Plant Cell Physiol. 39(3): 342-348 (1998)
JSPP © 1998
Biological Activities of an Abscisic Acid Analog in Barley, Cress, and Rice
Tadao Asami 1 , Masumi Robertson 2 , Shin Yamamoto1, Koichi Yoneyama3, Yasutomo Takeuchi3 and
Shigeo Yoshida1
1
2
3
Plant Functions Laboratory, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama, 351-01 Japan
CSIRO, Plant Industry, GPO Box 1600, Canberra, ACT2601 Australia
Weed Science Center, Utsunomiya University, Utsunomiya, 321 Japan
Biological activities of an abscisic acid (ABA) analog,
RCA-7a [l-(3-carboxyl-5-methylphenyl)-l-hydroxy-2,6,6trimethyl-4-oxo-2-cyclohexene], carrying a phenyl group in
the side chain are reported. ( + )-RCA-7a was approximately 3-fold less active than ( + )-ABA in the inhibition of aamylase induction by gibberellic acid (GA3) in embryo-less
barley grains. In aleurone layers, RCA-7a inhibited GA3-induced a-amylase mRNA accumulation and induced the accumulation of dehydrin mRNA, but less effectively than
ABA. It also activated a dehydrin gene promoter in barley
aleurone protoplast transient assays. A higher concentration of RCA-7a than ABA was required for similar levels
of inhibition of the growth of cress plants at 1 week after
sowing; however, RCA-7a remained effective for at least 3
weeks. In contrast, ABA rapidly lost its effectiveness and
became less effective than RCA-7a by 3 weeks. RCA-7a
also significantly retarded the growth of rice plants. These
greenhouse pot experiments showed that RCA-7a had
more useful characteristics than ABA for practical applications. These and our previous results indicate that RCA-7a
has an ABA-like activity in various biological processes in
plants and has potential as a novel plant growth regulator.
(Dashek et al. 1979, Sharkey and Raschke 1980, Gusta et
al. 1992, Balsevich et al. 1994). This instability represents a
significant drawback to the use of ABA in the field. Although numerous analogs of ABA have been synthesized
to circumvent this catabolism, only a few compounds with
modified side chain structures have shown ABA-like activities (Flores et al. 1988, Abrams and Milborrow 1991,
Grossnickle et al. 1996). In another approach, the methyl
group on the cyclohexenone ring was modified; these ABA
analogs were more potent inhibitors than ABA itself. For
example, inhibitory activities of (+)-8'-trifluoro- and methoxy-ABA analogs on the growth of rice seedlings were
30- and 5-fold higher than ABA, respectively (Todoroki et
al. 1994, 1995). In a GA3-induced a-amylase production
assay, the trifiuoro-ABA was 5 times more inhibitory than
ABA (Kim et al. 1995, Todoroki et al. 1995). 8'-Methylene
ABA (Abrams et al. 1997) and nonadeutero-ABA (Lamb et
al. 1996) showed more long-lasting effects than ABA in
cress seed germination and better persistence than ABA in
the medium of suspension cultures of corn cells. Because of
their enhanced effectiveness, these analogs might be useful
as synthetic plant growth regulators, although high synthetic costs may make them commercially impractical.
Key words: Abscisic acid — Aleurone cells — a-Amylase —
Dehydrin — Gibberellin — Plant growth regulator.
Previously, we reported novel ABA analogs carrying a
phenyl group substitution in the ABA side chain. In bioassays for ABA, some of these compounds exhibited activity, although higher concentrations (3- to 10-fold) were
required for equivalent activity (Asami et al. 1992). The
studies on structure-activity relationships revealed that
Abscisic acid (ABA) is involved in the control of many
processes in plants, such as the acceleration of abscission,
induction of dormancy, inhibition of rooting, and stimulation of stomatal closure (Zeevaart and Creelman 1988). In
addition, ABA has attracted considerable attention
because it is thought to play an important role in the
response to environmental stresses such as drought
(Chandler and Robertson 1994) and low temperature
(Chen et al. 1983, Heino et al. 1990, Anderson et al. 1994,
Robertson et al. 1995). Therefore, ABA has potential for
practical applications as a plant growth regulator. However, the side chain geometry (2Z, 4£) that is essential for
the activity of ABA is easily altered by light to form an inactive (2E, 4E) isomer (Milborrow 1970). Furthermore, ABA
is readily catabolized to phaseic acid (PA) (Milborrow
1969, Dunstan et al. 1992, Hampson et al. 1992), which is
unable to induce many of the processes controlled by ABA
COOH
COOH
(+)-abscisic acid
(-)-phaseic acid
COOH
COOH
(+)-RCA-7a
(-)-RCA-7a
Fig. 1 Structures of (+)-ABA, (+)- and (-)-RCA-7a used in
this study, along with (—)-phaseic acid.
342
ABA analog mimicking ABA action
RCA-7a [1 -(3-carboxyl-5-methylphenyl)-1 -hydroxy-2,6,6trimethyl-4-oxo-2-cyclohexene] (Fig. 1) was the most active
of these ABA analogs. RCA-7a can be prepared by a facile
and inexpensive "one-pot" synthetic method on a large
scale (Asami et al. 1994), which makes the development of
this chemical as a synthetic plant growth regulator commercially viable and encourages a detailed study of its effects
on various physiological responses. In this paper, we investigated whether RCA-7a had activity similar to ABA in a
range of in vitro assays, because there are many compounds which show an ABA-like activity in one assay
system but not in others. ABA can act as an inhibitor or retardant in some physiological processes; thus some test
compounds may appear to have ABA-like activities due to
their general toxic or inhibitory effects on different biological processes independent of ABA action. Examples include phenyl derivatives of ABA (Bittner et al. 1977, Ladyman et al. 1988, Yoshikawa et al. 1992) and 2-desmethyl
ABA (Hornberg and Weiler 1984, Yamashita et al. 1982).
To determine whether RCA-7a has an ABA-like activity in
plants, a "positive" biological response induced by ABA
should be included. Therefore, we measured expression of
dehydrin genes (Close et al. 1989, Robertson et al. 1995,
Lang et al. 1997), because it is up-regulated by ABA, as
well as the effect on GA3-induced a-amylase gene expression (Chandler et al. 1984), which is down-regulated by
ABA. If RCA-7a mimics ABA, it may be possible to develop this compound commercially as a plant growth regulator for retarding plant growth or preventing effects of environmental stress.
Materials and Methods
Chemicals—RCA-7a (for structure, see Fig. 1) was synthesized and optically resolved as previously described (Asami et al.
1994). S-(+)-ABA was a kind gift from Toray Co. Ltd. (Nihonbashi Chuo-ku, Tokyo, Japan), and (±)-ABA was purchased
from Junsei Chemical (Nihonbashi Chuo-ku, Tokyo, Japan) or
Sigma (Castle Hill, NSW, Australia).
Incubation of embryoless barley half-grains—Embryoless
half grains were prepared by removing the embryos and the distal
ends from barley (Hordeum vulgare L. cv. Senbonhadaka) grains.
Half-grains were surface sterilized with \% sodium hypochlorite
(w/v), and ten half-grains were incubated together in each 25 ml
Erlenmyer flask containing 2 ml of freshly prepared incubation
medium (1 mM acetate buffer (pH 5.4), 20 mM CaCl2). The samples were incubated at 25°C for 24 h in the presence of 0.1 fiM
GA3 together with 0, 0.3, 1.0, 3.0, 10, or 30//M (+)-ABA, 1.0,
3.0, 10, 30 or 100^M(+)-RCA-7a, (-)-RCA-7a, or (+)-RCA-7a
in triplicate for each concentration. After incubation, the halfgrains were homogenized in the incubation medium with an additional 3 ml of the 1 mM acetate/CaCl2 buffer. The homogenate
was centrifuged at 2,000 x g for 10 min. The supernatant (enzyme
solution) was used for a-amylase assay (see below).
Isolation of barley aleurone protoplasts—Barley grains, Hordeum vulgare cv. Himalaya, harvested at Canberra, Australia, in
1994 were used. The procedure used for barley aleurone proto-
343
plast isolation was essentially the same as described previously
(Lin et al. 1996) with minor modifications.
For assaying ABA suppression of GA3-induced a-amylase
activity, protoplasts were resuspended in an appropriate volume
of protoplast isolation medium (PIM) consisting of 10 mM Larginine HC1, 10 mM MES, 0.18 M KC1, 90 mM CaCl2 with 50
unit ml" 1 nystatin, and 150/igml"' cefotaxime. One milliliter of
suspended protoplasts per flask was used with various hormone
treatments. The samples were incubated in the dark at 25°C for
24 h in the presence of 1 /iM GA3 together with 0, 0.01, 0.03, 0.1,
0.3, 1, 3, 10 fiM (+)-ABA, (+)-RCA-7a, (-)-RCA-7a, or ( ± ) RCA-7a in triplicate for each concentration. After the incubation,
protoplasts were lysed completely by vortexing and microcentrifuged for 10 min at 4°C. Aliquots of the supernatant were used
for a-amylase assay.
a-Amylase activity assay—An appropriate volume of enzyme
extract from aleurone protoplasts or layers was mixed with 0.1%
starch (soluble) (Merck Japan Ltd., Shimomeguro, Meguro,
Tokyo) in 1 mM acetate buffer (pH 5.4) containing 2 mM CaCl2
and incubated at 37°C for 5 min in triplicate for each sample. The
reaction was stopped by adding 1% iodine-0.05 M HC1 solution.
The A^o of the solution was measured in a spectrophotometer.
The activity was expressed as % inhibition; the activity of aleurones treated with GA3 alone was designated as having 0% inhibition and that in control solution (no GA3) was designated as having 100% inhibition.
Barley aleurone protoplast transient assays—Protoplasts
were resuspended in 1 ml of incubation medium (IM) consisting of
0.5 M mannitol, 0.11 M glucose, 0.055 M sucrose, 14 mM L-arginine, 10 mM MES, and 0.32% B5-Gamborg salt, pH 5.5. They
were mixed gently with 100 ^g sheared salmon sperm DNA and
100 fig of Hv41(-935)-IGN plasmid DNA (Robertson et al. 1995),
and the suspension was left undisturbed for 1 min. Protoplast
transfection medium (PTM) consisting of 17.3% (w/v) polyethylene glycol 6000 (PEG), 10 mM Tris-HCl pH 9.0, 0.67 M mannitol, and 0.133 M Ca(NO3)2 was filter sterilized. Three volumes
of PTM were added to the protoplasts, mixed, and left at room
temperature for 20 min with occasional swirling. Then 40 ml of
IM was added in 10 ml aliquots with 2 min between additions. The
protoplasts were collected by centrifugation for 2 min at 50 x g
and washed twice in 30 ml of IM. Pelleted protoplasts were resuspended in an appropriate volume (generally 15 ml for protoplasts
isolated from 150 grains) of IM containing 50 unit ml" 1 nystatin,
150 fig ml"' cefotaxime, 20 mM CaCl2, 1.5 fig ml" 1 aprotinin and
1.5 fig ml" 1 leupeptin. One milliliter of the protoplasts was aliquoted into flasks, and samples were incubated in the dark at 25°C
for 24 h in the presence of 0, 10 fiM ( ± ) ABA, 100 fiM (+)-RCA7a, (-)-RCA-7a, or (±)-RCA-7a in triplicate for each treatment.
Fluorimetric assays of GUS activity were performed in triplicate
for each sample as described by Jefferson (1988).
RNA isolation and Northern analysis—Aleurone layers were
prepared from half grains of barley (Hordeum vulgare L. cv.
Himalaya) as previously described (Chandler et al. 1984). For GA3induced a-amylase gene expression, the layers were incubated in 2
ml medium (50 mM arginine, 2mM CaCl2) containing GA3 (1
fiM), GA3 (1 fiM) with (±)-RCA-7a (100 fiM) or GA3 (1 /iM) with
(±)-ABA (10^M) for 16 h. For ABA-induced dehydrin gene expression assay, the layers were incubated in 2 ml medium (50 mM
arginine, 2 mM CaCl2) containing 10 fiM (±)-ABA or 100 fiM
(±)-RCA-7a for 16 h. Total RNA was isolated as previously described (Close et al. 1989). The RNA was dissolved in 100//I of
RNAse free water. For RNA gel blot analysis, 2 fig of total RNA
was fractioned on a formaldehyde agarose gel (1.0% [w/v]) and
344
ABA analog mimicking ABA action
transferred to Hybond-N (Amersham, Castle Hill, New South
Wales). The RNA was covalently linked to the membrane by baking in a vacuum oven at 80°C for 2 h before hybridization.
The membrane was hybridised with a barley high-pi a-amylase cDNA clone pHV19 (Chandler et al. 1984) or a mixture of dehydrin cDNA clones B8 and B18 (Close et al. 1989) that had been
labelled with 32 P-dCTP by oligo-labelling (Bresatec, Adelaide,
South Australia). After prehybridization, the membrane was then
hybridized to labelled probes, washed, and exposed to X-ray film
as described (Lang et al. 1997).
Growth retardation activity on cress—The test compounds
were evaluated for their growth retardation activity in cress
(Lepidium sativum L.) using a pot test in a greenhouse. Locallypurchased cress seeds were imbibed in running tap water for 1 h
and sown in a plastic pot at 25 seeds per pot (15 cm i.d.) filled with
garden loam. At the time of planting, (±)-RCA-7a or (±)-ABA
was applied to the soil surface as 5 ml of acetone-water (1 : 99,
v/v) solution at 0, 1.25, 2.5, or 5 kg ha" 1 (kg ha" 1 =0.59 mg
pot" 1 ). The plants were grown in a greenhouse maintained at
about 25-35°C under natural daylight conditions and watered daily. One week after the treatment, the number of emerged seedlings
was counted. Three weeks after treatment, growth retardation activity was evaluated by counting the number of emerged seedlings
and by weighing their total fresh weight. Three pots were used for
each treatment.
Growth retardation activity on rice—The test compounds
were also evaluated for pre-emergence growth retardation activity
in rice plants under paddy conditions. Plastic pots (15 cm i.d.)
were filled with paddy soil and excessively watered to create paddy
conditions. Grains of rice (Oryza sativa L. cv. Koshihikari) were
germinated in an incubator at 30°C in the dark for 24 h and then 5
seedlings were transplanted into each pot to a depth of 1 cm.
Aqueous solutions of the chemicals at the dosages of up to 10 kg
ha" 1 were added to the irrigation water on the following day, and
the pots were placed in the greenhouse. The level of flooding was
maintained at 3 cm above the soil surface throughout the experiments. The shoot length was measured 3 weeks after treatment.
Three pots were used for each treatment.
Results
Chemical structure of ABA and analogs—The structures of ( + ) - and (—)-RCA-7a are shown in Fig. 1, and
their stereochemistry was determined according to circular
dichroism analysis (Koreeda et al. 1973). S-(+)-ABA had a
first positive Cotton effect and negative second Cotton
effect (Milborrow 1967), which was very similar to the profile of Cotton effects shown by ( + )-RCA-7a (data not
shown). Therefore, the absolute configuration at C-l' of
(+)-RCA-7a was S, as in Fig. 1.
Inhibition of GArinduced a-amylase expression—The
inhibition of GA3-induced a-amylase expression is a welldemonstrated effect of ABA and has been used to determine whether chemicals have ABA-like activity (Chrispeels
and Varner 1966, Chandler et al. 1984, Hill et al. 1995).
The effects of ABA and analogs on the inhibition of a-amylase expression were tested at 0.3, 1, 3, 10, 30, and 100//M.
Concentrations at which a-amylase activity was inhibited
by 50% (I50) were estimated from the graph. In half grains,
( + )-ABA reduced GA3-induced a-amylase activity
120
Concentration (M)
Fig. 2 Inhibitory activity of ABA and analogs on GA3-inducible
a-amylase expression in embryoless barley half-grains. Halfgrains were incubated in 1 ^M GA3 with (+)-ABA, (+)-RCA-7a,
(-)-RCA-7a or with (±)-RCA-7a for 24 h at concentrations indicated for ABA and analogs. Half-grains incubated in 1 /xM GA3
were designated as having 0% inhibition, and those which had the
same activity level as the control (no GA3) were designated as having 100% inhibition. The mean and SE of triplicate samples are
presented.
by 50% (Fig. 2). (+)-RCA-7a was about three times more
active than (—)-RCA-7a, but less active than ( + )-ABA, requiring 3//M for the 50% inhibition. The (±)-RCA-7a
showed an activity between that of two enantiomers.
a-Amylase activity of grains treated with GA3 with
either 1 ^M ( + )-ABA or 3//M ( + )-RCA-7a was reduced
by approximately 50% (Fig. 3). When both ( + )-ABA and
(+)-RCA-7a were added together to the incubation medium at these concentrations, the a-amylase activity was inhibited by nearly 100%.
Next, the activity of RCA-7a was compared to ABA using aleurone protoplasts to measure the inhibitory activities
of ABA and ABA analogs on GA3-induction of a-amylase
expression (Fig. 4). Concentrations tested ranged from 0.01
to 10/iM. Figure 4 shows that I50 was 0.04^M for (+)ABA, while it was 0.4 nM for both ( + ) - and (±)- RCA-7a
and 3^M for (—)-RCA-7a. Surprisingly, all three compounds inhibited the a-amylase induction in protoplasts at
lower concentrations than in half-grains; thus they appear
to be more effective in protoplasts than in half-grains. The
relative order of potency was very similar to that in halfgrains;
(+ )-ABA > (+ )-RCA-7a > ( ± )-RCA-7a > ( - )RCA-7a.
Northern blot analysis with a-amylase cDNA—Several
studies using barley grains have indicated that total a-amylase mRNA accumulates in the presence of GA3 and that
ABA analog mimicking ABA action
100
345
B
rJf
(+VABA
(+VflCA-7a (+)-ABA +
(+H?CA-7a
Chemicals used
Fig. 3 InruMoryacu\dtyof(+>ABAand(+>RCA-7aonGArinducible
a-amylase expression in embryoless barley half-grains. Halfgrains were incubated in 1 //M GA3 together with either 1 fiM (+)ABA or 3/^M (+)-RCA-7a for 24 h. Half-grains incubated in 1
fiM GA3 were designated as having 0% inhibition, and those
which had the same activity level as the control (no GA3) were designated as having 100% inhibition. The mean and SE of triplicate
samples are presented.
ABA inhibits this accumulation (Chandler et al. 1984,
Robertson et al. 1995). We tested (±)-RCA-7a for its
ABA-like activity by Northern blot analysis of a-amylase
mRNA accumulation (Fig. 5, panel A). GA3 at 1 nM stimulated a-amylase mRNA accumulation, and a 10-fold excess
of (±)-ABA counteracted the induction by GA3. The abun120
„
100-
I
80
60
40(+J-ABA
(+)-RCA-7a
(-)-flCA-7a
• A - (+)-RCA-7a
10"
10"7
10*
Concentration (M)
Fig. 4 Inhibitory activity of ABA and analogs on GA3-inducible
a-amylase expression in barley aleurone protoplasts. Protoplasts
were incubated in 1/iM GA3 with (+)-ABA, (+)-RCA-7a, ( - ) RCA-7a or with (±)-RCA-7a for 24 h at concentrations indicated
for ABA and analogs. Protoplasts incubated in 1 fiM GA3 were
designated as having 0% inhibition, and those which had the same
activity level as the control (no GA3) were designated as having
100% inhibition. The mean and SE of triplicate samples are
presented.
I
••
Fig. 5 The effect of(+)-RCA-7a on the accumulation of a-amylase (panel A) and dehydrin (panel B) mRNA in barley aleurone
layers, a-amylase: Layers were treated as follows: cont: control;
GA: GA3 (1/iM); GA + (±)-ABA: GA3 (1/iM) + (±)-ABA (10
(xM); GA+(±)-RCA-7a: GA3 (1 ,uM)-l-(±)-RCA-7a (100 /M) for
16 h. Northern blot was hybridised with an a-amylase cDNA.
Dehydrin. Layers were treated as follows: cont, control; ABA, 10
HM (±)-ABA; (±)-RCA-7a, 100/iM (±)-RCA-7a for 16 h. Northern blot was hybridised with a dehydrin cDNA. Lower panels:
stained gel showing rRNAs.
dance of a-amylase mRNA was also reduced in aleurone
layers treated with 1 fiM GA3 plus 100 ,uM (±)-RCA-7a.
Thus the analogs counteracted GA3-induction of a-amylase
mRNA, but less effectively than did ABA.
Dehydrin induction by ABA and analogs—ABA is
known to mediate rapid physiological responses to water
stress such as stomatal closure (Zeevaart and Creelman
1988) as well as slower responses such as dehydrin synthesis
(Close et al. 1989, Robertson et al. 1995, Lang et al. 1997).
Since exogenous ABA induces the accumulation of dehydrin in unstressed plants (Close et al. 1989), ABA-like activity of chemicals can be examined by measuring dehydrin
gene expression. ABA up-regulated dehydrin gene expression was used to evaluate ABA-like activity of RCA-7a
(Fig. 5, panel B). When isolated aleurone layers were incubated in the presence of 10^M (±)-ABA, high levels of
dehydrin mRNAs accumulated within 16 h. The layers incubated with 100 fiM (±)-RCA-7a also accumulated dehydrin mRNA, but to a lesser degree.
The effect of RCA-7a on the induction of dehydrin
gene expression was also tested in aleurone protoplasts by
measuring dehydrin promoter activity in transient assays
(Fig. 6). (±)-ABA (10//M) increased GUS activity about
20-fold over control levels. (+)-RCA-7a, (-)-RCA-7a and
(±)-RCA-7a also increased the GUS activity significantly,
but were less active than (±)-ABA even at concentrations
10-fold higher than (±)-ABA. Therefore, RCA-7a induced
dehydrin gene expression in both aleurone protoplasts and
layers as did ABA. The order of effectiveness for these com-
346
ABA analog mimicking ABA action
Table 1 Growth retardation of rice plants by ABA and
RCA-7a under paddy conditions
1000
800-
Compound
BOOH
(±)-ABA
(±)-RCA-7a
o
I£
f
400
(Control)
t
control
ABA
(+)-RCA-7a
(-)-RCA-7a
100 |iM
100 (iM
10|iM
(+)-RCA-7a
100 uM
Chemicals used
Fig. 6 Activation of the dehydrin promoter by (+)-ABA and analogs. Promoter activity was measured by GUS reporter gene activity in barley aleurone protoplast transient assays. Protoplasts were
treated with control, 10//M (±)-ABA, 100 fiM (+)-, ( - ) - or ( + ) RCA-7a for 24 h.
pounds was the same as that in the inhibition of a-amylase
induction bioassay; ( + )-ABA>( + )-RCA-7a^(±)-RCA7 a > ( —)-RCA-7a. The effectiveness of (+)-RCA-7a in
barley aleurone layers was about 1/3 to 1/10 of that of
( + )-ABA in the inhibition of GA3-induced a-amylase expression and far less than 1/10 of that of (-t-)-ABA in activating the dehydrin promoter activity.
Growth retardation activity in the pot tests—For practical use of RCA-7a as a synthetic plant growth regulator,
the duration of its activity is very important. Therefore, the
A
100
100
B0
5.0
7.5±0.2
2.5
8.1±0.3
—
12.2±0.4
-o-
RCA-7a
-o-
ABA
I
growth retardation activity of RCA-7a in cress was examined in pot tests conducted in a greenhouse. Since both
( + )- and (-)-RCA-7a had ABA-like activities in the previous bioassays, RCA-7a was used as a racemic mixture in
the pot tests. Both (±)-ABA and (±)-RCA-7a at 5 kg ha" 1
completely inhibited the emergence of cress 1 week after
sowing (Fig. 7, panel A). At lower dosages, (±)-ABA was
slightly more active than (±)-RCA-7a. For example, at
1.25 kg ha~', (±)-ABA inhibited emergence by 72%, while
(±)-RCA-7a inhibited it by 50%. However, the inhibitory
activity of (±)-ABA decreased significantly by 3 weeks
after the treatment, as shown in Fig. 7, panel B. In contrast, (±)-RCA-7a fully maintained its inhibitory activity.
For example, at 1.25 kg ha""1, emergence inhibition by
(±)-RCA-7a decreased from 50% to 42%, while that by
(±)-ABA decreased from 72% to 7% after 2 weeks. Total
fresh weight of cress harvested 3 weeks after sowing is
shown in Fig. 7, panel C. The total fresh weight of cress
treated with 1.25 kg ha" 1 (±)-RCA-7a was 32% of that of
untreated control plants, whereas the total fresh weight
c
mn
1UU
60
5
:
3 60
5
-o-
RCA-7a
-o-
ABA
T
l
\
§
o>
12.1±0.4
3.9±0.2
10
10
Shoot length was measured 3 weeks after treatment. Data are
means ±SE (n=15).
200
^60
8
S
Shoot length
(cm)
Dosage
(kg ha" 1 )
S 40
UJ
20
0
20
§
•^^—a
3
4
Dose (kg/ha)
\
0
2
3
X
4
5
Dose (kg/ha)
Fig. 7 The inhibitory effect of (±)-ABA and (±)-RCA-7a on the emergence of cress seeds and on the total fresh weight of emerged
cress seedlings. The plants were treated at 1.25, 2.5, and 5 kg ha"'. The number of emerged seedlings were counted I week after sowing
(panel A) and 3 weeks after sowing (panel B). Fresh weight of seedlings 3 weeks after sowing (panel C) is expressed as percent of the
weight of plants without any hormone treatment.
ABA analog mimicking ABA action
of (±)-ABA-treated cress was 71% of that of the control. These results showed that (±)-RCA-7a retarded the
growth of cress seedlings more strongly than did ABA. For
both germination and fresh weight reduction, 2.5 kg ha" 1
(±)-RCA-7a retained full inhibitory activity even after 3
weeks, while (±)-ABA lost most of its inhibitory activity
during the same time period.
(±)-RCA-7a also showed strong growth retardation
activity in rice plants, as shown in Table 1. The mean shoot
length of rice seedlings treated with 2.5 kg ha" 1 (±)-RCA7a was significantly less than the control, and it decreased
further with an increase in the application dose to 5.0 and
10 kg ha" 1 . In contrast, the mean shoot length of plants
treated with (±)-ABA was the same as the control; thus
(±)-ABA appears to be totally inactive even at the highest
dosage (10 kg ha" 1 ) tested in this assay. (±)-RCA-7a also
showed a similar growth retardation activity on transplanted rice plants but to a lesser extent (data not shown).
Discussion
RCA-7a showed ABA-like activity in inhibiting GA3induced a-amylase activity and a-amylase mRNA accumulation. However, these results alone are not sufficient to
speculate that RCA-7a has the same mechanism of action
as ABA and interacts with an ABA receptor. RCA-7a may
be a general inhibitor of metabolic processes. Some phenyl
compounds are reported to inhibit plant growth, transpiration, or GA3-induced a-amylase synthesis, but they do not
mimic ABA in all the assays for ABA action (Bittner et al.
1977, Ladyman et al. 1988, Yoshikawa et al. 1992). Alternatively, RCA-7a may inhibit steps in signal transduction
between GA perception and the expression of a-amylase
gene by a mechanism different from ABA. For example,
okadaic acid, a protein phosphatase inhibitor, blocked
GA3-induced a-amylase production and also greatly reduced the accumulation of a-amylase mRNA, but did not
lead to the accumulation of ABA-inducible mRNA (Kuo et
al. 1996). Therefore, to determine whether or not RCA-7a
mimics ABA, we examined the activity of RCA-7a on ABA
up-regulated expression of the dehydrin gene. The results
of both promoter activity and Northern analyses indicated
that RCA-7a has a dehydrin induction activity, and the
combined results show that RCA-7a acts as an agonist of
ABA, not as a so-called general toxin. The ABA-like activity of RCA-7a suggests that the analog may have the same
mechanism of action as ABA and may be recognized as active ABA in in vitro assay systems. In in planta assay systems, which were carried out over a longer period than the
in vitro assay systems described above, whether or not
RCA-7a mimics ABA was not clear, but RCA-7a was
shown to have a more prolonged ABA-like effect than
ABA. RCA-7a is likely to be resistant against inactivation
in the inhibition of cress seed germination bioassay.
347
ABA is hydroxylated at the 8'-position and is readily
catabolized into PA, which is ineffective as an inhibitor in
cress germination tests (Gusta et al. 1992). Therefore, one
of the reasons for the activity decrease of ABA in this pot
test could be a decrease of ABA concentration in plants
due to the catabolism of ABA to PA, and the prolonged
effects of RCA-7a could be due to the blockage of this
catabolic inactivation. Similarly, 8'-methoxymethyl-ABA
(Todoroki et al. 1994) and 8'-trifluoromethyl-ABA (Kim et
al. 1995, Todoroki et al. 1995) are anti-catabolic analogs of
ABA, and their activities are more long-lasting than those
of ABA in the lettuce seed germination test. Nonadeuteroand methylene-ABA also showed more persistent activity
than ABA in cress seed germination test due to their slower
rates of metabolism (Lamb et al. 1996, Abrams et al. 1997).
In contrast to these analogs, the 8'-methyl group of RCA7a is not chemically protected from enzymatic oxidation.
Assuming that the longer-lasting effect of RCA-7a in in
planta assay could be due to the slower rates of metabolism, the greater bulkiness of the phenyl group of RCA-7a
than the side chain of ABA, which could make it difficult to
access the 8'-methyl group of RCA-7a by an enzyme which
potentially oxidizes the 8'-methyl group of ABA, might be
the main reason for its long-lasting effect. In the rice
growth assay, RCA-7a retarded shoot elongation, whereas
ABA did not. The mechanism of action of RCA-7a in this
assay is also not clear, but one of the reasons for this result
could be a reduced catabolic inactivation of RCA-7a.
We have demonstrated that an ABA analog RCA-7a,
which has a phenyl ring instead of a side chain in ABA, has
an ABA-like activity in various biological responses. Since
RCA-7a can be prepared by a facile and inexpensive "onepot" synthetic method on a large scale and has a longlasting effect, it may be suitable for production as a plant
growth regulator. Furthermore, it may be possible to use
RCA-7a to confer tolerance for stresses such as chilling injury, wounding and dehydration.
This work was supported by special coordination funds from
the Science and Technology Agency of Japan and by a Grant-inAid for the Encouragement of Young Scientists (no. 07760123)
from the Ministry of Education, Science, Sports and Culture of
Japan.
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(Received October 8, 1997; Accepted January 10, 1998)