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Plant Physiol. (1999) 119: 1083-1090
The Mechanism of Rhythmic Ethylene Production in
Sorghum.
The Role of Phytochrome B and Simulated
Shading1
Scott A. Finlayson,
In-Jung Lee,
John E. Mullet, and
Page
W. Morgan*
Department of Soil and Crop Sciences (S.A.F., I.-J.L.,
P.W.M.), and Department of Biochemistry and Biophysics (J.E.M.), Texas
A&M University, College Station, Texas 77843-2474
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ABSTRACT |
Mutant sorghum (Sorghum
bicolor [L.] Moench) deficient in functional phytochrome B
exhibits reduced photoperiodic sensitivity and constitutively expresses
a shade-avoidance phenotype. Under relatively bright, high red:far-red
light, ethylene production by seedlings of wild-type and phytochrome
B-mutant cultivars progresses through cycles in a circadian
rhythm; however, the phytochrome B mutant produces ethylene peaks with
approximately 10 times the amplitude of the wild type. Time-course
northern blots show that the mutant's abundance of the
1-aminocyclopropane-1-carboxylic acid (ACC) oxidase mRNA SbACO2 is
cyclic and is commensurate with ethylene production, and that ACC
oxidase activity follows the same pattern. Both SbACO2 abundance and
ACC oxidase activity in the wild-type plant are very low under this
regimen. ACC levels in the two cultivars did not demonstrate
fluctuations coincident with the ethylene produced. Simulated shading
caused the wild-type plant to mimic the phenotype of the mutant and to
produce high amplitude rhythms of ethylene evolution. The circadian
feature of the ethylene cycle is conditionally present in the mutant
and absent in the wild-type plant under simulated shading. SbACO2 abundance in both cultivars demonstrates a high-amplitude diurnal cycle
under these conditions; however, ACC oxidase activity, although elevated, does not exhibit a clear rhythm correlated with ethylene production. ACC levels in both cultivars show fluctuations
corresponding to the ethylene rhythm previously observed. It appears
that at least two separate mechanisms may be involved in generating
high-amplitude ethylene rhythms in sorghum, one in response to the loss
of phytochrome B function and another in response to shading.
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INTRODUCTION |
The sorghum (Sorghum bicolor) cv 58M lacks a functional
PhyB protein due to a frame-shift mutation (Childs et al., 1997 ), which
causes this cultivar to constitutively exhibit a phenotype typical of
shade avoidance. This phenotype includes excessive shoot elongation,
low chlorophyll content, and early flowering, even under noninductive
photoperiods (Quinby, 1973; Pao and Morgan, 1986; Childs et al., 1991).
We have recently reported that although both the wild-type and
the mutant cultivars produce ethylene in a circadian rhythm, the
phyB-1 mutant produces ethylene with peak values
approximately 10 times higher than the wild type (Finlayson et al.,
1998). Furthermore, under conditions simulating high shade (low-irradiance, far-red-enriched light), the wild-type plant mimics
the phenotype of the phyB-1 mutant and produces diurnal ethylene rhythms with amplitudes similar to the mutant.
Ethylene biosynthesis in plants is likely to be regulated at two
possible points. Many studies have demonstrated the regulation of
ethylene biosynthesis by controlling the levels of its immediate precursor, ACC, and have shown that this regulation occurs at the level
of transcription (Abeles et al., 1992; Kende, 1993) and possibly
through phosphorylation-dependent control of ACC synthase
degradation (Spanu et al., 1994). Ethylene biosynthesis can also be
controlled by regulating the final step of the conversion of ACC to
ethylene by ACC oxidase (English et al., 1995). Previous studies
examining the phenomenon of ethylene rhythms in plants have implicated
both points in the control of ethylene production. Circadian ethylene
evolution in Stellaria longipes was demonstrated to
correlate with both the abundance of an ACC oxidase mRNA and in vitro
ACC oxidase activity (Kathiresan et al., 1996). Emery et al. (1997) and
Kathiresan et al. (1998) subsequently found that neither fluctuations
in ACC levels nor in ACC synthase mRNA abundances were likely to be
contributing to the rhythm. Conversely, Machácková et al.
(1997) ascribed diurnal ethylene production in Chenopodium
rubrum to rhythms in the activity of ACC synthase, as evidenced by
diurnal fluctuations in ACC levels. Circadian ethylene production in
cotton was correlated with the conversion of
S-adenosyl-L-methionine to ethylene, but
not ACC to ethylene, generating the similar conclusion that
fluctuations in ACC levels were responsible for the rhythm (Rikin et
al., 1984).
The objective of this study was to elucidate the mechanism responsible
for rhythmic ethylene biosynthesis by two sorghum cultivars under two
different light regimens. As an aid to understanding both how the
lesion in PhyB function derepresses cyclic ethylene production, and how
simulated high shade generates a similar rhythm in the wild-type plant,
we sought to examine the molecular and biochemical basis for the
production of these ethylene rhythms.
The accession numbers for the sequences described in this article are
AF079588 (SbACO1), AF079589 (SbACO2), and AF079590 (SbCABII).
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MATERIALS AND METHODS |
Plant Material
Two of the near-isogenic sorghum (Sorghum bicolor
[L.] Moench) maturity cultivars, 100M
(Ma1Ma1,
Ma2Ma2,
Ma3Ma3, and
Ma4Ma4 for the
maturity genes) and 58M
(Ma1Ma1,
Ma2Ma2,
ma3Rma3R,
and Ma4Ma4) (Quinby,
1967), were used. Ma3 has been redesignated as PHYB and
ma3R as phyB-1,
based on the determination that they encode the gene for phytochrome B
and a null mutant version of the gene, respectively (Childs et al.,
1997). Seeds were germinated vertically in the dark at 27°C on
moistened germination paper. After 40 h, four healthy, uniform
seedlings were carefully transferred in an upright orientation into
plastic 50-mL conical tubes containing 45 mL of fritted clay previously
saturated with one-quarter-strength Hoagland solution and drained. The
tube walls were covered with aluminum foil and placed in the dark for
24 h until the start of the first photoperiod. The plants received
photoperiods and temperature regimens as described, with small fans
ensuring constant air circulation. Lighting was provided by a
combination of F48T12/CW/HO fluorescent lamps (Phillips, Mahwah, NJ)
and 60-W incandescent lamps giving 200 µmol
m 2 s1 PAR with a
red-to-far-red ratio of 2.05. For dim, far-red-enriched light
treatments, irradiance was provided by four 60-W incandescent lamps
producing 29 µmol m2
s1 PAR with a red-to-far-red ratio of 0.75. Light measurements were made with a spectroradiometer (model no. 1800, Li-Cor, Lincoln, NE).
Cloning of ACC Oxidase, ACC Synthase, and CAB Genes
A sorghum  gt10 cDNA library prepared from green shoots of young
plants was screened with the rice ACC oxidase cDNA clone, X85747. Six
positive plaques were purified and cloned into the NOTI site of
pBluescript II KS(+). Inserts were sequenced using the ABI dye
terminator cycle sequencing ready reaction kit (Perkin-Elmer) and
analyzed on an ABI 377 (Perkin-Elmer). Using the primers CTG GAC TGG
GAG GAC ATC TT and CAT GTA CTT GGG GTA CGC CT, a PCR product was
amplified from sorghum shoot cDNA using hot start, touch down PCR.
Sequence analysis indicated that this product was different from the
previous clone obtained, and it was used to screen the sorghum cDNA
library. One positive plaque was purified and cloned into the NOTI site
of pBluescript II KS(+). The insert was sequenced as above.
An insert encoding a fragment of a type-II chlorophyll
a/b-binding protein of the PSII gene SbCABII was obtained
from the sorghum cDNA library and ligated into the NOTI pBluescript II KS(+) site. The sequence was obtained as described above.
RNA Preparation and Northern Blotting
At each time point, 16 seedlings of each cultivar were harvested
and immediately frozen in liquid nitrogen. The samples were stored at
 80°C until RNA was extracted. Samples were kept frozen with liquid
nitrogen and ground to a fine powder. The powder was then vortexed for
30 s in a test tube with 4 mL of Tris-saturated phenol and 4 mL of
100 mM Tris-HCl, pH 8.0, at 80°C. The tubes were then
placed on ice and 4 mL of chloroform:isoamyl alcohol (48:2, v/v)
was added and the tubes were vortexed again for 30 s. The mixture
was centrifuged for 20 min at 12,000g (4°C), after which
the aqueous (upper) phase was transferred to another tube. The RNA was
precipitated with 1 volume of 4 M LiCl at
20°C for 3 h, followed by centrifugation for 20 min at
12,000g (4°C). The RNA pellet was washed with 5 mL of cold
70% ethanol and repelleted by brief centrifugation at
12,000g, and the alcohol was removed. The pellet was dried
under a vacuum and resuspended in 400 µL of diethyl
pyrocarbonate-treated water. The RNA extracts were quantitated
in triplicate using a UV spectrometer, and the quantitation was
verified by running an ethidium bromide-stained agarose test gel and
noting the staining intensities of the rRNA bands. Denaturing agarose
gels (0.8% or 1.0%) were then run and the RNA was transferred to
charged nylon membranes, which were probed with random primed [32P]dCTP-labeled DNA as previously
described.
In Vitro ACC Oxidase Assays
At each time point, four groups of four seedlings of each cultivar
were harvested and immediately frozen in liquid nitrogen. The samples
were stored at 80°C and then homogenized to a fine powder in a
mortar kept frozen with liquid nitrogen. Approximately 0.05 g of
homogenized sample was weighed into 1.5-mL microfuge tubes, also kept
frozen with liquid nitrogen. These samples were then maintained at
80°C until extracted and assayed as described previously (He et
al., 1996) with slight modifications. These modifications included the
use of extraction and assay buffers with a pH of 6.9 and the use of 25 mM DTT during extraction and 50 µM
FeSO4 during the assay. The ethylene produced was
measured using a gas chromatograph (model 10S Plus, Photovac, Markham, Ontario, Canada).
Analysis of ACC Levels
The determination of ACC levels was based on the procedure of
McGaw et al. (1985) with modifications for high sample throughput. Replicate samples from the homogenization procedure given above were
weighed into 1.5-mL microfuge tubes (approximately 0.05 g). These
samples were also kept frozen in liquid nitrogen or at 80°C until
analyzed. Twenty nanograms of
2H4-ACC was added to each
sample, which was then extracted in 500 µL of 80% ethanol at 70°C
for 3 h. The samples were centrifuged at 14,000g for 7 min and the supernatant was transferred to another microfuge tube and
dried using a concentrating centrifuge. The residue was redissolved in
500 µL of 0.1 N acetic acid and partitioned with 200 µL
of chloroform. The acetic acid fraction was applied to a column of 500 µL of hydrogen-form Dowex AG 50X8 (Bio-Rad) prewashed with 1 mL of
0.1 N acetic acid in a 1-mL plastic pipettor tip. The
column was washed with 2.5 mL of 0.1 N acetic acid and then
eluted with 400 µL of 2 N NH4OH to
waste. A further 350 µL of 2 N
NH4OH was applied, collected, and dried. This in
turn was redissolved in 500 µL of 0.1 N
NH4OH and applied to 500 µL of acetate-form
Dowex AG1X8 (Bio-Rad) prewashed with 2 mL of 0.1 N
NH4OH in a column as above. The column was washed
with 2.5 mL of 0.1 N NH4OH, and then
eluted with 950 µL of 0.1 N acetic acid to waste. An
additional 450 µL of 0.1 N acetic acid was applied, collected, dried, and redissolved in 500 µL of 30 mM phthalic anhydride in glacial acetic acid.
The samples were heated at 110°C for 1.5 h, 500 µL of water
and 1.25 mL of ether were added, and the ether fraction was collected and dried. The sample was dissolved in 250 µL of 30% methanol and
200 µL was injected onto a HPLC system using a 3.9- × 300-mm µBondapak C18 column (Waters) with a linear gradient of 30% to 40%
methanol for 5 min, followed by 40% to 48% methanol over 5 min at 1.6 mL min1. Starting at a retention time of
8.4 min, an 800-µL fraction was collected, dried, and methylated with
diazomethane. The sample was analyzed on a HP 5890A GC coupled to a
5970 mass selective detector (Hewlett-Packard) using a 0.32-mm × 15-m DB-5 column (J&W Scientific, Folsom, CA) with on-column
injection and a temperature program of 50°C to 250°C at 50°C
min1, followed by 250°C to 270°C at 20°C
min1. The phthalimido-ACC-methyl ester
had a retention time of approximately 4.5 min, and ions 244.7 and 248.7 were monitored for quantitation.
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RESULTS AND DISCUSSION |
The Mechanism of Diurnal/Circadian Ethylene Production under
Unshaded Conditions
Six independent clones were obtained from a primary screening of a
sorghum cDNA library using the rice ACC oxidase X85747 as a probe. The
cloned inserts varied in length, but sequencing analysis revealed that
all represented the same gene. The longest insert included the entire
coding sequence of 951 bases, as well as 144 untranslated 5 and 349 untranslated 3 bases. The cloned product was named SbACO1 and the
conceptually translated product showed 49.2% identity to the rice ACC
oxidase gene AF049889 over the open reading frame. When used to probe
northern blots prepared from young sorghum seedlings, SbACO1 abundance
did not show time- or cultivar-dependent differences. The library was
again screened using a PCR-generated DNA fragment (described in
``Materials and Methods''). The primary screening identified several
positive plaques, which contained the same size insert (approximately
900 bases). One insert was cloned into pBluescript II KS(+) and
sequenced. Sequencing showed an insert of 872 bases, comprising 581 bases of coding sequence and 291 bases of 3-untranslated region. The
conceptual translation product of this gene fragment was 87.1%
identical to the rice ACC oxidase AF049889, and 58% identical to
SbACO1.
SbACO2 message abundance showed a strong diurnal rhythm coincident with
previously reported peak ethylene production in cv 58M (Fig.
1). This mRNA was expressed at a much
lower level in the wild-type cv 100M, and rhythmic expression could not
be detected. Additional analyses of cv 58M SbACO2 mRNA showed that the
variations in abundance were circadian, persisting in constant
light/temperature after initial entrainment (Fig.
2).
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| Figure 1.
Diurnal SbACO2 mRNA abundance from sorghum grown
under a 12-h/12-h photoperiod, 31°C/22°C thermoperiod. The first
sample was from 5-d-old plants (58M is phyB-1, 100M is
PHYB). White and black bars indicate light/warm and
dark/cool periods, respectively.
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| Figure 2.
Diurnal/circadian SbACO2 mRNA abundance from
sorghum grown under a 12-h/12-h photoperiod, 31°C/22°C thermoperiod
until 8 AM of d 6, then in constant light at 27°C. The
first sample was from 5-d-old plants (58M is phyB-1,
100M is PHYB). White and black bars indicate light/warm
and dark/cool periods, respectively.
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ACC oxidase enzyme activity in cv 58M also fluctuated in a circadian
rhythm, paralleling both SbACO2 abundances and previously observed
peaks in ethylene production (Fig. 3).
The levels of ACC oxidase activity in cv 100M were much lower than
those observed in cv 58M, and showed a weak, low amplitude cycle, which
is again commensurate with the observed SbACO2 abundances and ethylene production from this cultivar.
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| Figure 3.
Diurnal/circadian ACC oxidase activity from
sorghum grown under a 12-h/12-h photoperiod, 31°C/22°C thermoperiod
until 8 AM of d 6, then in constant light at 27°C. The
first sample was from 5-d-old plants (58M is phyB-1,
100M is PHYB); n = 4, means ± SE. White and black bars indicate light/warm and dark/cool
periods, respectively. FW, Fresh weight.
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Analyses of ACC levels in the two cultivars demonstrated the absence of
a clear-cut single peak rhythm in either cv 58M or cv 100M (Fig.
4). ACC levels were generally higher in
cv 58M, and a small peak early in the light period could be observed in this cultivar. However, a second smaller peak was also observed during
the dark period, and a larger spike at lights off was also apparent. cv
100M also showed an elevation in ACC content at the transition from
light to dark. ACC levels in this cultivar did not demonstrate a clear
rhythm but did gradually diminish over the duration of the
measurements.
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| Figure 4.
Diurnal ACC levels from sorghum grown under a
12-h/12-h photoperiod, 31°C/22°C thermoperiod until 8 AM of d 6, then in constant light at 27°C. The first
sample was from 5-d-old plants (58M is phyB-1, 100M is
PHYB); n = 4, means ± SE. White and black bars indicate light/warm and dark/cool
periods, respectively. FW, Fresh weight.
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Our conclusions from the analyses of SbACO2 mRNA abundance, ACC oxidase
enzyme activity, ACC substrate levels, and previously reported ethylene
measurements (Finlayson et al., 1998) are that circadian ethylene
rhythms in sorghum cultivars under nonshaded conditions are the
consequence of circadian fluctuations in ACC oxidase activity,
themselves a consequence of circadian fluctuations in SbACO2 mRNA
abundance. The lesion in phytochrome B, as a result of a frame-shift
mutation in the phyB-1 gene of cv 58M,
leads to overabundance of SbACO2 message, resulting in enhanced ACC oxidase activity and ethylene production in this cultivar. It is
possible that the slightly elevated ACC levels in cv 58M over the wild
type also contributed to the increased amplitude of ethylene production; however, the levels of substrate do not correlate well with
observed ethylene peaks and are therefore unlikely to be actually
driving the rhythm.
The Mechanism of Diurnal Ethylene Production under Dim,
Far-Red-Enhanced Simulated High-Shade Conditions
The diurnal abundance of SbACO2 mRNA from cv 58M seedlings grown
under conditions simulating extreme shading showed a rhythmic pattern
consistent with the pattern of ethylene evolution from the plants (Fig.
5). Under these growth conditions the
wild-type cv 100M also produced large peaks of ethylene (Finlayson et
al., 1998); and in this case, SbACO2 abundances were elevated over those from "normal light" growth conditions and showed strong diurnal fluctuations in abundance, similar to cv 58M. SbACO2 message abundance in both cultivars showed a very weak circadian pattern when
grown in simulated shade (Fig. 6).
Seedlings entrained for two cycles and then transferred to constant
shaded light/27°C temperature lost the capacity to produce ethylene
in rhythmic peaks as soon as the entraining stimuli were removed (Fig.
7). In contrast, SbCABII mRNA abundance
cycled with a strong circadian rhythm under this light/temperature
regimen, and showed a more robust rhythm than was observed in plants
grown under "normal light" (Fig. 8).
Apparently, the loss of circadian rhythmicity under this regimen is not
a general phenomenon, but is specific to the ethylene rhythm.
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| Figure 5.
Diurnal SbACO2 mRNA abundance from sorghum grown
under a simulated high-shade 12-h/12-h photoperiod, 31°C/22°C
thermoperiod. The first sample was from 5-d-old plants (58M is
phyB-1, 100M is PHYB). Gray and black
bars indicate shaded light/warm and dark/cool periods, respectively.
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| Figure 6.
Diurnal/circadian SbACO2 mRNA abundance from
sorghum grown under a simulated high-shade 12-h/12-h photoperiod,
31°C/22°C thermoperiod until 8 AM of d 6, then in
constant light at 27°C. The first sample was from 5-d-old plants (58M
is phyB-1, 100M is PHYB). Gray and black
bars indicate shaded light/warm and dark/cool periods,
respectively.
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| Figure 7.
Ethylene production from sorghum grown under a
simulated high-shade 12-h/12-h photoperiod, 31°C/22°C thermoperiod
until 8 AM of d 6, then given constant simulated high shade
at 27°C. The first sample was from 5-d-old plants (58M is
phyB-1, 100M is PHYB).
n = 5, means ± SE. Gray and black
bars indicate shaded light/warm and dark/cool periods, respectively.
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| Figure 8.
Diurnal/circadian SbCABII mRNA abundance
from sorghum grown under either "normal light" (A) or simulated
high shade (B) with a 12-h/12-h photoperiod, 31°C/22°C thermoperiod
until 8 AM of d 6, then in constant light at 27°C. The
first sample was from 5-d-old plants (58M is phyB-1,
100M is PHYB). White (gray) and black bars indicate
light (shaded light)/warm and dark/cool periods, respectively.
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Although SbACO2 mRNA abundances in both cultivars showed clear diurnal
cycles that correlated well with measured ethylene production, analysis
of in vitro ACC oxidase activity did not demonstrate either a circadian
or diurnal pattern (Fig. 9). The ACC
oxidase activity from cv 58M was generally higher than that observed
under "normal light," showed a rather late peak on the 1st d, and
then became chaotic. ACC oxidase activity from cv 100M was also
enhanced over "normal light" levels by approximately two to three
times, but did not show rhythmicity that would correlate with SbACO2
mRNA abundance or ethylene production under this growth regimen.
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| Figure 9.
Diurnal/circadian ACC oxidase activity from
sorghum grown under a simulated high-shade 12-h/12-h photoperiod,
31°C/22°C thermoperiod until 8 AM of d 6, then in
constant light at 27°C. The first sample is from 5-d-old plants (58M
is phyB-1, 100M is PHYB).
n = 4, means ± SE. Gray and black
bars indicate shaded light/warm and dark/cool periods, respectively.
FW, Fresh weight.
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When grown in a dim, far-red-enriched environment, the
phyB-1 mutant cv 58M exhibited strong peaks in ACC levels
coincident with ethylene produced (Fig.
10). Peak ACC levels were greater than
those observed with "normal light"; the transitory peak in ACC
levels observed at lights off under "normal light" was absent in
the simulated shade-grown plants. Wild-type sorghum also produced a
peak in ACC coincident with ethylene production on the 1st d measured;
however, the following day ACC levels began to fluctuate in an
oscillatory pattern of low amplitude. ACC levels in cv 100M grown under
the simulated-shade environment were higher than those in
"normal-light-grown" plants, especially toward the end of the experiment.
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| Figure 10.
Diurnal ACC levels from sorghum grown under a
simulated high-shade 12-h/12-h photoperiod, 31°C/22°C thermoperiod
until 8 AM of d 6, then in constant light at 27°C. The
first sample was from 5-d-old plants (58M is phyB-1,
100M is PHYB). n = 4, means ± SE. Gray and black bars indicate shaded light/warm and
dark/cool periods, respectively. FW, Fresh weight.
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The previous experiments that investigated circadian responses involved
a temperature shift to only 27°C on the 1st d of constant light,
instead of the 31°C used for diurnal days (22°C at night). The
importance of temperature in shade-enhanced ethylene production is
demonstrated in Figure 11, in which the
temperature shifted from 22°C at night to 31°C on the 1st d of
constant light. This regimen caused high-amplitude ethylene peaks in
both cultivars, with cv 58M showing weak circadian ethylene production
on the 3rd subjective d. Conversely, cv 100M produced a rather
low-amplitude ethylene peak in an identical experiment performed with
the lower temperature shift (to 27°C on the 1st d of constant light),
and under these conditions ethylene production by cv 58M was not
circadian (Fig. 7). When these results are compared with the analysis
of ACC levels under simulated shade (Fig. 10), it seems likely that the
failure of ACC levels in cv 100M to show a clear peak on d 2 (the 1st d
of constant light) may be a result of the plants not receiving a
sufficient temperature signal.
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| Figure 11.
Ethylene production from sorghum grown under a
simulated high-shade 12-h/12-h photoperiod, 31°C/22°C thermoperiod
until 8 AM of d 6, then given constant simulated high shade
at 31°C. The first sample was from 5-d-old plants (58M is
phyB-1, 100M is PHYB).
n = 5, means ± SE. Gray and black
bars indicate shaded light/warm and dark/cool periods, respectively.
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Under a simulated high-shade environment, SbACO2 mRNA abundances
continued to cycle in cv 58M and were strongly enhanced in cv 100M
(Fig. 5), but the fluctuations in mRNA abundance were not translated
into rhythmic ACC oxidase activity (Fig. 9). Diurnal ethylene
production under these conditions resulted from a pronounced rhythm in
ACC levels in both cultivars (Fig. 10).
| |
CONCLUSIONS |
From the analyses performed, it becomes apparent that two
mechanisms operate to produce rhythmic ethylene evolution in sorghum. Under relatively bright, high red:far-red light, cyclic patterns of ACC
oxidase activity in cv 58M drive high-amplitude circadian ethylene
rhythms. This ACC oxidase activity was also correlated with the
abundance of SbACO2 mRNA. Therefore, it is likely that the actual
transcription of SbACO2 was the primary event leading to rhythmic
ethylene production. The wild-type cultivar had both low SbACO2
abundance and low ACC oxidase activity, commensurate with the
low-amplitude ethylene evolution observed under these conditions.
When seedlings are grown under a dim, far-red-enriched light, both
cultivars produced high-amplitude ethylene rhythms; however, this
ethylene production was not circadian in cv 100M and was circadian in
cv 58M only when entrained with a pronounced temperature shift. Under
the simulated shade conditions, SbACO2 mRNA abundance continued to
cycle in cv 58M, and also showed high-amplitude, rhythmic abundance in
cv 100M; however, ACC oxidase activity did not follow the same pattern
in either cultivar. The generally higher levels of ACC oxidase activity
in cv 100M under the simulated high-shade conditions may have
contributed to the amplitude of ethylene production, but did not appear
to be the causative agent for the rhythm itself.
In many cases, high-amplitude cycles of mRNA abundance did not result
in similar cycles of protein abundance. For instance, circadianly
expressed transcripts cloned from Sinapis alba were observed
to cycle strongly, although the corresponding proteins showed little,
if any, rhythmicity (Heintzen et al., 1994a, 1994b). Even CAB, which
showed very robust cycling at the mRNA level, produced only minor
rhythms in protein levels (Piechulla and Gruissem, 1987). It is
surprising to observe that cycling SbACO2 transcript abundance does
translate into cycling enzyme activity under one light regimen, but not
under another. Possibly, there is a light-regulated posttranscriptional
regulation of SbACO2 translation or ACC oxidase activity.
Under a simulated high-shade growth environment, the mechanistic basis
for diurnal ethylene cycles in both cultivars appeared to shift to
rhythmic fluctuations in ACC levels, probably a result of ACC synthase
activity. Similar fluctuations in ACC synthesis are thought to control
diurnal ethylene rhythms in Chenopodium rubrum (Machackova
et al., 1997). Conversely, increased N-malonyl-ACC formation
at the expense of ACC levels inhibited ethylene formation in etiolated
bean seedlings exposed to red light, an effect reversible by far-red
light (Vangronsveld et al., 1988). Although actual enhancement of
ACC levels by far-red light was not demonstrated, it is conceivable
that the low red:far-red light in the simulated shade environment could
increase ACC levels by inhibiting N-malonyl-ACC synthesis.
Both a lesion in the plant's capacity to properly sense the light
environment and a simulated-shade regimen elicited a high-amplitude ethylene rhythm. The basic mechanisms generating the rhythm were quite
different, however, suggesting that these rhythms may play a
fundamentally important role in controlling the phenotype of the plant
in environments of varying light qualities and irradiances.
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FOOTNOTES |
1
This work was supported by the U.S. Department
of Agriculture National Research Initiative Competitive Grants Program
(grant no. 97-35304-4820 to P.W.M.), by the Texas Higher Education
Board (ATP grant no. 999902-87 to P.W.M.), by a Predoctoral Overseas Korean Government Scholarship to I.-J.L., and by the Texas Agriculture Experiment Station.
*
Corresponding author; e-mail p-morgan{at}tamu.edu; fax
1-409-845-0456.
Received July 27, 1998;
accepted November 30, 1998.
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ACKNOWLEDGMENTS |
We acknowledge with thanks the seed supplied for this study by
Dr. Bill Rooney (Texas A&M University, College Station) and the
molecular expertise of Drs. R.A. Creelman, K.L. Childs, and P. Ulanch.
We are also grateful to Dr. Jialing Lu for the use of the sorghum cDNA
library and to Dr. Hans Kende for his gift of the rice ACC oxidase gene
X85747.
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LITERATURE CITED |
Abeles FB, Morgan PW, Saltveit ME Jr (1992) Ethylene in Plant
Biology, Ed 2. Academic Press, New York
Childs KL,
Miller FR,
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Top
Abstract
Introduction