Plant Physiol. (1999) 119: 81-88
Patterns of Starchy Endosperm Acidification and Protease Gene
Expression in Wheat Grains following Germination1
Fernando Domínguez and
Francisco J. Cejudo*
Instituto de Bioquímica Vegetal y Fotosíntesis,
Centro de Investigaciones Científicas "Isla de la
Cartuja," Avda Américo Vespucio s/n, 41092 Sevilla, Spain
 |
ABSTRACT |
Cereal aleurone responses to
gibberellic acid (GA3) include activation of synthesis of
hydrolytic enzymes and acidification of the external medium. We have
studied the effect of the pH of the incubation medium on the response
of wheat (Triticum aestivum) aleurone cells to
GA3. De-embryonated half grains show the capacity for
GA3-activated medium acidification when incubation is
carried out at pH 6.0 to 7.0 but not at lower pHs. In addition, the
activating effect of GA3 on the expression of
carboxypeptidase III and thiol protease genes is more efficient when
the hormone treatment is carried out at neutral pH. In situ pH staining
showed that starchy endosperm acidification takes place upon imbibition
and advances from the embryo to the distal part of the grain. In situ
hybridization experiments showed a similar pattern of expression of a
carboxypeptidase III gene, which is up-regulated by GA3 in
aleurone cells. However, aleurone gene expression precedes starchy
endosperm acidification. These findings imply that in vivo GA
perception by the aleurone layer takes place at neutral pH and suggest
that the acidification of the starchy endosperm is regulated by
GA3 in germinated wheat grains.
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INTRODUCTION |
During the development of cereal grain endosperm cells
differentiate into two different tissues, the starchy endosperm and the
aleurone layer. Although they share a common origin, only the aleurone
cells remain alive in the mature grain, whereas the starchy endosperm
cells accumulate storage compounds (predominantly starch and proteins)
and are not alive in the mature grain (Fincher, 1989
).
Following germination, the storage nutrients accumulated in the starchy
endosperm are mobilized to provide nutrients for the growing seedling.
This degradative process is carried out by hydrolytic enzymes
(amylases, proteases, glucanases, and others) that are synthesized and
secreted by the aleurone layer. GAs that diffuse from the embryo to the
starchy endosperm (Appleford and Lenton, 1997) play an important
activating role in the production of hydrolytic enzymes (Fincher, 1989;
Jacobsen et al., 1995; Bethke et al., 1997; Bewley, 1997). An acidic pH
(4.9-5.1) is maintained in the starchy endosperm of germinated barley
grains (Mikola and Virtanen, 1980). The degradation that takes place in
the starchy endosperm of germinating cereal grains requires an acidic
pH for several reasons. Proteases participating in the mobilization of
storage proteins show optimal activity at acid pH (Jacobsen and Varner, 1967; Mikola and Mikola, 1980), the solubilization of storage compounds
of the starchy endosperm is also favored at acidic pH (Hamabata et al.,
1988), which facilitates the dissociation of endogenous inhibitors from
their respective hydrolases (Halayko et al., 1986). When the hydrolases
have produced sugars, peptides, and amino acids from starch and storage
proteins, the transport of these nutrients to the scutellum requires an
acidic pH (Sopanen et al., 1980; Salmekalio and Sopanen, 1989; Hardy
and Payne, 1991).
In the barley grain an accumulation of malic acid at late stages of
grain development is thought to be responsible for starchy endosperm
acidification (Macnicol and Jacobsen, 1992). However, in wheat
(Triticum aestivum) grains no acidification has been observed during grain development (Jacobsen et al., 1995). In previous
reports we described the content of proteolytic enzymes in both
developing and germinating wheat grains (Domínguez and Cejudo,
1995, 1996). Surprisingly, we found a similar amount of neutral and
acid proteases in the starchy endosperm of germinating grains, which
suggests the coexistence of neutral and acidic compartments in the
starchy endosperm of wheat grains following germination.
After Mikola and Virtanen (1980) found that isolated aleurone layers
from germinated barley grains could acidify the external medium,
several reports described differing effects of the hormones GA3 and ABA on this capacity of the aleurone
layer. Drozdowicz and Jones (1995) found an activating effect of
GA3 on the acidification capacity of dissected
barley aleurone layers; however, Heimovaara-Dijkstra et al. (1994)
found no activating effect of GA3 on barley
aleurone protoplasts. Hamabata et al. (1988) found that dissected wheat aleurone layers acidified the external medium, but acidification was
not affected by the presence of GA3. Sinjorgo et
al. (1993) found that a reduction of medium pH enhanced
GA3 response of isolated barley aleurone layers.
In this study we analyzed the effect of the pH of the incubation medium
on the response of wheat aleurone cells to GA3.
Our results show a more efficient response at neutral than at acidic pH. In agreement with this result, in situ pH staining of the starchy
endosperm and in situ hybridization of a carboxypeptidase III gene
showed that in vivo perception of GA by the aleurone cells takes place
at neutral pH in germinated grains.
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MATERIALS AND METHODS |
Plant Material
Wheat (Triticum aestivum cv Chinese Spring) grains or
de-embryonated half grains were sterilized in 2% (v/v) NaOCl for 20 min and washed twice with sterile water, once with 0.01 M HCl, and then thoroughly again with sterile
distilled water. Sterile grains were allowed to germinate at room
temperature on sterile filter paper soaked with water for as long as
6 d. Hormone treatments were carried out on sterile de-embryonated
half grains that were incubated on filter paper soaked with different
buffers: 20 mM sodium acetate or 20 mM citrate phosphate, pH 5.0 and 5.5, 20 mM sodium phosphate, pH 6.0, 6.5, and 7.0, or 20 mM Mops-KOH, pH 7.0. All buffers were
supplemented with 10 mM
CaCl2. When required, incubation medium was
supplemented with 5 µM
GA3, 25 µM ABA, or both.
Hormone treatments were carried out in the dark at 25°C. GA3 and ABA were purchased from Sigma.
pH and Titratable Acidity
The pH of the medium in which de-embryonated half grains had been
incubated was measured using a pH meter (model 071, Beckman). Titratable acidity was measured after a sample (0.5 mL) of the incubation medium was diluted 1:5 with distilled water and titrated with 0.1 N NaOH to pH 7.0 as previously reported by
Drozdowicz and Jones (1995).
In Situ pH Staining and in Situ Hybridization
Wheat grains were allowed to soak for up to 6 d. After the
shoots and roots were cut, grains were longitudinally dissected with a
razor blade. Dissected grains were then incubated for 15 min at room
temperature in a solution containing 0.1 g of the pH indicator
Bromcresol purple (Sigma) in 250 mL of water and 37 µL of 5 N NaOH. Dissected grains were then washed in water for 2 min, observed under a stereoscopic microscope, and photographed in dark
field.
In situ hybridization was carried out as previously reported (Cejudo et
al., 1992b). Wheat grains that had been soaked for 14, 24, or 48 h
were longitudinally dissected, and after the starchy endosperm was
removed they were immediately fixed in FAE (50% ethanol, 5% acetic
acid, and 3.7% formaldehyde) with an occasional vacuum. After
dehydration tissues were embedded in Paraplast Plus (Sigma). Sections
(10 µm thick) were hybridized with 35S-labeled
RNA. Riboprobes for in situ hybridization were labeled with uridine
5
-[
-thio][35S]triphosphate (Amersham).
2437 cDNA (Baulcombe et al., 1987) subcloned in pBluescriptII KS was
amplified by PCR using standard M13 reverse and forward primers. About
1 µg of the PCR product was used as the template to synthesize
35S-labeled RNA with T7 RNA polymerase (antisense
probe) or T3 RNA polymerase (sense probe). After the samples were
hybridized and washed, slides were coated with a Hypercoat LM-1 nuclear
emulsion (Amersham) and exposed at 4°C for 5 to 7 d before
development.
Northern-Blot Hybridization
Total RNA was extracted from liquid nitrogen-frozen tissue
(Baulcombe and Buffard, 1983), fractionated on agarose-formaldehyde gels, and blotted onto Hybond-N filters (Amersham) according to the
manufacturer's instructions. Hybridization was performed as described
by Sambrook et al. (1989) with 32P-labeled
probes. RNA loading was checked by hybridization of filters with
32P-labeled radish 18S rRNA (Grellet et al.,
1989).
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RESULTS |
Effect of GA3 and External pH on Medium Acidification
and Gene Expression by De-Embryonated Wheat Grains
To test whether aleurone cells are responsible for starchy
endosperm acidification and to evaluate the role that GA plays in this
process, we studied the effect of GA3 treatment
on incubation medium acidification by de-embryonated half grains that
were incubated at different initial pHs. Table
I shows that de-embryonated half grains
incubated for 5 d at an initial pH of 6.0 to 7.0 significantly lowered the pH of the external medium, and the acidification was enhanced by GA3 treatment (up to 1 pH unit at an
initial pH of 7.0). No acidification was observed at an initial pH of
5.0 to 5.5 independently of the presence of the hormone. The trend of incubation medium acidification with time was studied when the initial
pH was 7.0 by measuring either the variation of pH in the external
medium (Fig. 1A) or the titratable
acidity (Fig. 1B). GA3 treatment hastened
acidification, which was clearly observed after the 1st d of
treatment and continued during 5 d. These results confirm the
activating effect of GA3 on the acidification
capacity of wheat aleurone cells. The fact that this activating effect of GA3 was favored as pH approached neutrality
and did not take place at pH 5.0 to 5.5 shows that this
GA3 response of the aleurone layer is pH
dependent.
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Table I.
Effect of initial pH and GA3 treatment
on incubation medium acidification by de-embryonated wheat grains
De-embryonated half grains were incubated for 5 d at 25°C in the
dark on filter paper soaked with either of the following buffers: 20 mM sodium acetate, pH 5.0 or 5.5, or 20 mM
sodium phosphate, pH 6.0, 6.5, or 7.0. All buffers were supplemented
with 10 mM CaCl2. GA3 was added at
5 µM final concentration.
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| Figure 1.
Effect of GA3 treatment on external
medium acidification by de-embryonated half grains. Wheat
de-embryonated half grains were incubated on filter paper soaked with
20 mM sodium phosphate, pH 7.0, containing 10 mM CaCl2 and supplemented ( ) or not ( )
with 5 µM GA3. At the indicated days after
imbibition acidification was recorded by measuring the pH of the
external medium (A) or titratable acidity (B). Results are means ± SE of three independent determinations.
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The influence of the initial pH of the incubation medium on
GA3 activation of gene expression was also
analyzed. De-embryonated half grains were incubated for 2 d in the
presence of different hormones (GA3, ABA, or
GA3 plus ABA) at an initial pH of 5.0 and 7.0, and the accumulation of transcripts of two GA-regulated genes encoding
proteolytic enzymes, a thiol protease- (2529 cDNA; Cejudo et
al., 1992b), and a carboxypeptidase II- (2437 cDNA; Baulcombe et al.,
1987), was studied by northern-blot hybridization. A greater accumulation of both transcripts occurred when
GA3 treatment was carried out at pH 7.0, using
either phosphate buffer (I) or Mops buffer (II; Fig.
2). ABA treatment, which itself did not
show any positive effect on the level of transcript accumulation, did counteract the activating effect of GA3 at both
pHs, although this effect was more evident at pH 5.0, when the level of
transcript accumulation was lower.
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| Figure 2.
Effect of external pH on hormonal regulation of
carboxypeptidase III (2437 cDNA) and thiol protease (2529 cDNA)
transcript accumulation. Wheat de-embryonated half grains were
incubated for 2 d on filter paper soaked with 20 mM
sodium acetate, pH 5.0; 20 mM sodium phosphate, pH 7.0 (I);
or 20 mM Mops-NaOH, pH 7.0 (II), supplemented with 10 mM CaCl2. Hormone treatments were as follows:
lanes 1, no hormone; lanes 2, 5 µM GA3; lanes
3, 25 µM ABA; lanes 4, 5 µM GA3
plus 25 µM ABA. RNA samples (10 µg) were
electrophoresed, blotted onto Hybond-N filters, and hybridized with
32P-labeled 2529 cDNA (thiol protease) or 2437 cDNA
(carboxypeptidase III). Even loading was checked by rehybridizing
filters with 32P-labeled 18S rRNA.
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Since GA3 treatment of de-embryonated grains at
neutral pH promotes acidification of the incubation medium (Fig. 1), we
tested whether this GA3-promoted acidification
had any effect on GA3 activation of gene
expression. For this purpose the buffer concentration of the incubation
medium was increased to 100 mM sodium phosphate and
transcript accumulation after 1, 2, and 5 d of treatment with GA3 was analyzed by northern-blot hybridization
(Fig. 3). GA3 treatment of de-embryonated grains for 5 d in 100 mM
sodium phosphate lowered the external pH from 7.0 to 6.3; in contrast,
when treatment was carried out in 20 mM sodium phosphate
(pH 7.0), the pH of the incubation medium decreased to 4.4 (Table
I). In spite of this variation of the incubation medium pH,
GA3 treatment promoted a clear activation of
transcript accumulation in all of the conditions analyzed (Fig. 3,
lanes 1 and 2). To test for a possible effect of the high ionic
strength on the response of aleurone cells to GA3, hormone treatment was also carried out in
the presence of 20 mM sodium phosphate (pH 7.0)
supplemented with 100 mM NaCl. A similar level of
transcript accumulation in response to GA3 was
observed in these conditions (Fig. 3). It is worth mentioning that
after 5 d of GA3 treatment the mRNA of both
genes analyzed was almost undetectable in all of the conditions tested
(Fig. 3, lanes 5). This result suggests that regulation of protease gene expression in aleurone cells may also involve mRNA stability.
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| Figure 3.
Effect of buffer concentration on GA3
activation of carboxypeptidase III (2437 cDNA) and thiol protease (2529 cDNA) transcript accumulation at neutral pH. De-embryonated half grains
were incubated for 0, 1, 2, or 5 d on filter paper soaked with
sodium phosphate buffer (NaPhos) at the indicated concentration and pH
7.0, supplemented with 10 mM CaCl2 and 5 µM GA3. RNA samples (10 µg) were loaded,
electrophoresed, blotted onto Hybond-N filters, and hybridized with
32P-labeled 2529 cDNA (thiol protease) or 2437 cDNA
(carboxypeptidase III). Even loading was checked by rehybridizing
filters with 32P-labeled 18S rRNA.
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Pattern of Starchy Endosperm Acidification in Wheat Grains
following Germination
The observation that the pH of the incubation medium influences
the response of wheat aleurone cells to GA3
prompted us to study in situ the acidification of the starchy endosperm
in germinated grains. A tissue stain was devised using the pH indicator
Bromcresol purple to study in situ the variation of pH in the starchy
endosperm. This pH indicator shows brick red at pH 6.8 or above and
yellow at pH 5.2 or below, permitting distinction between neutral and acidic pH. Figure 4 shows the pattern of
acidification of the starchy endosperm of wheat grains following
germination. In grains that had been soaked for 3 d, most of the
starchy endosperm appeared brick red, indicating neutral pH. A yellow
stain was observed, however, in the area adjacent to the embryo, which
is indicative of acidic pH (Fig. 4A). Four days after imbibition, the
acidity advanced progressively toward the distal part of the grain
(Fig. 4B); by 6 d after imbibition, most of the starchy endosperm
showed acidic pH and was clearly degraded (Fig. 4C). From these results it may be concluded that the pH of the starchy endosperm is neutral in
the mature wheat grain and that acidification takes place during germination and seedling growth.
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| Figure 4.
pH staining of the starchy endosperm of wheat
grains following germination. Wheat grains that were soaked for 3 d (A), 4 d (B), or 6 d (C) were longitudinally sectioned and
stained with the pH indicator Bromcresol purple for 15 min. A yellow
color corresponds to pH 5.2 or below and a brick red color corresponds
to pH 6.8 or above. sc, Scutellum.
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Pattern of Expression of a Carboxypeptidase III Gene in
Germinating Grains
Based on the fact that the expression of genes encoding hydrolytic
enzymes in aleurone cells is activated by GA3
treatment, we used one of these genes, encoding a carboxypeptidase III
(Baulcombe et al., 1987), as a sensor of in vivo GA perception by the
aleurone cells following grain germination. The pattern of expression
of this gene was studied by in situ hybridization. When in situ
hybridization was carried out on sections of grains after 14 h of
imbibition with the 35S-labeled antisense probe,
expression was restricted to aleurone cells adjacent to the scutellum
(Fig. 5B, arrowhead). In grains that had
been soaked for 24 h (Fig. 5C) or 48 h (Fig. 5D), it was
observed that, as germination proceeded, carboxypeptidase III
expression in the aleurone layer progressed toward the distal part of
the grain. No signal above background was detected in control sections
hybridized with the 35S-labeled sense probe (Fig.
5A), i.e. starchy endosperm acidification and gene expression show a
similar pattern advancing from the embryo to the distal part of the
grain. However, 2 d after imbibition, when most of the starchy
endosperm showed neutral pH, the expression of the carboxypeptidase III
gene had progressed significantly toward the distal part of the grain
(Fig. 5D). Although it must be taken into account that the pH staining
of the starchy endosperm does not allow us to precisely determine the
environmental pH at the plasma membrane of aleurone cells, this result
shows that in vivo GA perception by the aleurone cells takes place when
the pH of the surrounding starchy endosperm is still neutral. This finding is in agreement with the above-mentioned results showing a
better response of aleurone cells to GA3 at
neutral than at acidic pH.
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| Figure 5.
Histological localization of carboxypeptidase III
mRNA (2437 cDNA) in germinating wheat grains. In situ hybridizations
were carried out with sense (A) or antisense 35S-labeled
riboprobes on longitudinal sections (10 µm thick) of grains that had
been soaked for 14 h (B), 24 h (C), or 48 h (D). al,
Aleurone layer; ep, epithelium; p, pericarp; sc, scutellum, se, starchy
endosperm. Bar corresponds to 100 µm.
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DISCUSSION |
The results presented in this report show a clear effect of the pH
of the incubation medium on the response of wheat aleurone cells to
GA3. The two aleurone responses to
GA3 that we analyzed, incubation medium
acidification and protease transcript accumulation, are favored at
neutral pH. In agreement with these results, the comparison of the
spatio-temporal pattern of starchy endosperm acidification (Fig. 4) and
carboxypeptidase III expression (Fig. 5) allows us to conclude that in
vivo GA perception by the aleurone cells takes place when the pH of the
surrounding starchy endosperm is neutral. This result was unexpected,
since it has been described that the starchy endosperm of
postgerminative cereal grains is maintained at acidic pH (Briggs, 1968;
Mikola and Virtanen, 1980). In fact, based on this observation, most
studies of GA3 regulation of gene expression in
wheat aleurone cells were carried out at pH 5.2 (Baulcombe and Buffard,
1983; Huttly and Baulcombe, 1989; Cejudo et al., 1992a, 1992b).
GAs are weak hydrophobic acids with pKa values of
4.0 to 4.2 (Hooley, 1994); therefore, at acidic pH an increased passive entrance to the aleurone cells, as undissociated molecules, would be
expected. There is consistent evidence that the site of GA perception
by the aleurone cells is the plasma membrane and not a receptor inside
the cells (Hooley et al., 1991; Gilroy and Jones, 1994). Therefore, it
would be expected that neutral pH, which increases the dissociated form
of GA and which is unable to passively enter the aleurone cell
membrane, is more efficient in promoting the aleurone response than
acidic pH. This view is supported by the results reported from this
study.
It appears that wheat and barley grains behave differently with regard
to starchy endosperm acidification. During late stages of barley grain
development, the starchy endosperm is acidified by the accumulation of
malic acid (Macnicol and Jacobsen, 1992). This is not the case in the
wheat grain, as shown by the pH-staining experiments described in this
report (Fig. 4). In addition, the in situ pH staining shows a clear
pattern of starchy endosperm acidification following wheat grain
germination. Initial acidification takes place in the area of the
starchy endosperm adjacent to the embryo, implicating the scutellum in
this process. In barley (Drozdowicz and Jones, 1995), it has been shown
that isolated scutella are able to acidify the external medium because
of the release of citric and succinic acids. We carried out a study of
the expression and localization of PEP carboxylase in germinating wheat
grains. Immunolocalization experiments showed an increase of PEP
carboxylase in the scutellar epithelium of grains 24 h after
imbibition (González et al., 1998) that might account for the
organic acid produced by the scutellum at early stages of
germination and, therefore, explain the acidification taking place near
the embryo.
The acidification of the starchy endosperm and the expression of
carboxypeptidase III following germination show a similar pattern. In
agreement with Mikola and Virtanen (1980) and Drozdowicz and Jones
(1995), we showed that the capacity of aleurone cells to acidify the
external medium is enhanced by GA3 (Table I; Fig. 1). These results suggest that the progression of acidification toward
the distal part of the grain involves aleurone cells and is a process
regulated by GA. Treatment of isolated barley aleurone layers with
GA3 results in accumulation of phosphoric and
malic acid in the external medium (Drozdowicz and Jones, 1995), which has been proposed to be the cause of acidification. We tested the
possibility that PEP carboxylase activity of aleurone cells might be
involved in acidification. Although these cells contain appreciable
amounts of PEP carboxylase (González et al., 1998), no effect of
GA3 was observed on its expression (M.C.
González and F.J. Cejudo, unpublished results), suggesting
that this enzyme is not involved in the acidification capacity of the
aleurone cells.
Upon wheat grain imbibition, GA1 and
GA3, which are synthesized in the scutellum and
diffuse to the starchy endosperm (Appleford and Lenton, 1997), promote
the metabolic activation of aleurone cells through a complex signaling
cascade that includes Ca2+, internal pH,
cGMP, and calmodulin as signaling molecules (for review, see Bethke et
al., 1997). This metabolic activation includes the synthesis and
secretion of hydrolytic enzymes and the acidification of the starchy
endosperm, which is essential for storage compound degradation since
most hydrolytic enzymes show maximal activity at acidic pH.
The different processes activated by GA in aleurone cells after grain
imbibition must be perfectly ordered and coordinated in time (Bethke et
al., 1997). To explain the pattern of starchy endosperm acidification
and carboxypeptidase III expression in germinated wheat grains, we
propose that the response of any single aleurone cell to GA depends on
its location in the wheat grain: aleurone cells proximal to the embryo
are the first to perceive GA and activate the synthesis of hydrolytic
enzymes and acidification of the surrounding starchy endosperm. As GAs
diffuse from the embryo (Appleford and Lenton, 1997), they
are perceived by aleurone cells located in the more distal part of the
grain, and acidification and production of hydrolases is extended to
this part of the grain.
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FOOTNOTES |
1
This work was supported by grant BIO97-1205 from
Comision Interministerial de Ciencia y Tecnologia, Ministerio de
Educación y Cultura, and grant no. CVI 118 from Junta de
Andalucía (Spain).
*
Corresponding author; e-mail fjcejudo{at}cica.es; fax
34-954-460065.
Received July 20, 1998;
accepted September 25, 1998.
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ACKNOWLEDGMENTS |
The photographic work of M.J. Cubas and Dr. J. García,
who prepared Figure 4, is appreciated. We thank SENASA for
providing cv Chinese Spring grains.
| |
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