Conditions
To study the regulation of RP-ACO genes under both
well-aerated and flooded conditions, we analyzed the
expression patterns of the R. palustris ACC
oxidase genes in the petioles and lamina of leaf 4 at several points in
time. Under aerated conditions, RP-ACO1 expression in the
petioles (Fig. 2A) and lamina (data not
shown) remained at a low level during the 24 h of the experiment. By contrast, during submergence a strong increase of the
RP-ACO1 transcript accumulation was found in both tissues
(Fig. 2B). The overall messenger concentration was higher in the
petiole than in the lamina, but showed a comparable expression pattern
in time. Restoring normal aerated conditions by lowering the water
level (desubmergence) 24 h after submergence caused a temporary
increase of the RP-ACO1 messenger concentration. After that,
the RP-ACO1 transcript levels decreased to a level
comparable to that before submergence (Fig. 2C).
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| Figure 2.
Accumulation of RP-ACO1 mRNA in
tissues of R. palustris during different treatments (15 plants per sample). A, Under aerated conditions in petioles of leaf 4. B, During submergence in elongating petioles and lamina of leaf 4. C,
In the shoot after 24 h of submergence and subsequent
desubmergence. Each lane in A and B contained 10 µg of total RNA and
20 µg was loaded in C. The RNA gel blot was reprobed with 28S rRNA as
a control for the loaded amount of total RNA.
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To localize the accumulation of RP-ACO1 transcripts in
submerged leaves, we performed RNA-blot hybridizations with RNA
isolated from segments of petioles and lamina of leaf 4 from submerged plants. Figure 3 shows that the level of
the RP-ACO1 transcript (1.3 kb) strongly increased upon
submergence, particularly in the apical parts of the petiole.
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| Figure 3.
Petiole and lamina segments of submerged R. palustris plants (50 plants were sampled for each time point).
The leaf at the top of the figure displays the segments of leaf 4 from
which total RNA was isolated. Ten micrograms per lane was loaded. The
RNA gel blot was reprobed with 28S rRNA as a control for the loaded
amount of total RNA, which is shown only at 24 h.
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The ACC oxidase enzyme activity in the complete shoot was measured with
an in vitro assay. Submergence induced ACC oxidase enzyme activity in
extracts of young R. palustris plants 12 h after the
start of the treatment. The maximum activity was reached after 24 h and remained on that level for at least 48 h (Fig. 4A). The messenger concentration in the
shoot of these plants showed a comparable pattern, although an increase
was observed after 6 h of submergence (Fig. 4B). In summary, the
concentration of the messenger was higher in the petioles than in the
lamina, and submergence induced RP-ACO1 transcript levels
and enzyme activity.
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| Figure 4.
A, ACC oxidase enzyme activity in shoot
homogenates of submerged R. palustris plants
(n = 7; 10 plants per sample; means ± SE). B, Accumulation of RP-ACO1 mRNA in the
shoot of plants that were used for the ACC oxidase enzyme activity
assay. The RNA gel blot was reprobed with 28S rRNA as a control for the
loaded amount of total RNA (10 µg).
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Modulation of RP-ACO1 Gene Expression by Gas
Treatments
Submergence causes a change in the endogenous gas composition.
When plants are exposed to flooding stress, the ethylene concentration generally increases and the oxygen level declines (Stünzi and Kende, 1989). Under these conditions petioles elongate at a rate faster
than leaf expansion. For this reason the impact of these changes on the
expression level of RP-ACO1 was investigated in petioles by
exposing R. palustris plants to 5 µL
L1 of ethylene and to 3% (v/v) oxygen.
Figure 5A shows that 5 µL L1 of ethylene induced a rapid increase in
RP-ACO1 expression within 2 h, which remained high at
least for further 24 h (data not shown). Low oxygen (3%)
treatment also induced gene activity, with an increase of transcript
accumulation observed after 6 h and remaining high at least for
48 h (Fig. 5B). These results indicate that not just submergence
but also high levels of ethylene or low oxygen concentrations were able
to induce and maintain accumulation of the RP-ACO1
transcript.
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| Figure 5.
A, Accumulation of RP-ACO1 mRNA in
petioles of R. palustris leaf 4 (15 plants per sample)
exposed to 5 µL L1 of ethylene. B, Accumulation of
RP-ACO1 mRNA in petioles of R. palustris
leaf 4 (15 plants per sample) exposed to 3% (v/v) oxygen. The RNA gel
blot was reprobed with 28S rRNA as a control for the loaded amount of
total RNA (10 µg).
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Both 3% (v/v) oxygen and submergence stimulated an increase of
RP-ACO1 messenger concentration, and submergence also caused an increase in ACC oxidase enzyme activity. The ethylene production rate, however, did not increase but during the first 24 h of these treatments remained similar to that under aerated conditions (Voesenek et al., 1993; Vriezen et al., 1997). To analyze further the effect of
oxygen on the ethylene production rate, we treated young R. palustris plants with several oxygen concentrations and measured the ethylene production after 1 h in the dark. The ethylene
production at oxygen concentrations below 5% (v/v) decreased to levels
below 35% of the production that was found at the ambient oxygen
concentration (Fig. 6). In addition,
plants that had been submerged for 24 h before this treatment were
capable of producing more ethylene than plants that were growing in air
at all times. The ethylene production of desubmerged plants in 5%
(v/v) oxygen was comparable to the ethylene production of nonsubmerged
plants growing in air.
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| Figure 6.
Ethylene production of whole R. palustris plants. Hatched bars represent the ethylene produced
per hour at several different oxygen concentrations, just after a 24-h
period of submergence. White bars represent the ethylene produced per
hour by untreated plants at these oxygen concentrations
(n = 3; means ± SE). FW, Fresh
weight.
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| |
DISCUSSION |
We isolated R. palustris cDNAs corresponding to
RP-ACO1 and RP-ACO2, ACC oxidase genes that
encode the enzyme responsible for the final conversion of the ethylene
biosynthetic pathway.
The concentration of the RP-ACO1 messenger
remained at a relatively low level under aerated conditions during the
day (Fig. 2A), whereas submergence strongly induced transcript
accumulation, particularly in the petiole (Figs. 2B and 3), the tissue
that elongates the most after submergence (Voesenek et al., 1990). This
suggests that localized gene expression and localized ethylene production in the petioles may be necessary for elongation during submergence. However, during this process, ethylene accumulates in the
whole plant to a much higher concentration than that needed to
stimulate the maximal elongation response (Banga et al., 1996), which
makes a precise localization of ethylene production unnecessary. On the
other hand, ethylene production at the site of elongation can be very
important when the plant grows partly under submerged conditions and
ethylene can diffuse freely to the environment through the aerenchyma
system.
In many species, ACC oxidase gene activity is positively regulated at
the transcriptional level by ethylene (Drory et al., 1993; Nadeau et
al., 1993; Kim and Yang, 1994; Tang et al., 1994; Peck and Kende, 1995;
Barry et al., 1996; Mekhedov and Kende, 1996). RP-ACO
transcript levels were also strongly induced in R. palustris
by ethylene treatment (Fig. 5A) or by submergence (Fig. 2B), which
causes accumulation of ethylene in the tissue. The ACC oxidase enzyme
activity measured in shoot homogenates doubled after 24 h of
submergence, probably due to the increased RP-ACO1 messenger
concentration (Fig. 4). However, according to Voesenek et al. (1993),
the ethylene production rate does not increase during submergence.
These data suggest that ethylene biosynthesis in submerged R. palustris plants was limited in one of the biochemical conversions
before the oxidation of ACC. The concentration of free ACC in the shoot
of submerged R. palustris plants proved nevertheless to
increase (Banga et al., 1996). This was also found in the intercalary
meristem of rice plants (Cohen and Kende, 1987; Zarembinski and
Theologis, 1997). It is therefore unlikely that during submergence the
ACC concentration limits ethylene production, suggesting that in
R. palustris ethylene production is instead limited by ACC
oxidase enzyme activity. The discrepancy between ACC oxidase enzyme
activity measured in vitro (in plant homogenates) and in vivo (as
ethylene released by the whole plant) shows that in vitro measurements
of enzyme activity should be taken as an indication of the enzyme
concentration.
In deepwater rice, submergence can cause oxygen shortage in the plant
tissue and this in turn induces ethylene biosynthesis, which is the
signal for increased cell elongation rate (Raskin and Kende, 1984;
Stünzi and Kende, 1989). On the contrary, low oxygen
concentrations did not increase ethylene production in R. palustris (Fig. 6), although we did find an increase of
RP-ACO1 mRNA levels after treating the plants with 3%
oxygen (Fig. 5B). Imaseki (1991) showed that hypoxia can reduce the
efficiency of the ACC oxidase enzyme activity.
Our data suggest that a low oxygen concentration induces an increase in
the ACC oxidase mRNA level, which causes an increased concentration of
ACC oxidase protein. Although this protein is less active at low oxygen
concentrations, the increased enzyme concentration ensures that there
is enough ethylene produced to stimulate petiole elongation during
submergence.
Restoring aerated conditions by desubmergence caused a rapid increase
in ethylene production (Fig. 6). This fast response could be attributed
to a greater activation of ACC oxidase enzyme activity by high levels
of oxygen (21%) and therefore by a fast conversion of the accumulated
ACC. The newly synthesized ethylene could be responsible for the
transient up-regulation of the transcription rate of RP-ACO1
in the first hours after desubmergence (Fig. 2C).
Ethylene triggers the signal transduction pathway responsible for stem
internode or petiole elongation (Metraux and Kende, 1983;
Jackson, 1985; Vriezen et al., 1997). During submergence in
both rice and R. palustris, ethylene accumulates to a level that is much higher than that required to stimulate the maximum elongation response (Metraux and Kende, 1983; Banga et al., 1996). Moreover, inhibition of ethylene action with silver ions abolishes the
elongation response under water (Banga et al., 1997). This implies that
the elongation rate is not limited by ethylene, but that ethylene is
necessary to initiate and maintain a fast elongation of petioles under
water. Thus, the elongation rate of R. palustris petioles
during submergence must be regulated by a factor either within or
downstream from the ethylene signal transduction chain.
In conclusion, ethylene production in R. palustris during
submergence is regulated by an increase in the ACC oxidase enzyme concentration that counterbalances the decreased enzyme activity caused
by submergence. Furthermore, most of the RP-ACO1 gene
expression was localized in the petioles, the tissue that elongates the
most during submergence.
| |
FOOTNOTES |
1
Present address: Department of Plant
Ecophysiology, Faculty of Biology, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
*
Corresponding author; e-mail wimv{at}sci.kun.nl; fax
31-24-3652490.
Received March 29, 1999;
accepted June 6, 1999.
| |
ACKNOWLEDGMENTS |
We wish to thank Ivo Rieu and Gerard Bögemann for
excellent technical assistance and Kees Blom for critically reading
this manuscript.
| |
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Abstract
Introduction