Annals of Botany 90: 623±630, 2002
doi:10.1093/aob/mcf239, available online at www.aob.oupjournals.org
Abscisic Acid Catabolism in Maize Kernels in Response to Water De®cit at
Early Endosperm Development
Z H A O L O N G W A N G , S T E F A N I A MA M B E L L I and T I M L . SE T T E R *
Department of Crop and Soil Sciences, Cornell University, Ithaca, NY 14853, USA
Received: 24 June 2002 Returned for revision: 24 July 2002 Accepted: 2 August 2002 Published electronically: 2 October 2002
To further our understanding of the greater susceptibility of apical kernels in maize in¯orescences to water
stress, abscisic acid (ABA) catabolism activity was evaluated in developing kernels with chirally separated (+)[3H]ABA. The predominant pathway of ABA catabolism was via 8¢-hydroxylase to form phaseic acid, while
conjugation to glucose was minor. In response to water de®cit imposed on whole plants during kernel development, ABA accumulated to higher concentrations in apical than basal kernels, while both returned to control
levels after rewatering. ABA catabolism activity per gram fresh weight increased about three-fold in response to
water stress, but was about the same in apical and basal kernels on a fresh weight basis. ABA catabolism activity was three to four-fold higher in placenta than endosperm, and activity was higher in apical than basal kernels. In vitro incubation tests indicated that glucose did not affect ABA catabolism. We conclude that placenta
tissue plays an important role in ABA catabolism, and together with ABA in¯ux and compartmentation, determine the rate of ABA transport into endosperms.
ã 2002 Annals of Botany Company
Key words: Zea mays, L., corn, maize, ABA catabolism rate, endosperm, placenta, grain, water de®cit.
INTRODUCTION
The phytohormone abscisic acid (ABA) regulates many
important physiological and developmental processes in
plants, as well as adaptive responses to imposed environmental stress (Zeevaart and Creelman, 1988). During seed
development, ABA plays several important roles, including
induction of storage protein and lipid synthesis, desiccation
tolerance and prevention of germination (Guerrero and
Mullet, 1986; Schmitz et al., 2000; Suzuki et al., 2000; Tian
and Brown, 2000). The effects exerted by ABA are a
function of tissue sensitivity as well as the extent to which
ABA accumulates in the target tissue (Corbineau et al.,
2000). In developing seeds, ABA is synthesized by embryo
tissues, but there is evidence that ABA is also transferred
from maternal tissues to the seed when a plant is subjected
to water de®cit (Ober and Setter, 1992; Jeschke et al., 1997).
ABA concentrations in plant tissues are maintained dynamically by opposing forces of synthesis, transport and
catabolism to inactive products. Although considerable
research has focused on ABA synthesis, much less has been
directed at comparisons of ABA transport and rate of
catabolism in various tissues and in response to environmental conditions.
Inactivation of ABA in plant tissues can occur via two
major pathways: oxidation and conjugation. Oxidation of
the 8¢-methyl group of ABA, catalysed by the P450 enzyme
ABA-8¢-hydroxylase, yields 8¢-OH-ABA (Balsevich et al.,
1994; Cutler et al., 1997; Krochko et al., 1998; Cutler and
Krochko, 1999). This product is unstable and rearranges to
phaseic acid (PA), which can be further reduced to
4¢-dihydrophaseic acid (DPA). Conjugation primarily
* For correspondence. Fax + 1 607 2552644, e-mail [email protected]
involves formation of ABA glucose ester (ABA-GE)
(Zeevaart and Creelmann, 1988). Different ABA inactivation pathways occur in different species, or even at different
developmental stages in the same species. ABA-GE is a
major metabolite in larch somatic embryo (Label and Lelu,
2000), Xanthium plants (Zeevaart, 1999), xylem sap of
sun¯ower (Hansen and DoÈrf¯ing, 1999), leaves and root
tissues in castor bean (Jeschke et al., 1997), and maize root
(Sauter and Hartung, 2000). However, in suspension
cultures of maize cells (Cutler et al., 1997) and somatic
embryos of white spruce (Dunstan et al., 1992), ABA was
quantitatively converted to PA. In addition, minor amounts
of several other metabolites, (+)-7¢-hydroxy-ABA, transABA and the cis- and trans-1¢,4¢-diols of ABA, have been
detected in some plant species (Balsevich et al., 1994;
Walton and Li, 1995; Zeevaart, 1999).
Studies involving water de®cit treatment, exogenous
ABA application, and viviparous mutants in which ABA
biosynthesis is compromised indicated that during early
kernel development ABA inhibits endosperm cell division
and kernel growth (Ober and Setter, 1990, 1992; Mambelli
and Setter, 1998). If endosperm cell division is halted at an
early stage, kernels abort and fail to set seed. If they
continue their development but with drastically decreased
rates of cell division, they form small shrivelled mature
kernels that do not contribute to yield (Artlip et al., 1995;
Setter et al., 2001). Water de®cit primarily affects
developmentally younger reproductive structures, in apical
positions along the ear, whereas basal and middle kernels
are relatively little affected. The physiological basis for the
differential response to water de®cit at apical vs. basal/
middle ear positions in maize is not known. Kernels in
apical regions of ears accumulate more ABA than those in
ã 2002 Annals of Botany Company
624
Wang et al. Ð ABA Catabolism in Maize Kernels
basal regions in response to water stress at the early stage of
kernel formation (Setter et al., 2001). This ABA accumulation correlates with endosperm cell division, indicating
that the higher accumulation of ABA might contribute to
increased likelihood of kernel abortion (Myers et al., 1990;
Ober and Setter, 1990, 1992; Ober et al., 1991; Artlip et al.,
1995; Mambelli and Setter, 1998). In maize kernels,
elevated ABA levels in apical kernels could be due to a
higher rate of ABA biosynthesis and import from maternal
organs, or a lower rate of ABA catabolism. In this study,
focus is on ABA catabolism activity in maize kernels, using
detached kernel explants which eliminate the possibility of
import, and permit introduction of [3H]ABA tracer. This
system was used to determine the major pathway of ABA
catabolism in maize kernels and to quantify its role in
placenta and endosperm tissues in differential accumulation
of ABA between apical and basal kernels.
MATERIALS AND METHODS
(+)-[ 3H]ABA preparation
(6)-2-cis, 4-trans-[G-3H]ABA (7´4 3 106 Bq ml±1, 1´11 3
1012 Bq mmol±1; Amersham Life Science, Piscataway, NJ,
USA) was methylated with excess, freshly made diazomethane, and then quenched with glacial acetic acid. (6)[3H]MeABA was dried with ¯owing N2, dissolved in
isopropanol-hexane (1 : 4 v/v; 20 mg Me-ABA ml±1) and
enantiomers were resolved by repeated 0´1 ml injections
onto an analytical chiral column (Chiralcel OD, 0´46 3
25 cm; Daicel Chemical Industries Ltd, Exton, PA, USA).
The mobile phase employed was isopropanol-hexane
(1 : 9 v/v) at a nominal ¯ow rate of 1 ml min±1. The eluates
were collected each 0´5 min, and 1 ml was analysed for
radioactivity in each fraction with liquid scintillation
counting. The (+) enantiomer eluted at 10 min and the (±)
enantiomer at 15 min. Each ester was converted to the
corresponding acid [85 % yield for (±)-ABA; 92 % for (+)ABA] by saponi®cation with 12 M NH4OH in the dark at
40 °C for 12 h, followed by acetic acid acidi®cation. Both
(+)-[3H]ABA and (±)-[3H]ABA were puri®ed by reversephase C18 HPLC, dried in vacuo and stored at ±20 °C.
Plant material
Maize (Zea mays, L. `Pioneer Brand 39K72'; Pioneer HiBred International, Des Moines, IA, USA) was sown in 12-l
pots containing a mix of vermiculite/perlite/peat (1 : 1 : 1).
Each pot was supplemented with 6 g pulverized limestone,
42 g powdered FeSO4, 35 g CaSO4, 1 g fritted trace
elements (Peters FTE 555; Scotts Co., Marysville, OH,
USA) and 3 g granular wetting agent (AquaGro G;
Aquatrols, Pennsauken, NJ, USA). Plants were grown in a
glasshouse under ambient light and temperature conditions
supplemented for 12 h d±1 with 1000 W metal halide lights
when solar photon ¯ux density (PFD) was less than
250 mmol m±2 s±1 of photosynthetically active radiation
(PAR) (400±700 nm wavelengths). Pots were thinned to
four plants per pot 3 weeks after planting, and watered with
an automatic system that contained 0´6 g l±1 of Peters
15±16±17 fertilizer (Scotts Co.) at 2 h intervals during the
day. Watering was suf®cient to leach excess nutrient salts.
Pollination was performed manually at 4 d after silk
emergence. Beginning at 1 and 6 days after pollination
(DAP), pots assigned to the water de®cit treatment were
weighed, then removed from watering system, and maintained at about 50 % of the initial wet weight of the pots and
plants. After 5 d of water de®cit, plants were fully rewatered
and returned to the watering system.
Samples
One to 6 d after the treatment (2±7 DAP), ears of both
water de®cient and well-watered (control) plants were
harvested, and kernels from apical and basal ear regions
were dissected to monitor endogenous ABA levels and for
the explant incubation study. The number of rings of kernels
from the base to the tip of the ear was counted and the apical
ear region was de®ned as the ®fth 6 two nodal rings of
fertilized ¯orets, counting basipetally from the apex. The
basal ear region was the 25th 6 two nodal rings of fertilized
¯orets from the apex.
Incubation
Maize kernels were dissected directly from the ear. After
glumes had been removed, kernels were cut off at the top of
pedicels, leaving the placental region intact with the
endosperm, pericarp and nucellus, and immediately incubated in conical tubes with placenta immersed in 10 ml
incubation medium (10 mM acetic acid±NaOH buffer,
pH 4´8, containing 300 Bq of (+)-[3H]ABA with or without
200 mmol l±1 of (6)-ABA). Preliminary tests performed by
varying the incubation time from 1 to 24 h showed that ABA
was taken up and metabolized linearly with time (data not
shown). ABA catabolism rate per gram fresh weight was
calculated as:
R [nmol h±1 g±1 f. wt] = Cr S±1 g±1 f. wt h±1,
where R is ABA catabolism rate, Cr is the sum of
radioactivity in PA, DPA and other ABA catabolites
identi®ed in the studies described below (Results), S is the
speci®c radioactivity of (+)-ABA in the tissue [Bq ABA/
mol ABA], and h is length of incubation (in hours) as
indicated in ®gure legends. After incubation, kernels were
rinsed three times in 1 ml distilled water. One batch of
kernels was dissected into placenta, and endosperm and
these isolated tissues were incubated in 200 ml incubation
medium (10 mM acetic acid±NaOH buffer, pH 4´8),
containing 300 Bq of (+)-[3H]ABA with 200 mmol l±1 of
(6)-ABA, to study the catabolism rate of different tissues in
the kernel. In each experiment, treatments were at least
duplicated, and experiments were repeated at least twice to
con®rm the results. Speci®c conditions for individual
experiments are described in the appropriate ®gures.
Metabolite extraction and analysis
After incubation, kernels were placed directly into icechilled extraction medium [80 % methanol, 1 % glacial
Wang et al. Ð ABA Catabolism in Maize Kernels
625
losses in ABA yield by liquid scintillation counting; yield
averaged >90 % and reported data are corrected for losses.
ABA was eluted with 200 ml of Solvent II [55 % (v/v)
methanol, 1% (v/v) glacial acetic acid]. ABA fractions were
dried in vacuo at <24 °C.
ABA enzyme-linked immunosorbant assay (ELISA)
F I G . 1. Time course of ABA accumulation in apical and basal kernels on
plants subjected to water de®cit stress (closed symbols) and control (open
symbols) treatments. Water de®cit treatments started at 1 day after
pollination (DAP). Pots were rewatered after 5 d water de®cit treatment
(6 DAP). Means 6 s.e.m. for three replicates are shown.
acetic acid (v/v); 10 mg l±1 butylated hydroxytoluene] with a
ratio of sample (g f. wt): extraction solution (ml) of 1 : 10.
Samples were homogenized in a 1´7 ml conical pestle and
mortar, and extracted for 12 h with shaking at room
temperature. After centrifugation at 5000 g for 10 min, the
supernatant was removed and the pellet was re-extracted
twice more, and supernatants were pooled, dried in vacuo
and dissolved in 100 ml 1 % (v/v) acetic acid before
chromatography by reversed phase HPLC. The column was
0´46 3 25 cm C18 (RSIL, 10 mm particle size; Alltech
Associates, Deer®eld, IL, USA). Solvent A was 1 % (v/v)
glacial acetic acid in water and solvent B was acetonitrile. A
two-step linear solvent gradient delivered at 1 ml min±1 was
used to separate the metabolites: injection with 12 % B then
a linear gradient to 20 % B in 18 min, and a linear gradient
to 88 % B in 3´5 min. Using this gradient, baseline
resolution of DPA, PA, abscisic acid glucose ester (ABAGE) and ABA was achieved. Metabolite identi®cation was
accomplished by co-chromatography with authentic standards (ABA from Sigma Chemical Co., St Louis, MO, USA;
ABA-GE from Apex Organic, Oxford, UK; DPA and PA
from M. Brenner, University of Minnesota, MN, USA).
ABA ¯ash chromatography
For endogenous ABA study, 100 ml aliquots of each
extract were dried in vacuo at <24 °C following addition of
[3H]ABA as a tracer (17 Bq for each) of yield in
chromatography. For the [3H]ABA incubation study, no
radioactivity was added during this step. Aliquots were
redissolved in 100 ml of Solvent I [20 % (v/v) methanol, 1%
(v/v) glacial acetic acid]. ABA was separated with reversephase chromatography on columns packed with 15 mg of
40 mm diameter C18-silica material (J.T. Baker Chemicals,
Phillipsburg, NJ, USA). Solvents were eluted by vacuum
(<35 kPa) and the ¯ow rate was limited to about 20 ml min±1.
Columns were pre-equilibrated with Solvent I prior to
samples being loaded in 100 ml; contaminants were eluted
with 400 ml Solvent I. Non-ABA fractions were analysed for
Samples from C18 ¯ash chromatography were dried in
vacuo and suspended in 100 ml Tris-buffered saline [50 mM
Tris±HCl, 10 mM NaCl, 1 mM MgCl2, 15 mM NaN3
(pH 7´5)]. ABA was analysed by competitive-binding
ELISA employing commercially available monoclonal
antibodies speci®c for (+)-ABA (Idetek, San Bruno, CA,
USA), essentially as described by Setter et al. (2001).
Preliminary study with a series of internal standards added
to extracts yielded parallel lines (Ober et al., 1991),
indicating an absence of interfering compounds.
RESULTS
To assess the time course of ABA accumulation during
stress, water was withheld at 1 d after pollination, and
kernels were sampled from apical and basal regions of ear
in¯orescences at various times after the imposition of water
de®cit (Fig. 1). Although both apical and basal kernels
rapidly accumulated ABA between 2 and 3 d after
withholding water, the extent of ABA accumulation was
about twice as high in apical than basal kernels. Maximal
ABA concentrations were reached at 3 d after withholding
water, corresponding to the time-frame when soil water was
depleted to the extent that leaves ®rst exhibited incipient
wilt symptoms (leaf rolling and glaucous appearance of
leaf-blade surface). ABA levels declined as the stress was
continued from days 3 to 5, and when pots were rewatered
ABA quickly returned to the baseline within 1 d. Other
studies, in which water stress was extended an additional
2±4 d, indicated that ABA did not return to control levels,
but remained elevated throughout a prolonged stress period
(data not shown).
To determine the involvement of ABA catabolism in
establishing steady-state ABA concentrations in these
tissues, kernels were sampled at various stages of stress
and recovery, and radiotracer analysis was conducted with
chirally separated (+)- and (±)-[3H]ABA. After incubation
of control kernels for 6 h, exogenously fed (+)-[3H]ABA
was metabolized to a variety of products (Figs 2 and 3). The
metabolites identi®ed included DPA, which eluted at 4 min,
ABA-GE (at 8´5 min), PA (at 10 min), 7¢-OH-ABA (at
12 min) and a small peak of unidenti®ed metabolite which
eluted at 19 min (Fig. 2). Unmetabolized ABA eluted at
23´5 min. In tissues fed the natural isomer, (+)-ABA,
radioactivity that co-eluted with ABA-GE and 7¢-OH-ABA
never exceeded 5 % of the recovered metabolites. In
contrast, the unnatural isomer (±)-[3H]ABA was mainly
metabolized to 7¢-OH-ABA, and no PA and DPA peaks
were detected among the recovered radiolabelled products.
After 6 h incubation, about 58 % of (+)-[3H]ABA fed to
kernels was converted to PA, whereas 30 % of (±)-[3H]ABA
was converted to 7¢-OH-ABA.
626
Wang et al. Ð ABA Catabolism in Maize Kernels
Consistent with the catabolism pathway ABA®PA®
DPA, (+)-[3H]ABA fed to kernels decreased with incubation time, whereas PA increased sharply after 2 h and
peaked at 9 h of incubation. DPA increased slightly after 9 h
and reached only 9 % of the recovered radioactivity after
24 h of incubation (Fig. 3). The radioactivity in ABA-GE,
however, was a minor proportion of the metabolites
recovered during 24 h of incubation, indicating that
conjugation to ABA-GE was not a major pathway for
natural (+)-ABA catabolism in maize kernels.
Studies of kernels fed (+)-[3H]ABA of high speci®c
radioactivity revealed that kernels from water-stressed
plants had substantially higher rates of ABA catabolism
than those from well-watered controls (Fig. 4). Waterstressed kernels steadily converted ABA to PA, reaching
about 60 % of radioactivity in PA at 6 h, compared with
about 20 % in control kernels. Catabolism rates estimated
with this system are affected by treatment differences in the
size of endogenous ABA pools with which the introduced
radiotracer mixes. This creates uncertainty of speci®c
radioactivity in the pools accessed by the catabolic
enzymes. To estimate ABA catabolism activity with
comparable ABA concentration and speci®c radioactivity
in both treatments, kernels were fed (+)-[3H]ABA containing 200 mmol l±1 ABA, thereby introducing suf®cient total
moles of ABA into endogenous pools so that speci®c
radioactivity in control and stress treatments was essentially
the same. This method of radiotracer analysis indicated that
ABA catabolism activity in water-stressed kernels was
about three-fold higher per gram fresh weight than that in
well-watered kernels in both apical and basal kernels
(Fig. 5A). However, if calculated on a per-kernel basis,
catabolism rates in basal kernels were higher than those in
apical kernels (Fig. 5B), due to the much larger size of basal
kernels in both treatments.
Studies of maize plants subjected to water stress in the
early post-pollination stage have indicated that maternal
tissues are the source of ABA which accumulates in
endosperm (Ober and Setter, 1992), and that ABA is
transported from leaves to sink organs via phloem (Ober
et al., 1990; Zhang et al., 1996). Hence, it is possible that
tissues in the transport pathway between the site of phloem
unloading in placenta to the site of net dry matter
accumulation in endosperm might play a role in modulating
the import and accumulation of ABA in endosperms. To
examine this pathway, kernels were dissected into three
tissue parts: placenta, pericarp and endosperm. Compared
with apical kernels, basal kernels retained a higher proportion of introduced radiolabel in the placenta, such that a
considerably lower proportion of fed radiolabel was transported into endosperm (Table 1). There was no difference in
the percentage of radiolabel from [3H]ABA transported into
pericarp tissue in apical and basal kernels.
F I G . 3. Time course of (+)-[3H]ABA catabolism to PA and DPA. Basal
kernels were excised from water-stressed plants at 9 days after
pollination and incubated on medium containing (+)-[3H]ABA for the
indicated times. Bars represent s.e.m., and are shown if they exceed
symbol size. n = 3±5.
F I G . 2. Elution pro®le of ABA metabolites separated by C18 HPLC from
water-stressed maize kernels. Basal kernels were harvested after 5 d of
water-de®cit treatment (6 d after pollination) and incubated for 9 h in
medium containing (+)-[3H]ABA or (±)-[3H]ABA. 1, DPA; 2, ABA-GE;
3, PA; 4, 7¢-hydroxy-ABA; 5, ABA.
F I G . 4. Effects of water-de®cit stress on [3H]phaseic acid accumulation
in maize kernels. Basal kernels incubated in 10 ml (+)-[3H]ABA medium
for 1±6 h. Means 6 s.e.m. for three replicates are shown.
Wang et al. Ð ABA Catabolism in Maize Kernels
A factor that may have contributed to the greater retention
of radiolabel in placenta of basal than apical kernels was the
greater activity of ABA catabolism in basal-kernel placenta.
In addition to the larger size of basal-kernel placenta,
(+)-[3H]ABA catabolism activity per gram fresh weight of
isolated basal-kernel placenta was higher than that of apicalkernel placenta (Fig. 6). Although ABA catabolism activity
per gram fresh weight in endosperm was higher in apical
than basal kernels (Fig. 6), on a whole kernel basis it was
higher in endosperms of basal kernels (data not shown).
Moreover, the activity in placenta predominated because at
627
6 DAP placenta were larger than endosperm, and placenta
had three to four-fold higher activity per gram fresh weight
than endosperm (Fig. 6). This suggests that placenta could
play a pivotal role in ABA supply to endosperm and other
kernel tissues.
To examine the catabolism of endogenous ABA in
kernels from water-stressed plants, kernels were explanted
at 6 DAP to in vitro medium and ABA was quanti®ed during
the time course of net ABA decrease (Fig. 7). Preliminary
studies with this system indicated that endogenous ABA
decrease was a function of the relative rates of synthesis and
catabolism. These studies showed that inclusion of tetcyclasis, an inhibitor of the P450 ABA catabolism enzyme
ABA 8¢-hydroxylase (Krochko et al., 1998), blocked ABA
decrease, and kernels responded to low water potential
osmoticum with enhanced ABA synthesis that could be
blocked with protein synthesis inhibitors (data not shown).
Water de®cit decreases photosynthesis and phloem
transport to kernels, so it is plausible that stress decreases
kernel sugar status (Zinselmeier et al., 1999; Setter et al.,
2001). To determine whether carbohydrate status modulates
the rate of ABA synthesis, kernels were explanted to in vitro
wells with their placenta immersed in control or glucosecontaining media. These tests indicated that net ABA
decrease was unaffected by supplemental carbohydrate
supply (Fig. 7A).
ABA decreased at about the same rate during in vitro
incubation in both apical and basal kernels (Fig. 7B);
however, apical kernels had a higher initial ABA concentration and this gap between apical and basal kernels was
not closed even after 8 h of incubation. Thus, apical kernels
had substantial capacity to deplete ABA, although not
suf®cient to prevent ABA accumulation to high levels
during stress.
DISCUSSION
F I G . 5. ABA catabolism in whole-kernel explants from apical and basal
region of maize ears from plants subjected to control and water stress
treatments. ABA catabolism activity per gram fresh weight (A) and per
kernel (B). Kernels were incubated for 6 h in 10 ml medium containing
200 mM (6)-ABA with (+)-[3H]ABA tracer. The sum of radioactivity in
ABA catabolites from HPLC separations was used to calculate ABA
catabolism rate, as described in Materials and Methods. Means 6 s.e.m.
for four replicates are shown.
When maize plants were subjected to water de®cit, kernels
in the apical region of the ear in¯orescence accumulated
more ABA than basal kernels (Fig. 1), as previously
reported (Ober et al., 1990; Artlip et al., 1995; Setter et al.,
2001). However, after prolonged drought, ABA levels
plateaued, and after rewatering at day 5 ABA levels rapidly
returned to normal (Fig. 1). This suggests that ABA
catabolism might be critical in maintaining steady-state
ABA levels in kernels.
TA B L E 1. Distribution of radiolabel in ABA and metabolites after 6 h of feeding (+)-[3H]ABA via exposed placenta tissue
immersed in media
Ear region
Placenta (%)
Pericarp (%)
Endosperm (%)
Apical
Basal
LSD0´05
45´8 6 0´6a
51´3 6 1´6b
4´3
38´6 6 0´8a
38´6 6 1´2a
5´1
15´6 6 1´3a
10´1 6 1´0b
3´0
Kernels were used at 6 d after pollination.
Means 6 s.e.m., n = 3.
Means within a column with the same superscript letter are not signi®cantly (P > 0´05) different.
628
Wang et al. Ð ABA Catabolism in Maize Kernels
F I G . 6. ABA catabolism activity in placenta and endosperm tissues from
apical and basal regions of ears on water-stressed maize plants. Isolated
tissues were incubated for 6 h in 200 mM (6)-ABA with (+)-[3H]ABA
tracer. The sum of radioactivity in ABA catabolites from HPLC
separations was used to calculate ABA catabolism rate, as described in
Materials and Methods. Means 6 s.e.m. for two replicates are shown.
ABA catabolism pathways
There are several metabolic pathways by which ABA can
be catabolized to inactive compounds in plant tissues
(Walton and Li, 1995; Zeevaart, 1999). The two major
metabolic pathways are oxidation to PA and DPA, and
conjugation of ABA to its glucose ester (ABA-GE)
(Zeevaart and Creelman, 1988). Although both pathways
exist in maize kernels, the present data showed that with
natural (+)-ABA, catabolism to PA and DPA is the
dominant pathway, and conjugation to ABA-GE is minor.
This contrasts with the situation in roots of maize and
sun¯ower where ABA-GE formation predominates such
that it represents a major transport form in xylem (Hansen
and DoÈrf¯ing, 1999; Sauter and Hartung, 2000). However,
in several other tissues of grass-family plants, catabolism to
PA predominates, including developing barley grains
(Naumann and Doerf¯ing, 1982) and maize suspensioncultured cells (Balsevich et al., 1994).
ABA catabolism in apical vs. basal kernels
Water stress was accompanied by an enhanced ABA
catabolism rate in kernels (Fig. 5A), consistent with
previous reports in barley and wheat aleurones (Uknes and
Ho, 1984), in potato and arabidopsis suspension cultures
(Windsor and Zeevaart, 1997), in bean leaves (Gergs et al.,
1993), and in embroyonic axes of chickpea (Babiano, 1995).
(+)-ABA 8¢-hydroxylase catalyses the ®rst step in the
oxidative catabolism of ABA and is considered to be the
pivotal enzyme controlling the rate of ABA catabolism.
Krochko et al. (1998) showed that (+)-ABA 8¢-hydroxylase
is induced by its substrate, (+)-ABA, which is elevated in
response to water stress. However, multiple responses may
be operating that affect the steady-state level of ABA.
Cutler et al. (1997) showed that, whereas ABA
8¢-hydroxylase was induced by (+)-ABA, such induction
was suppressed if maize suspension cells were pre-treated
with mannitol osmotic treatments. In the current work,
water stress, which substantially elevated kernel ABA
F I G . 7. Net catabolism of endogenous ABA during incubation of
explanted water-stressed maize kernels on in vitro media. A, Kernels
from water-stressed plants were explanted at 6 DAP to 50 ml medium
containing control or 50 mM glucose treatments. B, Kernels from apical
and basal regions of ears from water-stressed plants were explanted at
6 DAP to in vitro medium. Means 6 s.e.m. for eight replicates are
shown.
contents (Fig. 1), increased ABA catabolism rates to about
three times those of the controls (Fig. 5). In our explant
system for assaying ABA catabolism, kernels were placed in
a buffer containing fed [3H]ABA without osmoticum. Thus,
partial kernel rehydration during assay may have prevented
the stressed kernels from experiencing negative effects on
catabolism due to low water potential. By employing
exogenous ABA feeding, the assay system may have
induced ABA 8¢-hydroxylase beyond what was present in
situ, and partially masked treatment differences. Consistent
with this interpretation, ABA 8¢-hydroxylase was induced
progressively in maize suspension cells over a 15 h exposure
to 100 mmol l±1 ABA in cell suspension medium, with an
intermediate level of induction at 6 h (Cutler et al., 1997;
Krochko et al., 1998). However, ABA uptake in whole
kernel explants and in whole tissue segments was gradual
(data not shown), and hence the assay time frame may have
been suf®ciently short to avoid substantial induction of
ABA 8¢-hydroxylase by the [3H]ABA fed to kernels. Our
ability to detect substantially higher ABA catabolism
activity in water-de®cit kernels relative to controls (Fig. 5),
and the observation that comparisons of water stress vs.
control were similar when the assay system involved
feeding 200 mmol l±1 (6)-ABA (Fig. 5) vs. 0´3 pmol l±1
(+)-ABA (Fig. 4) supports this interpretation.
In maize ears, kernels in apical regions accumulate ABA
to a greater extent than those in middle and basal regions
(Ober and Setter, 1990; Ober et al., 1991; Setter et al.,
2001). Due to their import of solutes and water via the
phloem rather than xylem, kernel tissues remain at high
Wang et al. Ð ABA Catabolism in Maize Kernels
water potential during whole-plant water de®cit treatments
that substantially induce ABA synthesis in leaves and other
plant parts (Westgate and Thompson Grant, 1989; Ober and
Setter, 1990). Studies with ABA synthesis mutants indicated
that ABA in endosperms of stressed kernels is imported
from maternal tissues (Ober and Setter, 1992), yet the faster
growth and in¯ux of phloem solutes in basal than apical
kernels would, in turn, be expected to deliver a greater ¯ux
of maternally derived ABA to basal kernels. This led us to
postulate that ABA catabolism activity may be greater in
basal than in apical kernels. However, on a fresh weight
basis, ABA catabolism activity was essentially the same in
apical and basal kernels (Fig. 5A). Nor did the present study
®nd evidence that sugar supply affects the rate of ABA
catabolism (Fig. 7A). Indeed, ABA catabolism activity was
high relative to endogenous ABA levels (Figs 1, 5 and 6).
Thus, when ABA synthesis was decreased by rewatering
intact plants (Fig. 1) or incubating explants on in vitro media
(Fig. 7), the ABA concentration declined rapidly. But
despite the high capacity for ABA catabolism, the rapid
decline did not eliminate the difference between apical and
basal kernels in the ®rst 8 h of in vitro incubation (Fig. 7). A
similar situation was observed by Jia et al. (1996) in maize
leaves leading them to suggest that the rate of ABA
catabolism is partly a function of compartmentation and the
rate at which ABA is transported to the sites of ABA
catabolism enzymes. In the present system we found that the
placenta had particularly high ABA catabolism activity
(Fig. 6) and noted that it is positioned along the transport
pathway between phloem unloading and net importing
endosperm cells. It is possible that in its transport role, the
placenta may control ABA delivery to ABA catabolic
enzymes, and a high rate of catabolism in the placenta may
decrease the ¯ux of ABA reaching the endosperm. How
these opposing forces of ABA synthesis, catabolism and
compartmentation interact in determining ABA levels in
various tissues merits further investigation.
ACKNOWLEDGEMENTS
This project was supported by grants 00-35100-9279 and
95-37100-1606 from the NRI-CGP Program of the US
Department of Agriculture.
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