Journal of Experimental Botany, Vol. 56, No. 421, pp. 2935–2948, November 2005
doi:10.1093/jxb/eri290 Advance Access publication 3 October, 2005
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Abscisic acid regulation of heterophylly in Marsilea
quadrifolia L.: effects of R-(2) and S-(1) isomers
Bai-Ling Lin1,*, Huei-Jen Wang1, Jang-Shiun Wang1,†, L. Irina Zaharia2 and Suzanne R. Abrams2
1
Molecular and Cell Biology Division, Development Center for Biotechnology, 101 Lane 169 Konning Street,
Hsichih 22143, Taiwan
2
Plant Biotechnology Institute, National Research Council, 110 Gymnasium Place, Saskatoon, Canada S7N 0W9
Received 4 March 2005; Accepted 17 August 2005
Abstract
The plant hormone abscisic acid (ABA) induces a
developmental switch in the aquatic fern Marsilea
quadrifolia, causing the formation of aerial type characteristics, including the elongation of petioles and
roots, a change in leaf morphology, the expansion of
leaf surface area, and the shortening of the internodes.
A number of ABA-responsive heterophylly (ABRH) genes
are induced early during the transition. Using optically
pure isomers of ABA, it was found that both the natural
S-(1)-ABA and the unnatural R-(2)-ABA are capable
of inducing a heterophyllous switch and regulating
ABRH gene expression. When dose responses are
compared, the unnatural ABA gives stronger morphogenic effects than the natural ABA at the same concentration, it is effective at lower concentrations, and
its optimal concentration is also lower compared with
the natural ABA. Deuterium-labelled ABA enantiomers
were used to trace the fate of applied ABA and to distinguish the applied compound and its metabolites
from the endogenous counterparts. In tissues, the
supplied (1)-ABA was metabolized principally to dihydrophaseic acid, while the supplied (2)-ABA was converted at a slower rate to 79-hydroxy abscisic acid.
Treatment with either enantiomer resulted in increased
biosynthesis of ABA, as reflected in the accumulation
of endogenous dihydrophaseic acid. Taken together,
these results suggest two distinct mechanisms of
action for (2)-ABA: either (2)-ABA is intrinsically active, or its activity is due to the stimulation of ABA
biosynthesis.
Key words: Abscisic acid, deuterated analogue, developmental
switch, enantiomer, gene regulation, heterophylly, Marsilea
quadrifolia, mass spectrometry.
Introduction
Heterophylly in aquatic plants serves an adaptive advantage, and has been a subject of investigation for fundamental mechanisms in plant morphogenesis (Gifford and
Foster, 1988; Steeves and Sussex, 1989; Trewavas and
Jones, 1991). Controlled by the developmental programme,
environmental factors or both, plants produce distinct
types of leaves corresponding to changes in water level
and the seasons. In the laboratory, heterophylly is affected
by cultural parameters. When conditions are otherwise in
favour of producing the submerged type morphology, an
exogenous supply of the plant hormone abscisic acid
(ABA) at an optimal concentration induces aerial type
characteristics (Zeevaart and Creelman, 1988; Lin and
Yang, 1999). This effect is seen in a number of phylogenetically divergent aquatic plants. In Marsilea quadrifolia,
heterophylly is also affected by gibberellins (Allsopp,
1962), phytochrome (Gaudet, 1963), and blue light (Gaudet,
1965; Lin and Yang, 1999).
This raises the question whether applied ABA mimics
environmental factors or it is truly the endogenous signal
for the heterophyllous switch (Trewavas and Jones, 1991).
It has been shown previously that the blue light signal was
not mediated by ABA, but that a change in culture medium
composition was (Lin and Yang, 1999). Others demonstrated that osmotic stress and high photon fluence caused
* To whom correspondence should be addressed. Fax: +886 2 2695 7674. E-mail: [email protected]
y
Present address: National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli 350, Taiwan.
Abbreviations: ABA, abscisic acid; ABRH, ABA-responsive heterophylly; DPA, dihydrophaseic acid; LC-ES-MS-MS, liquid chromatography-electrospray
ionization-tandem mass spectroscopy; PA, phaseic acid; 79-OH ABA, 79-hydroxy abscisic acid.
ª The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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2936 Lin et al.
similar effects that could be correlated with ABA (Goliber,
1989; Goliber and Feldman, 1989). However, low cell
turgor and ABA seemed to induce aerial morphology via
different mechanisms (Deschamp and Cooke, 1983, 1984).
Heterophylly is the most morphologically distinct effect
of ABA during the vegetative phase. In M. quadrifolia
ABA has differential effects on organ development that are
both qualitative and quantitative (Liu, 1984; Lin and Yang,
1999; Hsu et al., 2001). It dramatically promotes growth in
leaves and roots, but inhibits growth in the internodes. The
responses are progressive and dose-dependent. ABA has to
be continuously present for the completion of the morphogenesis; when ABA is removed, growth and development
return to the default mode (Hsu et al., 2001).
Growth analyses indicate that, for heterophyllous induction, there is a developmental window of responsiveness; only the primordial tissues are affected by ABA (Hsu
et al., 2001). Since all organs of the mature M. quadrifolia
plant are derived from the shoot apical meristem, the tissues
responsive to ABA are localized in the shoot apex. From
shoot apices, 24 ABA-responsive early genes, the ABRHs,
were isolated; seven of them are immediate early genes
(Hsu et al., 2001). The ABA-induced growth characteristics and the ABRHs provide useful developmental and
molecular markers for the dissection of ABA-mediated
signal transduction.
Heterophylly studies in M. quadrifolia have been performed to date using commercially available ABA. This
ABA is a product of chemical synthesis and is racemic, i.e.
consisting of a 1:1 mixture of the enatiomers, namely the
natural hormone, S-(+)-ABA, and its mirror image, R-(ÿ)ABA, which has not been found in plant tissues. In
numerous previous studies the unnatural enantiomer has
been observed to elicit biological activity in some but not
all ABA responsive processes. Table 1 summarizes the
researchers’ interpretation of the results of the biological
activities that have been examined using optically pure
enantiomers. It shows that the structural requirement of
ABA action varies depending on the experimental system,
the developmental stage, the cell type, the concentration
used, and the time-course. For example, (+)- and (ÿ)-ABA
were found to be equally active in inhibiting germination of
wheat embryos (Milborrow, 1970; Walker-Simmons et al.,
1992; Rose et al., 1996), for inducing stomatal closure in
barley leaves (Cummins et al., 1971), and for interfering
with the synthesis and release of a-amylase induced by
gibberellin in barley half seeds (Sondheimer et al., 1971).
Unnatural (ÿ)-ABA was found less effective in inhibiting
the germination of barley seeds and excised ash embryos
(Sondheimer et al., 1971), in increasing freezing tolerance
in bromegrass suspension cell culture (Robertson et al.,
1994), and in inhibiting the growth of maize suspension
cells (Balsevich et al., 1994b). Such is also the case for the
regulation of gene expression. For example, both (+)- and
(ÿ)-ABA were found to induce the expression of dhn (rab)
and group 3 lea in imbibed wheat embryo, but the Em gene
was only induced by the natural ABA (Walker-Simmons
et al., 1992). In Arabidopsis thaliana cell cultures (+)ABA and not (ÿ)-ABA induced RAB18 gene expression
(Jeannette et al., 1999).
In a kinetic study of maize leaf elongation, Cramer et al.
(1998) found that the ABA effect on growth inhibition was
reversible. The leaf elongation rate was highly correlated
with the steady-state internal ABA concentration of the
growing zone. When compared on the basis of external
concentration, the (ÿ)-enantiomer of ABA had much less
effect than (+)-ABA due to its low level of accumulation.
From this analysis, these authors concluded that the leaf
elongation rate was equally sensitive to internal concentrations of (+)- or (ÿ)-ABA.
The concentration of ABA in plant tissues is the result of
a combination of transport, biosynthetic, and catabolic
processes (Cutler and Krochko, 1999). As shown in Fig. 1,
the metabolism of natural ABA (1) occurs principally
through oxidation of the 89-methyl group, resulting in
89-hydroxy ABA (2), which rearranges to phaseic acid (3,
PA). Further reduction of PA leads to dihydrophaseic acid
(4, DPA). Other metabolic processes include conjugation of
the acids as glucose esters, as well as the hydroxylation of
79-methyl (Hampson et al., 1992) and the 99-methyl group
of the ABA ring (Zhou et al., 2004). In plants, unnatural
ABA (5) is oxidized to (ÿ)-79-hydroxy abscisic acid (6, 79OH ABA) as the major metabolic product (Hampson et al.,
1992). While the biological activity of ABA is well documented, the effect of its metabolites has been less studied,
due to the scarcity of the compounds. However, there are
indications that the initial catabolites of ABA can have
pronounced hormonal activity (Hill et al., 1995; Zou et al.,
1995).
These results prompted an examination of the effects of
optically pure isomers of ABA on heterophylly in M.
quadrifolia. The main objective was to unravel the critical
issue of structural requirements in ABA signal transduction, to relate effects of pure enantiomers to gene expression in order to determine which genes are involved in
heterophylly. Previous studies showed that the continuous
presence of ABA in the culture medium was critical for the
maintenance of the heterophyllous switch (Hsu et al.,
2001), and so determining the ABA concentrations in the
tissues and the fate of supplied enantiomers may provide
clues to the underlying mechanism. By feeding deuteriumlabelled ABA enantiomers (Abrams et al., 2003) and
tracing their fate in plant tissues, it was possible to distinguish the applied ABA from the endogenous hormone
and to measure the changes in endogenous concentrations
of the hormone and the metabolites. This is the first paper
that reports the quantification of both supplied and endogenous ABA and its metabolites in plant tissues that could
be correlated with a developmental switch induced by
exogenous ABA.
Table 1. Comparison of biological effects of exogenously applied (+)- and (ÿ)-ABA
Effect
Biological activity
Equally effective
Inhibition
GA-induced synthesis and
release of a-amylase
GA-induced synthesis of
a-amylase
Synthesis and release of
a-amylase
Germination
Concentration used
(lM)
Reference
Embryo-free half seed
Barley
0.1–1
Sondheimer et al., 1971
Aleurone layers
Barley
0–20
Aleurone protoplasts
Embryo-containing half seed
Barley
0.1–1
Abrams et al., 1993;
Hill et al., 1995
Hill et al., 1995
Sondheimer et al., 1971
Dormant seed embryo
Wheat
1 and 10
Isolated seed embryo
Coleoptile
8–16 fronds plantlets
Microspore derived embryo
Suspension culture cells
Barley
Wheat
Duckweed
Oil seed rape
Carrot
1–5
?
0.125
1–10
0–30
Rose et al., 1996;
Walker-Simmons et al., 1992
Abrams et al., 1993
Milborrow, 1970
Smart et al., 1995
Wilmer et al., 1998
Windsor et al., 1994
Suspension culture cells
Amaranthus tricolor
0–35
Bianco-Colomas et al., 1991
Explant
Cell suspension culture
Callus
Cotton
Potato, Arabidopsis
Craterostigma
plantagineum
Oilseed rape
Oilseed rape
White spruce
White spruce
0.01–1.0 lg petioleÿ1
50
20
Sondheimer et al., 1971
Windsor and Zeevaart, 1997
Chandler et al., 1997
10
1–10
15
15 (1–3 h)
Wilmer et al., 1998
Wilmer et al., 1998
Dong and Dunstan, 1996
Dong and Dunstan, 1997
Wheat
10
Walker-Simmons et al., 1992
Suspension cell
Excised axis
Suspension cell
Leaf
8–16 fronds plantlets
Bromegrass
Bean
Bromegrass
Barley
Duckweed
25
17
25
0.1
0.125
Wilen et al., 1996
Sondheimer et al., 1971
Wilen et al., 1996
Sondheimer et al., 1971
Smart et al., 1995
Dormant embryo
Non-dormant embryo
Seed
?
?
0.3–5
Sondheimer et al., 1971
Sondheimer et al., 1971
Nambara et al., 2002
0.1–100
1, 2, 5 and 10
0.1–100
0.05–1
10
3.8–38
0.1–100
0.1–100
Gusta et al., 1992
Toorop et al., 1999
Nakano et al., 1995;
Shen et al., 1995;
Todoroki et al., 1994, 1995a, b
Hays et al., 1996
Balsevich et al., 1994b
Sondheimer et al., 1971
Shen et al., 1995
Nakano et al., 1995;
Todoroki et al., 1994, 1995a, b
Microspore-derived embryo
Microspore-derived embryo
Immature somatic embryo
Embryogenic suspension
culture
Embryo germination
Germination
Germination
Germination
Seed
Seed
Seed
White ash
White ash
Arabidopsis wild type,
chilled
Cress
Tomato, lettuce
Lettuce
Germination
Growth
Growth
Growth
GA3-induced synthesis and
release of a-amylase
Microspore-derived embryo
Suspension cell
Seedling
Seedling
Embryo-free half seed
Brassica napus
Maize
Barley
Radish
Barley
2937
Plant
ABA regulation of heterophylly in Marsilea quadrifolia
Germination
Growth
Growth
Growth
Carrier-mediated uptake of
(+)-ABA
Carrier-mediated uptake of
ABA
Promotion
Abscission assay
ABA 89-hydroxylase induction
Desiccation tolerance, callus
viability, LEA gene expression
Elongase activity
Erucic acid content
Gene expression- LMW hsps
Gene expression- non-Lea-like
genes: PgEM5, PgEMB23
Gene expression- dhn(rab),
lea (group 3)
Gene expression- dehydrin
Growth
Protein accumulation- RAB
Stomatal closure
Turion induction
(ÿ)-ABA less effective
Inhibition
Germination
Germination
Germination
Target tissue/stage
2938 Lin et al.
Table 1. (Continued)
Effect
Promotion
Biological activity
Target tissue/stage
Plant
Concentration used
(lM)
Reference
Elongation
Elongation
Third leaf
Second leaf
Maize
Rice seedlings
1–10
0.1–100
Stomatal opening
Epidermal strips
Spiderwort
10ÿ4–1
Transpiration rate
Transport of (+)-ABA by
uptake carrier
Stomatal closure
Freezing tolerance
Seedling
Suspension cell cultures
Wheat
Barley
1–1000
20
Cramer et al., 1998
Nakano et al., 1995;
Todoroki et al., 1994, 1995a, b
Nakano et al., 1995;
Todoroki et al., 1994, 1995a, b
Rose et al., 1996
Perras et al., 1994
Seedling
Suspension cell
Barley
Bromegrass
8
75; 25 and 75
79 and 89-hydroxylase induction
Cell suspension cultures
200
Gene expression- wheat Em::uidA
Gene expression- wheat Em::uidA
Gene expression- Lea-like genes:
PgEMB12, PgEMB14, PgEMB15
Gene expression- non-Lea-like
genes: PgEM5, PgEMB23
Gene expression- napin
Gene expression- napin, oleosin
Gene expression- GUS activity
Gene expression- GUS activity
Somatic embryo
Protoplast
Embryogenic suspension
culture
Embryogenic suspension
culture
Microspore-derived embryos
Microspore-derived embryos
Aleurone protoplasts
Fresh seeds, developing
transgenic seeds
Suspension cells
Black Mexican
sweet corn
White spruce
White spruce
White spruce
Transport by uptake carrier
(ÿ)-ABA no effect
Inhibition
Germination
Stomatal opening
Reduction
Promotion
Osmotic potential
Gene expression- Em
Gene expression- WCS120 gene
Gene expression- RAB18 gene
pH transition in medium
Sucrose uptake
Maltose accumulation
Radial water transport
Maturation
Excised embryo
Isolated leaf epidermal strips,
intact leaves
Suspension cell
Dormant seed embryo
germination
Suspension cell culture
Suspension cell culture
Suspension cell
Suspension cell
Suspension cell
Root
Somatic embryo
Cummins and Sondheimer, 1973
Robertson et al., 1994;
Wilen et al., 1996
Cutler et al., 1997
Bommineni et al., 1998
Dong et al., 1994
Dong and Dunstan, 1997
White spruce
24
>100
15 (3–48 h)
10 and 100 (24 h)
10 and 100 (24 h)
B. napus
B. napus
Barley
Tobacco
1, 10 and 30
1–30
0–10
0–1
Wilen et al., 1993
Hays et al., 1996
Hill et al., 1995
Jiang et al., 1996
Carrot
0–30
Windsor et al., 1994
Yellow cedar
Commelina comunis,
Tropaeolum majus
Bromegrass
Wheat
1
100
Schmitz et al., 2002
Milborrow, 1980
25
10
Wilen et al., 1996
Walker-Simmons et al., 1992
Barley
Arabidopsis
Maize
Bromegrass
Bromegrass
Maize
White spruce
20
10
10
25
25
0.1
15
Perras et al., 1994
Jeannette et al., 1999
Balsevich et al., 1994b
Wilen et al., 1996
Wilen et al., 1996
Sauter et al., 2002
Dunstan et al., 1992
Dong and Dunstan, 1997
ABA regulation of heterophylly in Marsilea quadrifolia
2939
(Jepson et al., 1991). To be sure there is no contaminated DNA, the
RNA preparation was further treated with DNase I. First strand
cDNAs were synthesized with oligo(dT15) priming, serially diluted in
water by a factor of ten, and then used as templates for PCR. The
gene-specific primers previously identified (Hsu et al., 2001) were
used for the detection of gene expression. PCR was carried out
according to Sambrook et al. (1989), using 95 8C for 30 s, the Tm of
gene-specific primers as the annealing temperature for 45 s, and 72 8C
for 45 s, repeated for 35 cycles, then followed by 72 8C for 5 min. The
RT-PCR products were resolved in 4% agarose gels (3% NuSieve
and 1% SeaKem, FMC, Newport, Maine, USA), and the ethidium
bromide-stained gel patterns were analysed in the Alpha Imager 1200
Documentation and Analysis System (Alpha Innotech, San Leandro,
California, USA). For comparing the level of specific gene transcripts
in shoot apices from plants in various treatments, the RT-PCR and gel
analysis were carried out in batches, with the PCR amplification
performed in serially diluted first strand cDNAs. A master reagent
mix was prepared for each step of these analyses. Only those batches
of RT-PCR showing consistent results in serially diluted templates
were considered valid for determining the regulation pattern of gene
expression. The comparative RT-PCR results were confirmed in at
least two separate runs with each RNA sample and using at least two
independent RNA sources.
Fig. 1. ABA and the main hydroxylated ABA metabolism products.
Materials and methods
ABA enantiomers
The work related to the chemical preparation of ABA and ABA
metabolites was carried out at the Plant Biotechnology Institute,
National Research Council, Saskatoon, Canada. Thus, S-(+)-ABA
and R-(ÿ)-ABA were chemically synthesized and resolved into
optically pure form by preparative HPLC resolution of racemic
methyl abscisate followed by hydrolysis of the resolved esters as
previously described (Dunstan et al., 1992). The (+)- and (ÿ)-[4,5d2]-ABA used in the feeding experiment, as well as the labelled
internal standards used in liquid chromatography-electrospray ionization-tandem mass spectroscopy (LC-ES-MS-MS) analysis were
either obtained by chemical synthesis (Abrams et al., 2003) or
isolated as biotransformation products of ABA from maize cell
suspension cultures (Zaharia et al., 2005).
Plant material and treatment
Aseptic cultures of Marsilea quadrifolia L. were established from
sporocarps and propagated by subculturing into two-node segments
(Liu, 1984; Lin and Yang, 1999). All plants used in this study were
clones of the same plant. The basal medium is a liquid mineral medium
(Laetsch and Briggs, 1961) supplemented with 3% sucrose. In all
treatments and the control, the pH in the growth medium was adjusted
to 6.4. ABA was added from a 10003 stock to the culture medium to
reach the indicated final concentration. The treatment of ABA was
applied 1 week after subculture. For the study of removal kinetics,
plants were treated with ABA for 2 weeks, then the culture medium
was removed and replaced with fresh basal medium without ABA.
Plants were then allowed to grow for another 10 d. All cultures were
kept in a growth room at 25 8C with a 16 h photoperiod. Illumination
was provided with fluorescent tubes emitting near sunlight spectrum
(FL40D-EX, Mitsubishi, Tokyo, Japan) at 40 lmol mÿ2 sÿ1.
RNA extraction and comparative RT-PCR
Total RNA was extracted from shoot apices using urea extraction
buffer (Chen and Dellaporta, 1994), and precipitated in 2 M LiCl
Analysis of endogenous and applied ABA and
ABA metabolites
This work was carried out at the Plant Biotechnology Institute,
National Research Council, Saskatoon, Canada. Samples were taken
each week during the 3-week incubation period. In the treatments and
the control, three replicates were sampled, each with shoot apices
pooled from eight plants. Plants were rinsed with sterile deionized
water. After gently removing the excess water, the apex was excised
from each plant and weighed. Samples were homogenized with a fine
glass rod in liquid N2. To each sample a 40 ll mixture of water:
acetonitrile (1:1, v/v) containing d4-ABA, d3-PA, d4-79-OH ABA and
d3-DPA (each at a concentration of 0.25 ng llÿ1) was added. Further,
a 1 ml mixture of isopropanol:glacial acetic acid (99:1, v/v) and
a small magnetic stir bar were added to each sample. After 12 h of
stirring in the dark, at room temperature, the samples were centrifuged at 1500 g for 10 min, the supernatant was transferred to a vial,
and a second portion of 1 ml extraction solvent mixture was added.
After stirring in the dark for another 4 h, the samples were centrifuged
and the supernatant was combined with the previous one. The organic
extract was dried under reduced pressure, then redissolved in 100 ll
methanol:glacial acetic acid (99:1, v/v) followed by 900 ml of
aqueous 1% glacial acetic acid. This mixture was further cleaned on
the OASIS cartridge (Waters OASIS extraction cartridge HLB 1 cc).
The fraction containing ABA and ABA metabolites was eluted with
1.5 ml methanol:water:glacial acetic acid (80:19:1, by vol.) and then
evaporated to dryness. The final residue was dissolved in 200 ll
(23100 ll portions) mixture of water:acetonitrile (80:20, v/v) with
0.07% glacial acetic acid, and then subjected to LC-ES-MS-MS
analysis, according to Ross et al. (2004) and Feurtado et al. (2004).
For statistical analysis, the significance of the differences between
sample means was calculated using the paired t-test.
Results
Both the natural (+)-ABA and the unnatural (ÿ)-ABA
induce heterophyllous switch and regulate ABRH
gene expression
In the authors’ laboratories, the cultural conditions were
adjusted to sustain the formation of the submerged type of
2940 Lin et al.
morphology (Fig. 2A). For the induction of heterophyllous
transition, the optimal concentration of the commercial
racemic ABA was 1 lM (Liu, 1984; Lin and Yang, 1999).
Initially, the optically pure ABA was tested at this concentration, and it was found that both natural (+)-ABA and
unnatural (ÿ)-ABA are capable of inducing the developmental switch (compare Fig. 2A, B, and C). Typical aerial
phenotypes are produced, the petioles and the roots elongate, the internodes shorten, the new leaves have expanded
surface areas, and their morphology resembles four-leaf
clover. Similar to the racemic ABA, the (+)- or (ÿ)-ABA
does not affect that part of the plant already developed prior
to the treatment; the effects are only seen in the newly
emerged organs, and are progressively stronger during the
development. However, following the initial growth stimulation in the leaves and roots, prolonged ABA treatment
triggers senescence, leading to developmental arrest and the
transition to the reproductive phase. This is evident in the
yellowing of the tissues, production of fewer nodes, and
sporocarp formation in the ABA treated plants (Fig. 2B, C).
In accord with the morphogenic responses, both ABA
enantiomers also regulate the early responsive ABRH gene
Fig. 2. The effects of the optically pure ABA enantiomers on heterophylly in M. quadrifolia. (A) An untreated plant. (B) A plant treated with 1 lM
S-(+)-ABA. (C) A plant treated with 1 lM R-(ÿ)-ABA. In (B) and (C), the plants were grown in basal medium for 10 d, then treated with ABA for
4 weeks. Note that both ABA enantiomers induced heterophyllous switch. The R-(ÿ)-ABA (C) had stronger effects than the S-(+)-ABA (B), resulting
in longer petioles and roots, highly clustered nodes, as well as earlier leaf senescence and growth arrest. Arrowheads indicate the position of the shoot
apex when ABA was added to the culture medium. Scale bars=1 cm.
ABA regulation of heterophylly in Marsilea quadrifolia
expression (Fig. 3). A number of ABRHs were examined,
including the immediate early genes, ABRH1, ABRH3,
ABRH4, ABRH5, and ABRH6, and the early secondary genes,
ABRH10, ABRH12, ABRH18, ABRH19, and ABRH24. These
genes are expressed at different levels and are regulated to
different extents. Both enantiomers show comparable levels
of regulation in most genes (Fig. 3). The encoded proteins
include homologues of transcription factors, ABRH4,
ABRH5, and ABRH12, homologues of signalling molecules, ABRH10 and ABRH18, a homologue of a membrane
protein, ABRH6, and the chloroplast encoded proteins,
ABRH19 and ABRH24 (Hsu et al., 2001). The regulation
of an array of genes and the variety of gene identity, including
regulatory and metabolic genes, suggest that both ABA
enantiomers are associated with triggering the heterophyllous
response in the morphogenic programme.
2941
In these studies, all the plants used were clones of the
same plant, propagated from axillary buds of two-node
segments (Liu, 1984; Hsu et al., 2001). At the time of
subculture, the axillary buds often already contained the
organ primordia for one to two nodes that, in response to
ABA, develop into intermediate morphology. To minimize
the residual effect on morphogenesis carried over in these
primordia, plants were routinely used that developed
three nodes, i.e. in these growth conditions 1 week after
subculture (see the parts to the left of the arrowhead in
Fig. 2B and C, and the nodes designated minus numbers in
Figs 4–6).
Time-course of growth responses: Following ABA application, the plants produce new nodes bearing organs with
striking changes in lengths compared with those existing
before the treatment and in untreated plants (Figs 2, 4).
The unnatural (ÿ)-ABA has stronger effects than
the natural (+)-ABA
Not only could both enantiomers induce the heterophyllous
switch, but it was also observed that, initially, the growth
responses of the plants were stronger when treated with
(ÿ)-ABA as compared to (+)-ABA (compare Fig. 2B and
C), although later the differences appeared to be less pronounced. Therefore a series of growth kinetics studies was
carried out to examine the time-course, the effect of ABA
removal, and the dose–response, using as references length
measurements of the various organs, including petioles,
internodes, and roots.
Fig. 3. Both the natural (+)-ABA and the unnatural (ÿ)-ABA regulate
the early responsive ABRH gene expression in the shoot apices of
M. quadrifolia. Plants were treated for 1 h with 1 lM ABA. ABA regulation is confirmed by a consistent pattern in comparative RT-PCR analyses using serially diluted RNA templates. Shown are representative gel
images of RT-PCR products. Clone C3-6 is not regulated by ABA and
serves as a control (Hsu et al., 2001). The RNA templates used are
150 ng, 15 ng, and 1.5 ng, respectively in (A), (B), and (C). The total
RNA templates are shown in a gel image in (D). C, control, (+)=(+)ABA-treated, (ÿ)=(ÿ)-ABA-treated.
Fig. 4. Time-course analyses of the effects of ABA enantiomers on
petiole elongation. (A) Measurements after 1 week of treatment. (B)
Measurements after 2 weeks of treatment. (C) Measurements after 3
weeks treatment. The concentration used was 1 lM. Arrows indicate the
position of the shoot apex at the time of ABA application, and the leaf
positions are labelled with reference to the treatment, i.e. 1 being the first
leaf produced after the treatment, 2 being the second, etc. Note
a progressive increase in petiole length during ABA treatment, and
(ÿ)-ABA has a stronger effect than (+)-ABA at each node. Immature
leaves in young nodes are not shown, therefore a smaller number of
leaves in the ABA-treated plants indicates an effect on growth arrest.
2942 Lin et al.
Fig. 6. Dose-response of petiole elongation to exogenously supplied
ABA enantiomers. (A) The responses to (+)-ABA. (B) The responses to
(ÿ)-ABA. Plants were grown in basal medium for 1 week, then treated
with ABA at the indicated concentration for 3 weeks. Immature leaves are
not shown.
Fig. 5. Growth kinetic responses to ABA application and removal. (A)
The effects on petiole growth. (B) The effects on internode growth. (C)
The effects on root growth. Plants were grown in basal medium for
1 week, treated with 1 lM of either ABA enantiomer for 2 weeks, then
allowed to grow in fresh basal medium for another 10 d. The organs were
measured at the end of the experimental period. A downward arrow
indicates the position of the shoot apex at ABA application, and an
upward arrow indicates that at ABA removal. ABA application induces
aerial type characteristics, i.e. longer petioles and roots, and shorter
internodes, whereas ABA removal reverses the effect. The effects are
progressive upon ABA addition and removal.
Figure 4 gives the measurements of petiole growth. The
effects are seen with both (+)-ABA and (ÿ)-ABA, but
the growth rates are higher in (ÿ)-ABA. Table 2 compares
the linear regression fit data for petiole growth curves,
showing at least 2-fold difference in growth rate between
treatments with (+)- and (ÿ)-ABA at all time points.
The time-course study also shows that, in (ÿ)-ABA
treatments, the growth arrest has an early onset, such that
fewer leaves are produced, compared with the untreated
plants and those treated with (+)-ABA. While the growth
persists in (+)-ABA treatments, some of the leaves produced later in (+)-ABA reach the length of the earlier leaves
produced in (ÿ)-ABA (Fig. 4). Hence it appears that the
difference in the growth effects of the two ABA enantiomers becomes less distinct with prolonged treatment. The
effects on root elongation and internode shortening are
similarly progressive, as shown below.
The time-course of ABA effects was observed in more
than a dozen experiments, either with the (+)- and (ÿ)-ABA
alone, or for a side-by-side comparison with the effects of
their metabolites (H-J Wang, B-L Lin, unpublished results).
Although the absolute values of organ measurements varied
within a range, the progressively increasing or decreasing
patterns of ABA responses were seen repeatedly.
Reversibility kinetics: It has previously been found that
ABA has to be present continuously in order to complete
the morphogenesis for heterophyllous switch (Hsu et al.,
2001). When ABA is removed, intermediate morphology
appears in organs that were already formed, yet still
developing during the transition. To understand the action
of optically pure ABA isomers, the growth kinetics responding to ABA removal were studied. Figure 5 shows
that the progressive effect of ABA on organ development
not only occurs during ABA treatment, but follows ABA
removal as well. Moreover, the unnatural ABA has a distinctly stronger effect than the natural ABA on petiole
growth (Fig. 5A). The rates of increase and decrease
following ABA addition and removal, respectively, are
ABA regulation of heterophylly in Marsilea quadrifolia
3–4-fold higher in the treatments with (ÿ)-ABA than in
those with (+)-ABA (Table 2).
The growth effects on internodes and roots are less
distinguishable between the ABA enantiomers (Fig. 5B,
C). The nodes are essentially clustered at the shoot apex, and
the internode lengths are calculated from the total length
of the rhizome fragment. Root growth extends for a long
period of time and has been observed to continue for a month
(Y-C Chang, R-W Chen, B-L Lin, unpublished results).
During the experimental period in this study, 2 weeks for
ABA treatment and 10 d after ABA removal, most roots did
not complete growth to reach the maximal length.
Dose responses: The progressive and differential responses
of the organs suggest a difference in the sensitivity of various developmental stages and of the tissues. This, together
with the different intensity in the responses between
ABA enantiomers, prompted an examination of the dosage
effects. Concentrations spanning five orders of magnitude,
from 10ÿ8 M to 10ÿ4 M, were used. Organ measurements
2943
were taken at the end of a 4-week treatment. Figure 6 shows
that (+)-ABA was effective in promoting petiole elongation
from 10ÿ6 M to 10ÿ4 M, with 10ÿ5 M being the optimal concentration. Whereas (ÿ)-ABA was effective from 10ÿ8 M
to 10ÿ4 M, and the optimal concentration was between
10ÿ7 M and 10ÿ6 M.
Table 3 summarizes the dose responses. In each organ,
the effective concentration and the optimal concentration of
(+)- and (ÿ)-ABA differ by one to two orders of magnitude.
The unnatural ABA is effective at lower concentrations, the
effective range is wider, and the optimal concentration is
lower, compared with natural ABA. A low dosage effect
was also noticed, i.e. the opposite response is produced at
a concentration between the ineffective concentration and
the lower end of the effective range. Among the organs, the
root responds at lower concentrations than the leaf and the
rhizome. In addition, the optimal concentration in the root
shifts to a different level during the course of development,
indicating biphasic responses.
Table 2. Comparison of growth rates of petioles in ABA-treated plantsa
Treatment
Time-course
Control
(+)-ABA
(ÿ)-ABA
b
Reversibility kineticsc
Week 1
Week 2
Week 3
0.2660.27
0.5360.15
0.6060.16
4.9561.84
5.0161.89
5.5662.58
9.4064.18
13.6361.34
16.0362.26
Application
Removal
0.5160.03
0.3060.09
3.2860.20
ÿ3.6560.33
13.9061.22
ÿ11.4060.64
a
The slopes of the growth curves are shown as mean 6SE. The time interval for node development and leaf emergence is similar throughout the
course of development in the treatments and the control. The growth rate was calculated using node development as a time unit.
b
The lengths of the petioles were measured at the end of each week. Paired t-test results for the difference in growth rates between the groups treated
with (+)- and (ÿ)-ABA are P <0.1 for week 1, P <0.02 for week 2, and P <0.001 for week 3 (n=3).
c
Plants were treated with 1 lM ABA for 2 weeks, then in fresh basal medium without ABA for 10 d. Petiole length was measured at the end of the
experimental period. Paired t-test results for the difference in growth rates between (+)- and (ÿ)-ABA treated groups are P <0.005 for ABA application,
and P <0.007 for ABA removal (n=3).
Table 3. Comparison of the effective concentrations of (+)-ABA and (ÿ)-ABA on the induction of heterophyllous switch in
M. quadrifolia
Plants were treated with the optically pure ABA enantiomer at various concentrations, ranging from 10ÿ8 M to 10ÿ4 M. The criteria of heterophyllous
induction include the change in leaf morphology, the elongation of the petiole and the root, and the shortening of the internode. The measurements of
the organs were taken after 3 weeks of treatment.
Organ
ABA isomer
Concentration (M)
Ineffective
Petiole
Internode
Root
a
ÿ8
Low dosage effecta
ÿ7
Effective range
ÿ6
ÿ4
(+)
(ÿ)
(+)
(ÿ)
(+)
10
–
10ÿ8
–
10ÿ8
10
–
10ÿ7
10ÿ8
–
10 ;10
10ÿ8;10ÿ4
10ÿ6;10ÿ4
10ÿ7;10ÿ4
10ÿ7;10ÿ4
(ÿ)
–
–
10ÿ8;10ÿ4
Optimal
10ÿ5
10ÿ7;10ÿ6
10ÿ5;10ÿ4
>10ÿ7 c
Earlyd
10ÿ5
Earlyd
10ÿ7;10ÿ6
b
b
b
Lated
10ÿ6
Lated
10ÿ5;10ÿ4
The responses are the opposite, i.e. the shortening of the petiole and the root, and the elongation of the internode.
The effects of these concentrations are not significantly different.
c
The nodes are clustered at the shoot apex, and the internode lengths were calculated by dividing the total length by the number of nodes.
d
Biphasic responses, with distinct optimal concentration in each phase.
b
b
2944 Lin et al.
Applied (ÿ)-ABA accumulates in the heterophyllous
responsive shoot apices
Feeding of labelled compounds and mass spectrometric
studies were carried out to understand the correlation between
growth response and ABA concentration in the heterophyllous responsive shoot apices. The scheme to synthesize
optically pure (+)- and (ÿ)- ABA analogues was developed
previously, with deuterium atoms replacing the protons on the
trans double bond of the side chain, positions which are not
involved in known metabolism processes and are not exchangeable in the plant or the medium (Abrams et al., 2003).
These compounds are useful for tracing the fate of exogenously applied ABA and also enable exogenous ABA to be
distinguished from endogenous ABA. To correlate with the
developmental studies, plants were supplied with 1 lM
of either isomer of deuterated ABA, and analysed weekly
for 3 weeks. Measurements of ABA and ABA metabolites
are listed in Table 4. In addition, it was found that, after
a week, the added ABA in the medium reached a relatively
steady concentration, which persists during the 3-week
period of the experiments: the contents of (+)-[4,5-d2]-ABA
and (ÿ)-[4,5-d2]-ABA in the medium were similar, 1466
35 ng mlÿ1 (0.55 lM) and 138616 ng mlÿ1 (0.52 lM),
respectively. As expected, because the experiments were
conducted in the light, the ABA in the medium equilibrated
to a 1:1 mixture with 2-trans ABA.
In both treated and untreated plants, it was found that the
concentration of endogenous ABA in the heterophyllous
responsive shoot apex tissues was within a 2–3-fold difference, in the range of 0.1–0.3 nmol gÿ1 FW (Table 4).
Paired t-tests comparing measured values of endogenous
ABA between the experimental groups in each week and
between the same groups in different weeks showed no
significant differences (P <0.05, n=3) except in week 2 the
value in (+)-ABA treated tissues was significantly lower
than that of the control (mean difference 3.50), and in week 3
the values in both (+)- and (ÿ)-ABA treated tissues were
significantly higher than that of the control (mean difference
2.58 and 4.08, respectively). In the first two weeks, the
concentration of applied (+)-[4,5-d2]-ABA in these tissues
was in the concentration range of the endogenous ABA, and
in the third week, it became 8–10-fold, reaching 1.2 nmol
gÿ1 FW (mean difference 8.14, P <0.01, n=3). By contrast,
starting in the first week, the concentrations of applied (ÿ)[4,5-d2]-ABA in the tissues were significantly higher than
the concentrations of endogenous ABA (P <0.01, n=3), and
the measured values ranged from 1.3–2.7 nmol gÿ1 FW
during the 3-week period. This concentration is 4-fold to
over 10-fold that of the endogenous ABA, and is more than
2–5-fold of the concentration of applied (ÿ)-[4,5-d2]-ABA
that remains in the medium. Thus the exogenously supplied
(ÿ)-ABA accumulates in the responsive shoot apex tissues.
It was also found that both treated and untreated tissues
contain endogenous ABA metabolites, namely PA and DPA,
while 79-OH ABA and the ABA glucose ester were not
detected. In the first and the second weeks, PA was detected
only in (ÿ)-ABA-treated shoot apices, whereas DPA was
detectable in the tissues treated with either enantiomer but not
in the control. In the third week, concentrations of endogenous
ABA metabolites were significantly higher in (ÿ)-ABAtreated tissues than in those treated with (+)-ABA (P <0.05,
n=3). These results suggest that upon treatment, endogenous
ABA was produced and further metabolized in the plant
tissues. Moreover, for this stimulation of endogenous ABA
synthesis, (ÿ)-ABA has a stronger effect than (+)-ABA. In
addition, in treated plants, derivatives of the applied ABA
enantiomers were detected; (+)-[4,5-d2]-ABA was metabolized to PA and DPA, while (ÿ)-[4,5-d2]-ABA was biotransformed to 79-OH ABA (Table 4).
Discussion
Issues on ABA recognition
In M. quadrifolia the unnatural ABA enantiomer, (ÿ)ABA, not only induces a heterophyllous switch, but also
Table 4. Contents of ABA and ABA metabolites in heterophyllous responsive shoot apex tissues during a time-course of ABA
treatmenta
ABA isomer
Concentration (nmol gÿ1 FW)
Endogenous (d0)
Week 1
Week 2
Week 3
a
Control
(+)
(ÿ)
Control
(+)
(ÿ)
Control
(+)
(ÿ)
Labelled (d2)
ABA
PA
DPA
0.2760.00
0.2360.04
0.3060.11
0.2360.04
0.1560.00
0.1960.08
0.1160.00
0.2360.08
0.3060.08
n.d.
n.d
0.2960.07
n.d.
n.d.
0.0460.00
0.1860.11
0.1160.04
0.2960.11
n.d.
1.8860.50
1.8860.71
n.d.
2.4160.82
4.0160.85
0.7860.32
2.8760.50
5.5061.67
ABA
PA
DPA
79-OH ABA
0.1160.08
1.8060.38
n.d.
14.5465.04
n.d.
n.d.
0.1560.04
2.7460.94
0.7460.00
13.6363.24
n.d.
0.8260.53
1.2060.19
1.2860.11
0.0460.04
12.3961.58
n.d.
0.5060.04
Values are shown as mean 6SE of three replicates with eight shoot apices in each. n.d.: not detected.
ABA regulation of heterophylly in Marsilea quadrifolia
produces stronger effects than the natural (+)-ABA. Like
natural ABA, (ÿ)-ABA causes growth responses that are
progressive, reversible, dose-dependent, and organ-specific.
Moreover, (ÿ)-ABA is effective at lower concentrations
than (+)-ABA, and the optimal concentration of (ÿ)ABA is also lower, by one to two orders of magnitude,
depending on the organs. So far (ÿ)-ABA has not been
identified in plants; what has been seen in these experiments does not occur in nature. This raises the issue of
ABA perception and how the action of (ÿ)-ABA ties in
with that of the natural (+)-ABA.
These data show that heterophyllous development has
a distinct quantitative nature, and ABA affects individual
morphogenic traits quantitatively. For each experiment
reported here a set of plants was simultaneously treated
with racemic ABA in order to effect a comparison. It was
found that the extent of differential growth responses
caused by racemic ABA, in every aspect, falls between
those of the (+)-ABA and (ÿ)-ABA (data not shown).
Similar results have been reported in other systems (Smart
et al., 1995). Dosage analyses further indicate that, for
heterophyllous induction in M. quadrifolia, the optimal
concentration and the effective range of (+)- and (ÿ)-ABA
differ with organs and developmental stages. This complexity associated with a developmental switch suggests
a cell-autonomous nature of the ABA response.
The differential growth responses to ABA during heterophyllous induction include promoting effects, such as the
elongation of roots and petioles, the formation of lateral roots,
and the expansion of the leaf surface. Other aspects of the
developmental switching include inhibitory effects, for example, shortening of internodes, senescence, and growth
arrest. The effects of (ÿ)-ABA are stronger than those of (+)ABA in every parameter when individual organs are compared at specific developmental time points. However, if
growth responses were measured as a sum of multiple tissues
or over an accumulated period of time, different conclusions
might result from comparing the effects of (+)- and (ÿ)-ABA.
For example, regardless of the morphological change,
when the growth response is compared using total leaf
number, both (+)- and (ÿ)-ABA would appear to be
inhibitory at the end of the experimental period used here.
Similarly, when the growth response is measured using
total plant mass after 2 months of treatment, (ÿ)-ABA
would appear less effective than (+)-ABA in promoting
growth or would have little effect compared with the
untreated plants. These results further indicate that the
heterophyllous switch consists of multiphasic developmental transition. Collectively, these observations provide useful reference for further investigations on the molecular
mechanism of ABA recognition and signalling.
Endogenous versus exogenous ABA
The deuterated ABA analogues, used as standards for mass
spectrometry and metabolism studies, allowed a distinction
2945
of the level and the fate of endogenous and exogenous ABA.
Regardless of the treatment, endogenous ABA in heterophyllous-inducible shoot apices appeared to remain in
a more or less constant range of 0.2060.07 nmol gÿ1 FW,
or approximately 0.2 lM. For switching the developmental
path, (ÿ)-ABA is effective at as low as 0.01 lM for both
petioles and roots, and at 0.1 lM for the internodes, whereas
(+)-ABA is effective when supplied at 1 lM for shoots and at
0.1 lM for roots. Following the induction via the treatment
of 1 lM of either ABA enantiomer, the supplied ABA
accumulates at 4–10-fold the concentration of endogenous
ABA. These data indicate that both the exogenous ABA
level and the internal tissue concentration of ABA associated
with the developmental trigger are probably attainable by the
tissues under normal growth conditions and, therefore, are
within the physiological range.
The study of Smart et al. (1995) addressed similar
questions on the role of (+)- and (ÿ)-ABA on turion
induction in the water plant Spirodela polyrrhiza L. These
workers fed racemic, (+)- and (ÿ)-ABA to the fronds and
found that (ÿ)-ABA had as strong or a stronger effect on
growth inhibition and turion formation than the natural
compound or the racemic mixture. This is comparable to
what has been observed in M. quadrifolia. They measured
natural ABA using an ELISA assay in fronds treated with
(+)-ABA and were able to demonstrate that the concentrations of internal ABA required to induce turion formation was within a physiological range. The measurement of
total ABA in fronds using high performance liquid chromatography/gas chromatography-electron capture detection showed that the concentrations of ABA [total of both
(+)- and (ÿ)-ABA] in the fronds treated with (ÿ)-ABA
were much higher than the ABA concentrations in the
fronds treated with (+)-ABA. They concluded that the
accumulation was due to the (ÿ)-ABA enantiomer. A tight
correlation has been consistently observed between the
exogenous concentration of ABA and turion formation.
However, with measurements in fronds, they could not find
a simple internal concentration of ABA required for
triggering the switch. This is possibly because the ABAresponsive tissue may represent only a fraction of the total
plant material used for quantitative ABA analysis, as
alluded to by these authors and shown to be the case for
M. quadrifolia (Hsu et al., 2001).
It was found that supplied (ÿ)-ABA accumulates in
heterophyllous-responsive shoot apices at a higher concentration than the supplied (+)-ABA (Table 4). This may be
because (ÿ)-ABA is metabolized more slowly than (+)ABA, as has been accounted for in cell suspension cultures
of bromegrass and maize (Abrams et al., 1989; Balsevich
et al., 1994a, b). Indeed, the concentration of the metabolite, 79-OH ABA, represents only a fraction (approximately 30%) of the accumulated (ÿ)-ABA. By contrast,
metabolites of the supplied (+)-ABA exist in the tissues at
10-fold to over 100-fold the concentration of the hormone
2946 Lin et al.
itself. Similarly, the metabolites of endogenous ABA are at
high levels, approximately 10–20 times the concentration
of endogenous ABA, which remains at a constant range
(Table 4). However, the high levels of metabolites of (+)ABA in tissues, both endogenous and exogenous, do not
readily correlate with the physiological responses. Based on
these observations, it is conceivable that either (ÿ)-ABA
itself or one of its metabolites, or both, can be recognized
by the cellular component(s) of the signalling pathway and
function similarly to (+)-ABA.
enantiomer remained effective to induce the heterophyllous
switch, despite the inhibitor’s general toxicity to plant growth
(H-J Wang, B-L Lin, unpublished results). Based on the
findings in this work and the literature precedents, it cannot be
ruled out that (ÿ)-ABA itself is intrinsically active. It remains
to be tested whether some of the metabolites also have a role
for the induction. The work is now in progress.
The role of ABA metabolism
We are indebted to Su-Chuan Chiang for exceptional technical
assistance. We thank Rung-Wu Chen and Hung-Chi Liu for help
with the plant measurements, and Hung-Chi Liu also for help with
preparing the figures and tables. We would also like to thank Dr
Andrew Ross and Steve Ambrose for help with recording mass
spectrometry data, as well as Ken Nelson for his technical assistance.
This research was supported by the Cooperative Research Program
of the National Science Council, Taiwan and the National Research
Council, Canada, grant No. NSC 90-2311-B-169-002 and NSC
91-2311-B-169-002 to BLL. HJW is a recipient of a post-doctoral
fellowship from National Science Council, Taiwan.
In maize suspension cultures, (+)-ABA but not (ÿ)-ABA
has been shown to induce (+)-ABA hydroxylation (Cutler
et al., 1997), and in potato and Arabidopsis both enantiomers induce ABA oxidation (Windsor and Zeevaart,
1997). The effect of inducing oxidation of ABA in M.
quadrifolia would be to reduce internal, supplied and
endogenous, (+)-ABA levels in (+)-ABA-treated tissues.
If the M. quadrifolia ABA hydroxylation enzymes metabolize the natural (+)-ABA more rapidly than the (ÿ)-ABA
as occurs in maize and bromegrass (Abrams et al., 1989;
Balsevich et al., 1994a, b), the (ÿ)-ABA concentration
would decline at a slower rate.
The authors have no data on the uptake and export of
ABA enantiomers. However, it was found that applied
ABA was metabolized in tissues of M. quadrifolia. On the
other hand, the endogenous DPA concentration increases
and accumulated in (ÿ)-ABA-treated plants to higher levels
than in those treated with (+)-ABA after 3 weeks. This
indicates more endogenous ABA was synthesized in the
(ÿ)-ABA-treated tissues. Measurements of the metabolites
and the hormone give a complete picture of the amount of
ABA that had been synthesized and affected the system
during the experiment. Thus the more persistent (ÿ)-ABA
causes increased biosynthesis of (+)-ABA that may contribute to triggering the switch at a lower concentration of
(ÿ)-ABA versus (+)-ABA.
In other biological responses elicited by (ÿ)-ABA (Table
1) it is possible that the ABA-like effects observed are the
results of increased ABA biosynthesis and catabolism. This
may also be the case in the study on turion formation (Smart
et al., 1995). In the effects on gene expression and growth
inhibition in wheat embryos (Walker-Simmons et al.,
1992), differential gene expression was observed while
both enantiomers were equally effective germination inhibitors, indicating that (ÿ)-ABA may have been acting by
means other than by stimulating ABA biosynthesis. The
effects of external stimuli such as supplied (+)- and (ÿ)ABA on endogenous hormone synthesis need to be considered for each individual system.
This work demonstrates that applied (ÿ)-ABA stimulates
ABA biosynthesis and suggests that it may contribute to
at least part of the activity observed. Studies to block
ABA biosynthesis using fluridone showed that either ABA
Acknowledgements
References
Abrams SR, Reaney MJT, Abrams GD, Mazurek T, Shaw AC,
Gusta LV. 1989. Ratio of (S)- to (R)-abscisic acid from plant
cell cultures supplied with racemic ABA. Phytochemistry 28,
2885–2889.
Abrams SR, Lamb N, Hill RD, Abrams GD, Yin XS, Fernets W,
Laycock G, Gusta LV. 1993. ABA analogs for regulation of
germination in malting barley. In: Walker-Simmons MK, Ried JL,
eds. Preharvest sprouting in cereals, 1992. St Paul, MN: American
Association of Cereal Chemists, 278–286.
Abrams SR, Nelson K, Ambrose SJ. 2003. Deuterated abscisic acid
analogs for mass spectrometry and metabolism studies. Journal of
Labelled Compounds and Radiopharmaceuticals 46, 273–283.
Allsopp A. 1962. The effects of gibberellic acid on morphogenesis in
Marsilea drummondii A. Br. Phytomorphology 12, 1–10.
Balsevich JJ, Abrams SR, Lamb N, König WA. 1994a. Identification of unnatural phaseic acid as a metabolite derived from
exogenously added (ÿ)-abscisic acid in a maize cell suspension
culture. Phytochemistry 36, 647–650.
Balsevich JJ, Cutler AJ, Lamb N, Friesen LJ, Kurz EU, Perras
MR, Abrams SR. 1994b. Response of cultured maize cells to (+)abscisic acid, (ÿ)-abscisic acid, and their metabolites. Plant Physiology 106, 135–142.
Bianco-Colomas J, Barthe P, Orlandini M, Le Page-Degivry MT.
1991. Carrier-mediated uptake of abscisic acid by suspensioncultured Amaranthus tricolor cells. Plant Physiology 95, 990–996.
Bommineni VR, Bethune TD, Hull KL, Dunstan DI. 1998.
Expression of uidA under regulation of the ABA-inducible wheat
Em promoter in transgenic white spruce somatic embryos. Journal
of Plant Physiology 152, 455–462.
Chandler JW, Abrams SR, Bartels D. 1997. The effect of ABA
analogs on callus viability and gene expression in Craterostigma
plantagineum. Physiologia Plantarum 99, 465–469.
Chen J, Dellaporta S. 1994. Urea-based plant DNA miniprep. In:
Freeling M, Walbot V, eds. The maize handbook. New York:
Springer-Verlag, 526–527.
Cramer GR, Krishnan K, Abrams SR. 1998. Kinetics of maize
leaf elongation. IV. Effects of (+)- and (ÿ)-abscisic acid. Journal
of Experimental Botany 49, 191–198.
ABA regulation of heterophylly in Marsilea quadrifolia
Cutler AJ, Krochko JE. 1999. Formation and breakdown of ABA.
Trends in Plant Science 4, 472–478.
Cutler AJ, Squires TM, Loewen MK, Balsevich JJ. 1997. Induction of (+)-abscisic acid 89 hydroxylase by (+)-abscisic acid in
cultured maize cells. Journal of Experimental Botany 48,
1787–1795.
Cummins WR, Sondheimer E. 1973. Activity of the asymmetric isomers of abscisic acid in a rapid bioassay. Planta 111,
365–369.
Cummins WR, Kende H, Raschke K. 1971. Specificity and
reversibility of the rapid stomatal response to abscisic acid. Planta
99, 347–351.
Deschamp PA, Cooke TJ. 1983. Leaf dimorphism in aquatic
angiosperms: significance of turgor pressure and cell expansion.
Science 219, 505–507.
Deschamp PA, Cooke TJ. 1984. Causal mechanisms of leaf dimorphism in the aquatic angiosperm Callitriche heterophylla.
American Journal of Botany 71, 319–329.
Dong J-Z, Dunstan DI. 1996. Characterization of three heat-shockprotein genes and their developmental regulation during somatic
embryogenesis in white spruce [Picea glauca (Moench) Voss].
Planta 200, 85–91.
Dong J-Z, Dunstan DI. 1997. Characterization of cDNAs representing five abscisic acid-responsive genes associated with somatic
embryogenesis in Picea glauca, and their responses to abscisic
acid stereostructure. Planta 203, 448–453.
Dong J-Z, Pilate G, Abrams SR, Dunstan DI. 1994. Induction of
a wheat Em promoter by ABA and optically pure ABA analogs in
white spruce (Picea glauca) protoplasts. Physiologia Plantarum
90, 513–521.
Dunstan DI, Bock CA, Abrams GD, Abrams SR. 1992. Metabolism of (+)- and (ÿ)-abscisic acid by somatic embryo suspension
cultures of white spruce. Phytochemistry 31, 1451–1454.
Feurtado JA, Ambrose SJ, Cutler AJ, Ross ARS, Abrams SR,
Kermode AR. 2004. Dormancy termination of western white pine
(Pinus monticola Dougl. Ex D. Don) seeds is associated with
changes in abscisic acid metabolism. Planta 218, 630–639.
Gaudet JJ. 1963. Marsilea vestita: conversion of the water form
to the land form by darkness and by far-red light. Science 140,
975–976.
Gaudet JJ. 1965. The effect of various environmental factors on the
leaf form of the aquatic fern Marsilea vestita. Physiologia
Plantarum 18, 674–686.
Gifford EM, Foster AS. 1988. Morphology and evolution of
vascular plants, 3rd edn. New York: WH Freeman and Company.
Goliber TE. 1989. Endogenous ABA content correlates with photon
fluence rate and induced leaf morphology in Hippuris vulgaris.
Plant Physiology 89, 732–734.
Goliber TE, Feldman LJ. 1989. Osmotic stress, endogenous
abscisic acid, and the control of leaf morphology in Hippuris
vulgaris L. Plant, Cell and Environment 12, 163–171.
Gusta LV, Ewan B, Reaney MJT, Abrams SR. 1992. The effects of
abscisic acid and abscisic acid metabolites on the germination of
cress seed. Canadian Journal of Botany 70, 1550–1555.
Hampson CR, Reaney MJT, Abrams GD, Abrams SR, Gusta LV.
1992. Metabolism of dextro-abscisic acid to dextro-79-hydroxyabscisic acid by bromegrass cell cultures. Phytochemistry 31,
2645–2648.
Hays DB, Rose P, Abrams SR, Moloney MM. 1996. Biological
activity of optically pure C-1 altered abscisic acid analogs in
Brassica napus microspore embryos. Journal of Plant Growth
Regulation 15, 5–11.
Hill RD, Liu JH, Durnin D, Lamb N, Shaw A, Abrams SR. 1995.
Abscisic acid structure–activity relationships in barley aleurone
layers and protoplasts. Plant Physiology 108, 573–579.
2947
Hsu TC, Liu HC, Wang JS, Chen RW, Wang YC, Lin BL. 2001.
Early genes responsive to abscisic acid during heterophyllous
induction in Marsilea quadrifolia. Plant Molecular Biology 47,
703–715.
Jeannette E, Rona JP, Bardat F, Cornel D, Sotta B, Miginiac E.
1999. Induction of RAB18 gene expression and activation of K+
outward rectifying channels depend on an extracellular perception
of ABA in Arabidopsis thaliana suspension cells. The Plant
Journal 18, 13–22.
Jepson I, Bray J, Jenkins G, Schuch W, Edwards K. 1991. A rapid
procedure for the construction of PCR cDNA libraries from
small amounts of plant tissue. Plant Molecular Biology Reporter
9, 131–138.
Jiang L, Abrams SR, Kermode AR. 1996. Vicillin and napin
storage-protein gene promoters are responsive to abscisic acid in
developing transgenic tobacco seed but lose sensitivity following
premature dessication. Plant Physiology 110, 1135–1144.
Laetsch WM, Briggs WR. 1961. Kinetin modification of sporeling
ontogeny in Marsilea vestita. American Journal of Botany 48,
369–377.
Lin BL, Yang WJ. 1999. Blue light and abscisic acid independently
induce heterophyllous switch in Marsilea quadrifolia. Plant
Physiology 119, 429–434.
Liu BLL. 1984. Abscisic acid induces land form characteristics
in Marsilea quadrifolia L. American Journal of Botany 71,
638–644.
Milborrow BV. 1970. The metabolism of abscisic acid. Journal of
Experimental Botany 21, 17–29.
Milborrow BV. 1980. A distinction between the fast and slow
responses to abscisic acid. Australian Journal of Plant Physiology
7, 749–754.
Nakano S, Todoroki Y, Hirai N, Ohigashi H. 1995. Synthesis and
biological activity of 79-, 89- and 99-alkyl analogs of abscisic acid.
Bioscience, Biotechnology, and Biochemistry 59, 1699–1706.
Nambara E, Suzuki M, Abrams SR, McCarty DR, Kamiya Y,
McCourt P. 2002. A screen for genes that function in
abscisic acid signalling in Arbidopsis thaliana. Genetics 161,
1247–1255.
Perras MR, Abrams SR, Balsevich JJ. 1994. Characterization of an
abscisic acid carrier in suspension-cultured barley cells. Journal of
Experimental Botany 45, 1565–1573.
Robertson AJ, Reaney MJT, Wilen RW, Lamb N, Abrams
SR, Gusta LV. 1994. Effects of abscisic acid metabolites and
analogs on freezing tolerance and gene expression in bromegrass
(Bromus inermis Leyss) cell cultures. Plant Physiology 105,
823–830.
Rose PA, Lei B, Shaw AC, Barton DL, Walker-Simmons MK,
Abrams SR. 1996. Probing the role of the hydroxyl group of
ABA: analogues with a methyl ether at C-19. Phytochemistry 41,
1251–1258.
Ross ARS, Ambrose SJ, Cutler AJ, Feurtado JA, Kermode AR,
Nelson K, Zhou R, Abrams, SR. 2004. Determination of
endogenous and supplied deuterated abscisic acid in plant tissues
by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry with multiple reaction monitoring.
Analytical Biochemistry 329, 324–333.
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning:
a laboratory manual, 2nd edn. Cold Spring Harbor: Cold Spring
Harbor Laboratory Press.
Sauter A, Abrams SR, Hartung W. 2002. Structural requirements
of abscisic acid (ABA) and its impact on water flow during radial
transport of ABA analogues through maize roots. Journal of Plant
Growth Regulation 21, 50–59.
Schmitz N, Abrams SR, Kermode AR. 2002. Changes in ABA
turnover and sensitivity that accompany dormancy termination of
2948 Lin et al.
yellow-cedar (Chamaecyparis nootkatensis) seeds. Journal of
Experimental Botany 53, 89–101.
Shen J, Niwa T, Marumo S. 1995. Comparison of the biological
activity of natural (S)-(+)-ABA and non-natural (R)-(ÿ)-ABA.
Zhiwu Shengli Xuebao 21, 166–174.
Smart CC, Fleming AJ, Chaloupkova K, Hanke DE. 1995. The
physiological role of abscisic acid in eliciting turion morphogenesis. Plant Physiology 108, 623–632.
Sondheimer E, Galson EC, Chang YP, Walton DC. 1971.
Asymmetry, its importance to the action and metabolism of
abscisic acid. Science 174, 829–831.
Steeves TA, Sussex IM. 1989. Patterns in plant development.
Cambridge: Cambridge University Press.
Todoroki Y, Hirai N, Koshimizu K. 1994. 89- and 99-methoxyabscisic acids as antimetabolic analogs of abscisic acid. Bioscience,
Biotechnology, and Biochemistry 58, 707–715.
Todoroki Y, Hirai N, Koshimizu K. 1995a. 89,89-Difluoro- and
89,89,89-trifluoroabscisic acids as highly potent, long-lasting analogs of abscisic acid. Phytochemistry 38, 561–568.
Todoroki Y, Hirai N, Koshimizu K. 1995b. Synthesis and biological activity of 19-deoxy-19-fluoro- and 89-fluoroabscisic acids.
Phytochemistry 40, 633–641.
Toorop PE, Bewley JD, Abrams SR, Hilhorst HWM. 1999.
Structure–activity studies with ABA analogs on germination and
endo-b-mannanase activity in tomato and lettuce seeds. Journal of
Plant Physiology 154, 679–685.
Trewavas AJ, Jones HG. 1991. An assessment of the role of ABA in
plant development. In: Davies WJ, Jones HG, eds. Abscisic acid:
physiology and biochemistry. Oxford: Bios Scientific Publishers,
169–188.
Walker-Simmons MK, Anderberg RJ, Rose PA, Abrams SR.
1992. Optically pure abscisic acid analogs: tools for relating
germination inhibition and gene expression in wheat embryos.
Plant Physiology 99, 501–507.
Wilen RW, Fu P, Robertson AJ, Abrams SR, Low NH, Gusta LV.
1996. An abscisic acid analog inhibits abscisic acid-induced
freezing tolerance and protein accumulation, but not abscisic
acid-induced sucrose uptake in a bromegrass (Bromus inermis
Leyss) cell culture. Planta 200, 138–143.
Wilen RW, Hays DB, Mandel RM, Abrams SR, Moloney MM.
1993. Competitive inhibition of abscisic acid-regulated gene
expression by stereoisomeric acetylenic analogs of abscisic acid.
Plant Physiology 101, 469–476.
Wilmer JA, Abrams SR, Helsper JPFG, van der Plas LHW.
1998. Role of the ring methyl groups in abscisic acid activity in
erucic acid accumulation in oilseed rape (Brassica napus L.).
Journal of Plant Growth Regulation 17, 19–23.
Windsor ML, Zeevaart JAD. 1997. Induction of ABA 89-hydroxylase by (+)-S-, (ÿ)-R- and 89,89,89-trifluoro-S-abscisic acid in
suspension cultures of potato and Arabidopsis. Phytochemistry
45, 931–934.
Windsor ML, Milborrow BV, Abrams SR. 1994. Stereochemical
requirements of the saturable uptake carrier for abscisic acid in
carrot suspension culture cells. Journal of Experimental Botany
45, 227–233.
Zaharia LI, Galka MM, Ambrose SJ, Abram SR. 2005. Preparation of deuterated abscisic acid metabolites for use in mass
spectrometry and feeding studies. Journal of Labelled Compounds
and Radiopharmaceuticals 48, 435–445.
Zeevaart JAD, Creelman RA. 1988. Metabolism and physiology of
abscisic acid. Annual Review of Plant Physiology and Plant
Molecular Biology 39, 439–473.
Zhou R, Cutler AJ, Ambrose SJ, et al. 2004. A new abscisic acid
catabolic pathway. Plant Physiology 134, 361–369.
Zou J, Abrams GD, Barton DL, Taylor DC, Pomeroy MK, Abrams
SR. 1995. Induction of lipid and oleosin biosynthesis by (+)abscisic acid and its metabolites in microspore-derived embryos of
Brassica napus L. cv. Reston. Plant Physiology 108, 563–571.