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Plant Physiol. (1999) 120: 361-370
The Sensitivity of Barley Aleurone Tissue to Gibberellin Is
Heterogeneous and May Be Spatially Determined1
Sian Ritchie,
Andrew McCubbin,
Genevieve Ambrose,
Teh-hui Kao, and
Simon Gilroy*
Department of Biology, The Pennsylvania State University, 208 Mueller Laboratory, University Park, Pennsylvania 16802 (S.R., G.A.,
S.G.); and Department of Biochemistry and Molecular Biology, The
Pennsylvania State University, 403 Althouse Laboratory, University
Park, Pennsylvania 16802 (A.M., T.K.)
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ABSTRACT |
In cereals, gibberellin (GA) enhances
the synthesis and secretion of hydrolytic enzymes from aleurone cells.
These enzymes then mobilize the endosperm storage reserves that fuel
germination. The dose-response curve of aleurone protoplasts to GA
extends over a range of concentrations from 10 11 to more
than 106 M. One hypothesis is that
subpopulations of cells have different sensitivities to GA, with each
cell having a threshold concentration of GA above which it is switched
on. The dose-response curve therefore reflects a gradual recruitment of
cells to the pool exhibiting a full GA response. Alternatively, all
cells may gradually increase their responses as the GA level is
increased. In the present study we found that at increasing GA
concentrations, increasing numbers of barley (Hordeum
vulgare) cells showed the enhanced amylase secretion and
vacuolation characteristic of the GA response. We also observed that
the region of aleurone tissue closest to the embryo contains the
highest proportion of cells activated at the GA concentrations thought
to occur naturally in germinating grain. These data indicate that an
aleurone layer contains cells of varying sensitivities to GA and that
recruitment of these differentially responding pools of cells may
explain the broad dose response to GA.
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INTRODUCTION |
The aleurone layer of the barley (Hordeum vulgare)
grain secretes hydrolases that mobilize endosperm reserves during
germination (for review, see Fincher, 1989 ; Jones and Jacobsen, 1991).
The synthesis and secretion of these hydrolases (principally
 -amylases) is under hormonal regulation. GA stimulates the synthesis
and secretion of -amylase and ABA reverses this effect (Fincher, 1989; Jones and Jacobsen, 1991). Therefore, barley aleurone has been
used extensively as a model system for the study of signal transduction
in response to GA and ABA. However, the molecular basis of GA and ABA
signal transduction remains poorly understood (Bethke et al., 1997;
Ritchie and Gilroy, 1998a).
The GA enhancement of -amylase production increases with the time of
exposure to the hormone. Thus, once exposed to GA, the cells of the
aleurone layer show detectable stimulation of -amylase secretion
after a lag time of approximately 8 h (Varner and Chandra, 1964),
and this increase continues for 2 to 3 d. This GA-induced enhancement of -amylase secretion with time is thought to be due to
the recruitment of more and more cells to a secreting population rather
than to a gradual increase in the secretory activity of all of the
cells (Hillmer et al., 1993).
In addition to a time-dependent increase in secretion, GA also elicits
the stimulation of secretion over a broad range of concentrations.
Increasing [GA] induces increasing -amylase synthesis and
secretion. Two possible models for the cellular basis of this dose-response curve can be proposed (Bradford and Trewavas, 1994). Increasing GA levels could recruit more and more cells to the secreting
population, with each cell having an all-or-nothing secretory response.
This would be analogous to the explanation of the time-related increase
in secretion induced by GA proposed by Hillmer et al. (1993). An
alternative explanation is that increasing levels of GA gradually
increase the output of all of the secreting cells.
These two models suggest two very different modes of cellular
regulation. The all-or-nothing recruitment model implies a hormonal response system in which GA throws a limited number of molecular "switches" that engage the cellular machinery, leading to activated hydrolase synthesis and secretion. Cells with different [GA]
activation thresholds might have subtly different receptors with
different GA affinities, different receptor numbers, or different
requirements for receptor occupancy to elicit a response (Rodbard,
1973). However, once the receptor is triggered, the full activity of
the GA signal transduction and response machinery would be elicited. In
the second model, GA would continuously modulate the activity of each cell's secretory machinery over the entire dose-response range of the
GA response. Thus, a single cell would have to possess a GA-receptor
system capable of monitoring [GA] ranging from
1011 to 108
M, as well as a signal transduction and response
system capable of setting an appropriate intermediate secretory
activity for each [GA].
The recruitment model of the GA-response time course proposed by
Hillmer et al. (1993) suggested to us that a similar mechanism might
underlie the dose-response curve of aleurone to GA. This idea implies
heterogeneity in hormone sensitivity within cells of a single aleurone
layer, with groups of cells exhibiting a different threshold
concentration of GA above which they are activated (Bradford and
Trewavas, 1994). Thus, as GA levels are increased, more cells are
activated. This population-based threshold model has been tested at the
level of tomato seed germination. The addition of increasing
concentrations of GA (107 to
104 M) to GA-deficient tomato seeds
resulted in increasing numbers of germinated seeds, and hastened the
rate at which germination proceeded (Ni and Bradford, 1993). These
effects of GA on germination indicated that each seed had a threshold
GA concentration below which germination failed to occur. Likewise,
increasing the applied [GA] beyond this threshold resulted in a
faster rate of germination. These observations have led to the concept
of "GA time," in which a GA response is proposed to include
components of the concentration of GA and the rate of the GA response.
The rate of response depends on how much the [GA] is above the
response threshold (Bradford and Trewavas, 1994).
The threshold/response model was originally proposed to explain the
hormone and inhibitor responses in mammalian endocrine cells (Rodbard,
1973). This model of hormone responsiveness predicted that the behavior
of a tissue reflects the average response of a heterogeneous population
of cells. We report that individual aleurone cells and protoplasts
respond to increasing GA concentrations in an all-or-nothing manner,
which is consistent with such a threshold model of activation. As the
level of GA increases, more cells exhibit the GA response, suggesting
that this hormone acts to recruit cells with different thresholds for
activation. In addition, the two ends of the aleurone layer are shown
to have significantly different sensitivities to the endogenous levels
of GA released from the embryo. These results suggest that position
within the grain may be one factor that determines the GA sensitivity
of an aleurone cell, and that developmental factors may play a role in
determining this property.
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MATERIALS AND METHODS |
Plant Material and Aleurone Cell Preparation
Barley (Hordeum vulgare cv Himalaya, provided by the
Department of Agronomy, Washington State University, Pullman) grains were de-embryonated and prepared for aleurone layer or protoplast isolation as described by Deikman and Jones (1985) and Hillmer et al.
(1993). Aleurone layers or protoplasts were incubated for 16 h
with 10 mM CaCl2 and
GA3, ABA, or GA3 plus ABA
at various concentrations. -Amylase secretion was assayed as
described by Bush and Jones (1988). For preparation of the different
sections, de-embryonated seed halves were cut into either proximal and
distal regions (relative to the embryo), or left and right sides of the grain. To control for variations in the size of the pieces used, the
layers for each sample were weighed after removal of the starch and
also at the end of each treatment. Proximal and distal tissues were
routinely produced with similar weights (±3%) from an individual seed. Although dorsal/ventral sections of aleurone layer were also
taken, these proved difficult to prepare reproducibly. In addition, the
ventral section contained a large region of suture tissue that is very
different from the rest of the aleurone layer (Cochrane and Duffus,
1980; Olsen et al., 1992). Because of these difficulties, we restricted
our analysis to the proximal/distal and left/right regions.
Embedding Protoplasts for Monitoring -Amylase Secretion
from Individual Protoplasts
Single aleurone protoplasts were embedded in a gel matrix
according to the method of Gilroy and Jones (1994). The gel matrix contained 3% (w/v) ultra-low-melting-point agarose (Sigma) and 3%
(w/v) soluble potato starch (Baker Chemical, Philadelphia, PA) in
Gamborg's B5 medium supplemented with 0.5 M mannitol.
Single-cell secretion assays were carried out as described previously
(Hillmer et al., 1993).
Zymograms and IEF Immunoblotting
Glycerol was added to a final concentration of 10% (v/v) to
samples of incubation medium from layers treated for 16 h with various concentrations of GA3 (as described in
the figure legends). Polyacrylamide IEF gels (1 mm) were cast onto the
hydrophobic side of film (Gelbond PAG, FMC Bioproducts, Rockland, ME).
The gels consisted of 5% (w/v) acrylamide/bis (37.5:1), 10% (v/v) glycerol, 0.93% (v/v) Ampholine (Amersham Pharmacia Biotech,
Piscataway, NJ), pH 3.5 to 10.0, 0.067% (v/v) Ampholine, pH 4.0 to 6.0, 0.067% (v/v) Ampholine, pH 5.0 to 7.0, and 0.044% (w/v) Glu,
giving an overall pH range of 3.5 to 10.0. Polymerization was initiated by the addition of 600 µL of 1% (w/v) ammonium persulfate, 20 µL
of
N,N,N ,N-tetramethylethylenediamine,
and 20 µL of 1% (w/v) riboflavin in a final volume of 40 mL. Five
microliters of each sample was loaded onto application wicks at
positions equivalent to a final pH of 6.0. The gels were run at a
constant 1 W/cm gel for 30 min, after which time the wicks were removed
and the gel was run for a further 45 min using an IEF apparatus (LKB
Multiphor, Amersham-Pharmacia Biotech).
After focusing, a piece of chromatography paper was pressed evenly onto
the gel surface, and the paper was peeled back with the gel attached
and removed from the backing sheet. The gel was equilibrated for 10 min
in 0.3% (v/v) acetic acid and then blotted onto PVDF membranes
(Immobilon-P, Millipore) in the same buffer for 1 h at 100 V using
a TransBlot Cell (Bio-Rad). Immunodetection of -amylase was carried
out using an -amylase antibody, as described previously (Ritchie and
Gilroy, 1998b). For detection of amylase activity after focusing, the
gels were incubated in 50 mM
KH2PO4, pH 5.7, 5% (w/v)
starch, and 20 mM CaCl2 for 20 min at
room temperature with gentle shaking. The starch solution was poured
off and the gels were washed with water. The remaining starch was
stained for 5 min using 0.03% (w/v) I2, 0.3%
(w/v) IKI, and 0.05 N HCl. The stain was washed off with
water and regions of amylase activity were revealed as cleared areas of
the gel devoid of stained starch. Gels and immunoblots were imaged
using a scanner (ColorOne, Apple Computer, Cupertino, CA) and
densitometry was performed using image-analysis software (Spectrum,
IPLabs Signal Analytics, Vienna, VA).
(1 3,14)- -Glucanase Assays
Samples of medium from layers treated for 16 h with various
GA3 concentrations were centrifuged for 5 min at
16,000g (model 415C Eppendorf centrifuge, Brinkmann) to
remove particulate matter. A 50-µL sample was then added to 500 µL
of 0.1 M succinate and 1 mM
EGTA, pH 5.8, containing 250 µg of -glucan from barley (Sigma), vortexed, and left at room temperature for 1 h. Then, 250 µL of 1 mg/mL Congo red dye (Sigma) was added, the mixture was vortexed and
centrifuged for 5 min at 16,000g, and the supernatant was removed. The Congo red precipitated the -glucan and a pellet was
formed after centrifugation (Wood, 1982). Because the amount of Congo
red remaining in the supernatant is inversely proportional to the
amount of -glucan in the sample, the more
(13,14)--glucanase activity in the sample, the more Congo red
remaining in the supernatant. The amount of dye in the supernatant was
assayed spectrophotometrically (model Du7500, Beckman) at
A550.
A calibration curve was constructed using a range of -glucan
concentrations from 100 to 300 µg per assay (see Fig. 4A), and the
activity of (13,14)--glucanase from Bacillus
subtilis (Fluka) was used as the standard enzyme activity. To
determine whether the -amylases produced by aleurone would interfere
with this assay as a result of contamination of the -glucan with
polymer containing -bonds, up to 10 µg of purified Bacillus
licheniformis -amylase (Sigma) was added to the assay, but did
not yield detectable glucan hydrolysis. Similarly, when 100 µg of the
amylase/subtilisin inhibitor (Sigma) was added to 1 mL of medium from
25 aleurone layers that had been treated for 16 h with 5 µM GA3, it reduced the
activity of amylase in the sample by 60%. This
amylase/subtilisin-inhibitor treatment had no detectable effect on the
glucanase activity measured in the same sample using this Congo red
assay, suggesting that the -amylase present in the samples from
aleurone would not contribute significantly to the
(13,14)--glucanase activity monitored. Thus, the glucanase
assay appears selective for monitoring glucanase activity in the
background of aleurone -amylase.
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| Figure 4.
Analysis of secreted -amylase isoforms in
response to different [GA] from aleurone layers isolated from distal
and proximal portions of the grain relative to the embryo. Samples are
from one representative experiment of four carried out as described for
Figure 3B, in which aleurone layers were treated with different [GA]
for 16 h. A, IEF zymogram showing negatively staining bands of
-amylase activity. B, Immunoblot detecting isoforms of -amylase.
The figures beneath B show the densitometry of representative high-pI
(a) or low-pI (b) bands normalized to 100% for 107
M GA treatment.
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Visualizing Vacuolation
Aleurone layers were incubated in 10 µM BCECF-AM
(Molecular Probes, Eugene, OR) prepared from a 1 mM stock
in DMSO (final DMSO concentration, 0.2%, v/v) for 1 to 2 h, after
which time the BCECF was loaded into the vacuoles (Swanson and Jones,
1996). BCECF fluorescence was visualized using an inverted
epifluorescent microscope (Diaphot 300, Nikon) using a ×40, dry, 0.7 numerical aperture fluor objective, with 480 nm excitation, a 500-nm
dichroic mirror, and >530 nm emission. Images were captured using a
cooled CCD (charge-coupled device) camera (CH250A, Photometrics,
Tucson, AZ), and the size of vacuoles was measured using image-analysis software. For each vacuole the microscope was focused to the plane of
the largest cross-sectional area before the image was digitized and the
vacuole size was measured. The degree of aleurone cell or protoplast
vacuolation was assigned to stages I to IV as defined by Bush et al.
(1986). Aleurone cells show a progressive vacuolation in response to GA
from many small vacuoles (stage I) to extensive vacuolation (stage IV).
Vacuolation to stage IV is thus indicative of a maximal GA response.
Individual aleurone cells are between 25 and 30 µm in diameter. Cells
with a large vacuole (>20 µm in diameter) were scored as having
reached stage IV. Cells were scored as stage III if one or more
vacuoles were 15 to 20 µm, and as stage II if one or more vacuoles
were 7 to 15 µm. Cells with small vacuoles only (<7 µm) were
scored as stage I.
Vacuolation of protoplasts was also scored based on the stages
described by Bush et al. (1986); vacuolar development is easily visible
using light microscopy. At stage I the cytoplasm is very dense and
vacuoles are barely visible. Once the GA response is initiated, the
vacuoles enlarge and become much more visible. Protoplasts of stage III
or IV (as defined by Bush et al., 1986) were scored as vacuolated,
clearly exhibiting the GA response.
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RESULTS |
Individual Aleurone Protoplasts Do Not Respond to GA as a
Homogeneous Population
We first asked whether all or only some aleurone cells respond to
increasing [GA] by increasing their levels of secretion. Therefore,
we monitored the GA response in isolated aleurone cell protoplasts.
Aleurone protoplasts respond similarly to GA, as do cells of the
aleurone layer (Hillmer et al., 1993). However, the use of protoplasts
allowed us to analyze the GA response of individual cells using a
series of single-cell response assays developed for use with
protoplasts. GA-induced secretion can be assayed from single aleurone
protoplasts using thin starch films in which a secreting protoplast
digests the starch in its immediate vicinity. This region of starch
digestion is then visualized as a cleared "halo" after the starch
in the gel is stained blue with iodine reagent (Hillmer et al., 1993).
Another single-cell measure of the GA response is the development of
prominent vacuolation in protoplasts (Bush et al., 1986; Gilroy, 1996).
Figure 1 shows that at low concentrations
of GA only a small percentage of cells exhibited secretory activity or
vacuolation by 48 h of GA treatment, whereas at increasing GA
concentrations more cells exhibited a detectable GA response. These
observations are consistent with increasing GA levels recruiting more
cells to a full GA response. However, this result could also arise from protoplasts being differentially damaged or desensitized during the
process of cell wall removal. Therefore, we repeated these assays in
intact aleurone cells, in which there would be no potential artifacts
due to protoplast formation.
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| Figure 1.
Dose-response curve of aleurone protoplasts to GA
assayed by single-cell secretion ( ) and vacuolation ( ). Freshly
released protoplasts were treated for 48 h with various [GA],
after which single-cell secretion assays were carried out and
vacuolation was assessed. The data show mean percentages ± SE from three separate experiments. For single-cell
secretion, at least 60 protoplasts were examined per treatment per
experiment. For vacuolation, at least 300 protoplasts were examined.
Protoplasts were classified as exhibiting GA-induced vacuolation if
they were at stage III or IV (as defined by Bush et al., 1986) and as
secreting if they had digested a clear zone of at least 100 µm in
diameter in the single-cell secretion assay. n.a., No GA added.
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Vacuole Development in Aleurone Tissue in Response to GA
The cells in an intact aleurone layer cannot be assayed by the
single-cell secretion assay described above for protoplasts. However,
as with protoplasts, development of vacuolation can be used to monitor
the GA response in intact aleurone layers. Vacuole size was determined
using the fluorescent dye BCECF-AM, which is readily taken up into the
vacuole of aleurone cells (Swanson and Jones, 1996). We recorded
fluorescence images of layers incubated in the BCECF-AM and visualized
vacuole size in the cells of intact aleurone layers after treatment
with various concentrations of GA. Cells were assigned to one of four
categories as described by Bush et al. (1986). As shown in Figure
2A, at stage I the cells contained small
vacuoles characteristic of untreated aleurone cells, at stages II and
III the cells had increasingly larger vacuoles, and at stage IV the
volume of the cell was almost entirely occupied by one or two large
vacuoles. This increasing vacuolation is characteristic of
GA-responding cells. Figure 2, B to E, shows that, similar to
protoplasts, as the [GA] was increased the proportion of vacuolated
cells in the aleurone layer also increased.
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| Figure 2.
The effect of GA on the vacuolation of aleurone
cells. A, Fluorescence micrographs of aleurone cells loaded with
BCECF-AM showing vacuolation from stages I to IV. Bar = 10 µm. B
to E, Comparison of stages of vacuolation of cells proximal (black
bars) and distal (white bars) to the embryo treated with a range of
[GA] for 16 h. Layers were loaded with BCECF-AM as described in
"Materials and Methods," and vacuole size was monitored by
fluorescence microscopy. The data represent means ± SE for three experiments (n  3 aleurone
layers and 150 cells per experiment). n.a., No GA added.
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In conducting these assays we noted a spatial component to the
GA-regulated vacuolation patterns in the aleurone layer. As shown in
Figure 2, B to E, in the absence of added GA the proportions of
vacuolated cells were the same at both ends of the layer. Similarly, at
saturating GA concentrations (>106
M) all regions of the layers appeared similar. However,
between 109 and 106
M GA, although the proportions of cells showing
vacuolation to stages II, III, and IV increased with increasing [GA],
this increase was greater in the proximal region of the layer (nearest
the embryo in the intact grain) than in the distal region (farthest
from the embryo in the intact grain). To ensure that the difference in
the responses of the two ends of the layer was not due to differential damage during the preparation of the layer, we used the vital dye
fluorescein diacetate (Huang et al., 1986) to determine the number of
dead cells in freshly stripped layers, and found no difference in the
proportion (95%) of living cells between the two ends of the layer.
We also determined the number of vacuolated cells after 5 d of
treatment with GA. Under these conditions all viable, responsive cells
should have completed their response to GA. At this time there was no
difference in the proportion of stage IV cells (92% ± 5%) at either
end of the layers. Finally, we examined the vacuolation of aleurone
cells directly beneath the testa pericarp. After imbibition the testa
pericarp can be removed with forceps from an intact half seed,
revealing the aleurone cells beneath. These cells were not subjected to
the starch-removal protocol of a standard aleurone tissue preparation,
which would affect cells on the other side of the three-cell-thick
aleurone layer. The range and degree of vacuolation and GA dose
response of these aleurone cells just below the seed coat were
identical to those of the innermost layer of cells examined (Fig. 2),
the cells on the other face of the aleurone layer. The proximal/distal
difference in vacuolation was also evident in aleurone cells just below
the testa pericarp (data not shown). These results suggested that the
difference in the response to GA of the proximal and distal regions of
the aleurone layer was not an artifact of tissue preparation.
-Amylase and (13,14)--Glucanase Secretion Differs
between Proximal and Distal Aleurone Tissue
Vacuolation is only one measure of the GA response of the aleurone
layer. Therefore, we asked whether the spatial difference in
responsiveness was seen in other aspects of the GA response. We
compared the GA dose-response curves of the left versus the right side
of the aleurone layer, and proximally versus distally (depicted
schematically in Fig. 3) with respect to
GA-induced -amylase production. Figure 3A shows that the left and
right sides of the aleurone layer did not differ in their GA
dose-response curves. However, as shown in Figure 3B, the proximal part
of the aleurone showed a statistically significantly higher level of secreted amylase activity than the distal part (in the range of 3 × 1011 to 1 × 108 M GA; P < 0.05 by
t test). Outside of this range the responses were
identical.
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| Figure 3.
Dose-response curve of aleurone layers to GA
assayed by secreted -amylase activity. A, Comparison of the
-amylase secreted in response to different [GA] by aleurone tissue
from the left () and right () sides of the grain relative to the
embryo. B, Comparison of the -amylase secreted in response to
different [GA] by aleurone tissue proximal ( ) and distal ( ) to
the embryo. Layers were prepared and cut as depicted in the diagrams
included with the graphs. After treatment with various concentrations
of GA for 16 h, secreted -amylase was assayed. Data are
means ± SE from at least three separate experiments.
n.a., No GA added.
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The -amylase synthesized and secreted by aleurone cells of cereals
is made up of many different isoforms that are enhanced to different
degrees by GA (Jacobsen et al., 1970; Jacobsen and Higgins, 1982;
Lazarus et al., 1985; Rogers, 1985; Huttly et al., 1988; Karrer et al.,
1991). Therefore, we separated the isoforms produced at each end of the
layer at particular GA concentrations by IEF to determine whether
different -amylases might be secreted by the proximal and distal
tissues. Figure 4 shows that when
visualized either by zymogram (Fig. 4A) or immunoblot (Fig. 4B), the
low-pI -amylase isoforms did not appear to vary significantly with
GA level. However, in the range of 1011 to
108 M GA, the increase in
production of high-pI -amylase isoforms was more prominent at lower
GA levels in the proximal tissue. This is shown quantitatively below
Figure 4B as densitometry of representative high- and low-pI bands
scanned from the immunoblot.
We next assayed another enzyme activity up-regulated by GA,
(13,14)--glucanase II (Mundy and Fincher, 1986; Stuart et al., 1986), to determine if the difference in GA response between the proximal and distal regions of the layer was specific to -amylase. An assay was developed based on the property of -glucan binding to,
and precipitating, the dye Congo red (Wood, 1982). As shown in Figure
5A, there was a nearly linear
relationship between precipitation of Congo red and the amount of
-glucan, in the range of 150 to 250 µg/assay. Therefore, we used
this relationship to assay for (13,14)--glucanase activity in
the aleurone samples. Figure 5B shows the GA dose-response curve of
proximal and distal tissue assayed for (13,14)--glucanase
secretion. This proximal/distal dose-response curve is similar to that
of the -amylase graph (Fig. 3B) in that the proximal layers showed a
statistically significantly (P < 0.05 by t test)
greater response than the distal cells at 1010
to 108 M GA, whereas outside
of this range the levels of secreted (13,14)--glucanase were
similar.
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| Figure 5.
(13,14)--Glucanase assay and secreted
(13,14)--glucanase from aleurone layers proximal and distal to
the embryo in response to different [GA]. A, Standard curve of the
relationship between the amount of -glucan added per assay and
unprecipitated Congo red as expressed as
A550 of the assay supernatant. Data are
means ± SE from one representative experiment of
three. Note that as the glucan level increased more Congo red was
precipitated. B, Assay of (13,14)--glucanase activity secreted
from aleurone layers proximal () and distal () to the embryo and
treated with different [GA] for 16 h using the Congo red
precipitation assay. Data are means ± SE from three
separate experiments. n.a., No GA added.
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Proximal Aleurone Protoplasts Show Enhanced Sensitivity to GA
As noted previously, aleurone protoplasts responded to GA in the
same manner as aleurone layers. Therefore, we looked at protoplasts isolated from proximal and distal regions of the aleurone layer at
48 h after treatment with various GA concentrations to determine if the difference in GA responsiveness was also evident in protoplasts. As shown in Figure 6, protoplasts
isolated from proximal regions showed a statistically significant
increase in response to GA relative to those isolated from distal
regions (P < 0.05 by t test), confirming the
results from the aleurone layers. It is important to note, however,
that protoplasts require higher GA levels to yield the same response as
intact layers. This is in keeping with previous reports indicating that
protoplasts respond qualitatively identically to intact cells but that
the speed of their response is slower (Jacobsen and Beach, 1985;
Hillmer et al., 1993; Bethke et al., 1997). In addition, the
dose-response curve of protoplasts to GA is broader than that of layers
(Fig. 3). This altered dose response may reflect a subtle change in the
GA-response system upon protoplast formation. Alternatively, the more
rapidly responding cells of the intact layer may have more fully
completed their GA response at the time of the assay than the slower
protoplasts. Assaying the protoplasts while they were still progressing
toward a maximal GA response could have led to the broader apparent
dose-response curve, because even the less-sensitive cells of the layer
would have completed an obvious GA response at the time of assay. In
contrast, the less-sensitive pool of protoplasts (i.e. the
slower-responding group) would still be developing the full response
and so would appear as a broadening in the dose response of the
protoplast population.
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| Figure 6.
Dose-response curves to GA of protoplasts prepared
from aleurone layers proximal () and distal () to the embryo. A,
Secreted -amylase activity. Data are means ± SE
from three separate experiments. B, Vacuolation of protoplasts. Freshly
isolated aleurone protoplasts were treated with various [GA] and
after 48 h secreted -amylase activity and the degree of
vacuolation were assessed. Percentages are shown from three replicates
of at least 200 protoplasts per treatment per experiment. n.a., No GA
added.
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The ABA Response of Proximal and Distal Layers
During seed development the levels of ABA increase dramatically
and then later decline as the seed reaches maturity (King, 1976). ABA
also inhibits the GA response of aleurone cells. Therefore, we wanted
to determine if higher residual levels of ABA could account for the
decreased sensitivity to GA in the distal versus the proximal tissue.
We first determined whether proximal and distal tissue showed marked
differences in ABA sensitivity. Figure 7
shows that at 1 × 109 M ABA
there was no inhibition of -amylase secretion from the proximal or
distal layers elicited by 1 × 106
M GA. Increasing the ABA concentration from 3.3 × 109 to 1 × 107 M resulted in an increasing
inhibition of -amylase production, but this inhibition was not
different between the proximal and distal tissues. Thus, the proximal
and distal tissues did not exhibit a pronounced difference in ABA
sensitivity at saturating (1 × 106
M) GA levels.
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| Figure 7.
Dose-response curve of aleurone layers to ABA as
assessed by ABA suppression of GA-stimulated -amylase secretion.
Aleurone layers isolated from regions proximal () and distal ()
to the embryo were treated with 1 × 106
M GA and different [ABA] for 16 h, after which time
secreted -amylase was assayed. Data are means ± SE
from three separate experiments.
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Figure 3 shows that at much lower GA levels (1 × 109 M), proximal tissue secreted
more -amylase than distal tissue. Therefore, we exposed proximal
tissue treated with 1 × 109 M
GA to a range of ABA concentrations to determine if we could reduce the
level of -amylase produced to that of the distal tissue. Figure
8 shows that at between 1 × 1011 and 1 × 108
M GA, 3.3 × 109
M ABA inhibited the -amylase produced by the
proximal tissue to a level resembling that produced by the distal
tissue. This suggests that if the lower response to GA of the distal
tissue is due to endogenous ABA levels, these would likely be
approximately 3.3 × 109
M. We reasoned that if this was true, incubation of the
distal tissue in a medium containing 3.3 × 109
M ABA should have no effect on the GA response,
because the tissue must already contain this concentration of ABA.
Mixing two solutions of equal [ABA] will not alter the final [ABA]
of either, and thus incubation of aleurone tissue already containing
3.3 × 109 M ABA in a
solution of 3.3 × 109
M will not alter the [ABA] of the tissue.
Figure 8 shows that there was a statistically significant decrease
(P < 0.05 by t test) in -amylase activity
secreted by the distal tissue treated with 1 × 109 M GA and 3.3 × 109 M ABA, which is
inconsistent with the idea that distal tissue already has an internal
concentration of 3.3 × 109
M ABA.
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| Figure 8.
The effect of ABA on the inhibition of the GA
response of proximal and distal aleurone tissues. Proximal and distal
aleurone layers were prepared and treated with various [GA] with or
without 3.3 × 109 M ABA (as indicated)
for 16 h, after which time secreted -amylase activity was
assayed. Data are means ± SE from duplicate samples
from three separate experiments.
|
|
| |
DISCUSSION |
Our results support the model for the GA response of the barley
aleurone layer whereby different populations of aleurone cells have
different thresholds for the GA response (Bradford and Trewavas, 1994).
This suggests that GA does not exert a continuous, graduated control of
cellular functions but, rather, that above a critical threshold its
signal is transduced via a molecular switch(es) that triggers
subsequent GA responses. Such a switch presumably lies close to the
initial GA-perception event. Our observations concur with the threshold
model originally developed by Rodbard (1973) for mammalian cells, which
has also been suggested to be applicable to plant cells (Bradford and
Trewavas, 1994). Underlying these models for a threshold basis of
dose-response curves is a requirement for the cell-to-cell variation in
hormone sensitivity that we observed in both aleurone protoplasts and
the intact aleurone layer. Therefore, the aleurone layer contains cells
with thresholds for GA activation that span the entire dose-response
range of the tissue, and the effect of increasing hormone concentration is to recruit cells with progressively lower sensitivities to GA to the
secreting population. Responsiveness to a broad range of hormone
concentrations is not unique to aleurone tissue; for example,
inhibition of root elongation is sensitive to auxin through approximately 3 orders of magnitude in concentration (Evans et al.,
1994). Therefore, a threshold-based recruitment phenomenon could
explain many plant hormone-response systems. Testing the generality of
this model in plants must await collection of data on hormone
sensitivity at the single-cell level for these other hormone responses.
The differential sensitivity to GA is reflected spatially in the
aleurone layer. The proximal end of the aleurone layer (that nearest
the embryo in the intact seed) shows a higher sensitivity to GA than
the distal region. We assayed for GA responsiveness in three ways:
secreted -amylase activity, (13,14)--glucanase activity,
and vacuolation. In all three assays the proximal cells showed a
greater response to GA concentrations in the range of 1010 to 108 M than cells
from the distal region of the aleurone layer. The proximal and distal
tissues had the same non-GA-treated -amylase activity and the same
maximum capacity for -amylase secretion at saturating levels of GA
(1 × 106 M). This
means that there are probably no inherent differences in the metabolic
or secretory capacity of these regions, but more likely a genuine
difference in the proportions of GA-sensitive cells.
From Figure 4 it is clear that the high-pI -amylase isoforms are the
cause of the differences in total secreted -amylase activity. This
group of -amylase isoforms is thought to be much more highly induced
by GA than the low-pI isoforms (Fig. 4; Jacobsen and Higgins, 1982;
Rogers, 1985), so the difference in isoforms produced provides further
evidence that the distal cells do not have a lower capacity for
secretion but, rather, that regulation of specific GA-sensitive genes
is responsible for the difference. Studies of the production and
secretion of (13,14)--glucanases in barley seed tissue have
demonstrated that the aleurone layer produces only isoform II (Stuart
et al., 1986), and that secreted activity is enhanced significantly by
the application of GA. Therefore, the differential levels of secreted
(13,14)--glucanase activity seen in Figure 5 suggest a
differential GA response, with the proximal region responding at lower
GA levels with enhanced (13,14)--glucanase secretion.
There was no apparent difference in sensitivity to ABA between the ends
of the aleurone layer (Fig. 7). The method we used to assess ABA
sensitivity was inhibition of GA-stimulated -amylase activity at
saturating GA levels (1 × 106
M). We used this saturating [GA] to ensure that the
levels of -amylase production were the same throughout the layer
before ABA treatment. Therefore, we cannot rule out the possibility
that at lower [GA] there is a differential response to ABA between the proximal and distal regions of the layer. However, such analysis of
the ABA response would be complicated by the differential GA response
between the ends of the layer under these subsaturating [GA]. Another
aspect to the ABA response of the aleurone layer is that a range of
genes are up-regulated after ABA application (Bethke et al., 1997). It
will be interesting to see if measurement of the transcription of these
ABA-regulated genes in the proximal and distal tissue reveals spatial
heterogeneity in ABA sensitivity in this alternative aspect of the ABA
response of the aleurone layer.
It is possible that the spatial patterning of the GA responsiveness in
the aleurone layer could reflect the patterns set up during aleurone
development. During seed formation, the aleurone starts to form from
the endosperm cells around the ventral groove, and then in other
locations around the periphery of the endosperm. The left and right
sides and the dorsal region of the aleurone layer originate separately
(Kowles and Phillips, 1988; Bosnes et al., 1992). Therefore, the
patterns of aleurone development might predict a dorsal/ventral or
left/right difference in aleurone layer physiology, but not the
proximal/distal differences seen in this study.
An alternative explanation for the differences in GA responsiveness
between the different ends of the layer might be different residual
levels of ABA between the ends. Levels of ABA increase during cereal
seed development, and then decline as maturation occurs (King, 1976).
ABA inhibits the GA response and so higher residual ABA levels in
distal tissues could desensitize this region to GA. We think this
hypothesis is unlikely because the distal region of the layer still
responds to 3.3 × 109 M ABA. This
is the concentration required to inhibit the proximal tissue to the
level of the distal tissue and thus the level predicted to already be
in the distal tissue (Fig. 8). This interpretation assumes that no
active ABA uptake processes are accumulating ABA to levels above the
3.3 × 109 M in the medium.
However, ABA levels have also been measured in aleurone tissue (Napier
et al., 1989), and in fully mature seeds there was no detectable ABA in
the aleurone despite the immunoassay used being sensitive to
concentrations of 1 × 109 M.
Taken together, these observations suggest that different levels of ABA
are unlikely to account for the proximal-to-distal difference in GA
sensitivity in the aleurone.
The heterogeneity of cereal aleurone cells in response to GA has
already been suggested by several kinds of data. Immunolocalization of
-amylase in aleurone layers revealed that of the three layers of
cells, the central layer gave the most intense signal after 16 h
of treatment with 1 × 106
M GA (Jacobsen and Knox, 1973). In addition, both
wheat aleurone cells and barley aleurone protoplasts showed several
different patterns in the cytoplasmic calcium increase in response to
5 × 106 M GA (Bush, 1996;
Gilroy, 1996). Hillmer et al. (1993) noted that after 48 h of
treatment with 5 × 106 M GA,
individual aleurone protoplasts were gradually recruited to secrete
-amylase, suggesting a heterogeneous population of cells. Schuurink
et al. (1997) used a range of microscopic techniques to show patches of
GA-responding cells in layers of a dormant barley variety. Finally,
Hoecker et al. (1995) found spatial patterning in transcription factor
activities in aleurone. In maize aleurone the transcription factor VP1
is involved in ABA-related repression of -amylase production
(Hoecker et al., 1995). In a line that contains a somatically unstable
mutant allele of VP1, a mosaic pattern of precociously activated
aleurone cells was found (Hoecker et al., 1995). The aleurone nearest
the embryo contained more of these mutant sectors than areas away from
the embryo, suggesting that these regions are somehow inherently
different.
During germination of the cereal grain, GA is released from the embryo
into the starchy endosperm. Thus, in a whole-seed scenario the aleurone
cells nearer the embryo will be exposed to GA sooner than those farther
away. It has been demonstrated using the accumulation of both
-amylase (Sugimoto et al., 1998) and (13,14)--glucanase (McFadden et al., 1988) mRNA as markers for the GA response that there
is a wave of activation that travels through the seed after imbibition.
Our results suggest that this wave of activation is imposed not only by
the GA level but also by the proximal/distal gradient in aleurone
sensitivity to GA. Endogenous GA levels in the germinating seed (2-3 d
after imbibition) have been estimated at 1 ×109 M to 20 ×109 M (Paleg et al., 1962; Cohen
and Paleg, 1967; Radley, 1967; Murphy and Briggs, 1973; Gaskin et al.,
1984), the range in which GA shows quantitative regulation of
-amylase production (Fig. 3) and the proximal/distal difference. The
proximal/distal differential GA sensitivity is therefore likely to
operate in the intact germinating grain, and most likely reflects a
steady and continuous decline in GA sensitivity, from highly sensitive
cells near the embryo to less-sensitive cells in the distal regions of
the aleurone layer. Such a gradient may help to impose a steady and
progressive mobilization of endosperm reserves starting near the embryo
and progressing distally, and so ensure an extended supply of fuel for
the processes of seed germination and early seedling growth.
The question remains regarding the cause of differential sensitivity
between cells. At a molecular level, differential sensitivity could
arise from different cells having different receptor types with varying
affinities for GA, receptors with altered requirements for the time of
receptor occupancy, or GA receptors that are posttranscriptionally or
posttranslationally modified in some way (Rodbard, 1973; Hausdorff et
al., 1990; Freedman and Lefkowitz, 1996). Alternatively, differential hormone sensitivities could be generated by cells containing different numbers of receptors (Rodbard, 1973). In the latter scenario, a GA
receptor need not bind GA over the broad concentration range known to
elicit tissue responses in plants, because it would be its abundance in
the cell that determines each cell's threshold for response. Yet it
may be a component downstream of the receptor that is variable and so
leads to heterogeneity in the hormone response. There are some clues to
the components of the signal transduction elements in the GA response
of aleurone (Ritchie and Gilroy, 1998a), but we await identification of
a GA receptor to probe the molecular basis of the sensitivity
modulation in the aleurone cell.
| |
FOOTNOTES |
1
This work was supported by grants from the U.S.
Department of Agriculture (no. 98-3503-6669 to S.G.) and the National
Science Foundation (no. IBN-9603993 to T.K.).
*
Corresponding author; e-mail sxg12{at}psu.edu; fax
1-814-865-9131.
Received October 15, 1998;
accepted February 1, 1999.
| |
ABBREVIATIONS |
Abbreviation:
BCECF-AM, 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxy
methyl ester.
| |
ACKNOWLEDGMENTS |
We thank Elison Blancaflor and Sarah Swanson for critical
reading of the manuscript.
| |
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Top
Abstract
Introduction