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Plant Physiol. (1998) 116: 1249-1258
Expression and Localization of Phosphoenolpyruvate
Carboxylase in Developing and Germinating Wheat Grains1
María-Cruz González,
Lidia Osuna,
Cristina Echevarría,
Jean Vidal, 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 (M.-C.G.,
F.J.C.); Departamento de Biología Vegetal, Facultad de
Biología, Universidad de Sevilla, Spain (L.O., C.E.); and Institut de Biotechnologie des Plantes, Unité, Recherche Associé,
Centre National de la Recherche Scientifique D 1128, Bâtiment
630, Université de Paris-Sud, Centre d'Orsay cedex, France
(J.V.)
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ABSTRACT |
Phosphoenolpyruvate
carboxylase (PEPC) activity and corresponding mRNA levels were
investigated in developing and germinating wheat (Triticum
aestivum) grains. During grain development PEPC activity
increased to reach a maximum 15 d postanthesis. Western-blot experiments detected two main PEPC polypeptides with apparent molecular
masses of 108 and 103 kD. The most abundant 103-kD PEPC subunit
remained almost constant throughout the process of grain development
and in the scutellum and aleurone layer of germinating grains. The
less-abundant 108-kD polypeptide progressively disappeared during the
second half of grain development and was newly synthesized in the
scutellum and aleurone layer of germinating grains. PEPC mRNA was
detected throughout the process of grain development; however, in
germinating grains PEPC mRNA accumulated transiently in the scutellum
and aleurone layer, showing a sharp maximum 24 h after imbibition.
Immunolocalization studies revealed the presence of the enzyme in
tissues with a high metabolic activity, as well as in the vascular
tissue of the crease area of developing grains. A clear increase in
PEPC was observed in the scutellar epithelium of grains 24 h after
imbibition. The data suggest that the transiently formed PEPC mRNA in
the scutellar epithelium encodes the 108-kD PEPC subunit.
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INTRODUCTION |
PEPC (EC 4.1.1.31) catalyzes the  -carboxylation of PEP using
HCO3 as a substrate in a
reaction that yields oxaloacetate and Pi (for review, see Lepiniec et
al., 1994 ; Chollet et al., 1996; Vidal and Chollet, 1997). This enzyme
has been extensively studied in C4 and CAM
plants, where it plays an essential role in photosynthetic C
metabolism. C3 plants contain less, yet
appreciable, amounts of PEPC, which is found in virtually all organs
and may play multiple physiological roles (Latzko and Kelly, 1983). One
such role is the so-called anaplerotic function, which consists of the
replenishment of oxaloacetate in the tricarboxylic acid cycle whenever
the demand for C skeletons for amino acid biosynthesis is high (Huppe
and Turpin, 1994).
The connection of PEPC to N metabolism and its contribution to N
assimilation has been well established by previous studies. Feeding
maize (Zea mays) leaves with
NH4+ or
NO3 increased the levels of
C4 PEPC protein and mRNA via cytokinin-dependent stimulated transcription of the C4 PEPC gene
(Sugiharto and Sugiyama, 1992; Suzuki et al., 1994). The supply of
NO3 to illuminated,
N-deficient wheat (Triticum aestivum) leaves markedly
enhanced the PEPC kinase activity/PEPC phosphorylation status, thereby
causing an activation of the enzyme and a decrease in the sensitivity
to the feedback inhibitor l-malate (Van Quy et al., 1991;
Van Quy and Champigny, 1992; Duff and Chollet, 1995). PEPC in soybean
(Glycine max) nodules seems to be modulated by photosynthate
availability, strongly supporting the view that PEPC phosphorylation
might be contributing to control the C/N balance in the nodules (Zhang
et al., 1995). PEPC in nodules, in algae, and in leaves of
C3 plants is strongly inhibited by the amino
acids aspartate and glutamate (Huppe and Turpin, 1994); a similar
inhibitory effect of these amino acids on PEPC activity of germinating
castor bean (Ricinus communis) cotyledons has also been
reported (Podestá and Plaxton, 1994b).
There is increasing evidence for the presence of PEPC in seeds of
different plant species (Blanke and Lenz, 1989). Two different full-length PEPC cDNA clones, gmppc16 and gmppc1, have been isolated from developing soybean seeds (Sugimoto et al., 1992; Vazquez-Tello et
al., 1993). The deduced proteins resemble more closely
C3-type PEPCs and possess the N-terminal
phosphorylation domain found in other PEPCs sequenced so far.
Furthermore, gmppc16 is expressed not only in seeds but also in leaves,
stems, and roots. Therefore, soybean contains a small multigene family
of PEPC sequences, some members of which (at least two) are expressed
in seeds. Sangwan et al. (1992) proposed that the large increase in
PEPC activity during maturation of castor seeds might be involved in
generating malate for the synthesis of storage lipids. The increased
malate flux would also be a source of additional C skeletons for N
assimilation in cotyledons of germinating castor seeds (Podestá
and Plaxton, 1994b).
The presence of PEPC in barley (Hordeum vulgare) kernels was
reported as early as 1973 (Duffus and Rosie, 1973). Isolated aleurone
layers from barley and wheat grains show capacity for endosperm
acidification (Mikola and Virtanen, 1980; Hamabata et al., 1988)
following the accumulation of organic and phosphoric acids (Drozdowicz
and Jones, 1995). Concomitant increases in malate and PEPC activity
during the last stages of barley grain development pointed to the
involvement of PEPC in the process of acidification that takes place in
the endosperm during development (Macnicol and Jacobsen, 1992).
Using an electron-microscopic immunolabeling technique, Araus et al.
(1993) detected a substantial amount of PEPC in the protein bodies of
immature durum wheat (Triticum durum) grains, where it might
contribute to amino acid and protein biosynthesis during grain
development. The presence of PEPC protein and mRNA has also been
reported in germinating sorghum (Sorghum bicolor) seeds
(Khayat et al., 1991).
Recently, we have showed that wheat grain PEPCs possess the
characteristic N-terminal phosphorylation domain and that the predominant 103-kD form was susceptible to phosphorylation, both in
desalted crude extracts and in situ, indicating that the machinery for
PEPC phosphorylation is present and functional in these grains (Osuna
et al., 1996). The present work was undertaken to investigate further
the distribution and fate of the enzyme and its mRNA in the different
parts of the grain during development and germination.
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MATERIALS AND METHODS |
Wheat (Triticum aestivum cv Chinese Spring) plants were
cultivated under controlled-environment conditions in a greenhouse under a 16-h day/8-h night cycle at 22 to 25°C. Grains were harvested at different stages of development, frozen in liquid
N2, and kept at  80°C until used. Mature
grains were sterilized in 2% (v/v) NaOCl for 20 min and washed twice
with sterile water, once with 0.1 m HCl, and then
thoroughly with sterile water. Grains were allowed to germinate at room
temperature on sterile filter paper soaked with water for periods of up
to 4 d. The rabbit polyclonal antibodies against native sorghum
(Sorghum bicolor) C4 PEPC were prepared as reported by Osuna et al. (1996). All reagents were of
analytical grade and were purchased from Sigma.
PEPC Activity Assay
PEPC activity was determined from crude extracts of grains at
different stages of development. Extracts were prepared from 5 to 10 grains/mL of buffer A containing 0.1 m Tris-HCl, pH 7.5, 20% (v/v) glycerol, 1 mm EDTA, 10 mm
MgCl2, 10 µg/mL chymostatin, 10 µg/mL
bestatin, 10 µg/mL leupeptin, 1 mm PMSF, 1 µg/mL
microcystin-LR (L and R are the two variable amino acids in the
structure of microcystin), and 14 mm 2-mercaptoethanol. To
analyze PEPC activity from germinating grains, aleurone layers,
scutellum, and starchy endosperm were carefully dissected from 10 grains at the indicated times after imbibition and washed thoroughly
with 0.1 m Tris-HCl, pH 8.0. PEPC activity was assayed
using the NADH-malate dehydrogenase coupled assay as previously
reported (Osuna et al., 1996). Protein determinations were carried out
by the method of Bradford (1976) using a kit (Bio-Rad).
PEPC Immunocharacterization
Extracts (75 µL containing 2.7 mg protein/mL) from aleurone
layers of germinating grains were incubated overnight at 4°C in the
presence of increasing amounts of preimmune serum or sorghum C4-PEPC IgG, which was added in a total volume of
50 µL of TBS. After this incubation, protein A (5 mg) was added,
incubated for 15 min at 4°C, and centrifuged for 15 min at
13,000g, and PEPC activity was assayed in the supernatant.
Aliquots of the tissue extracts were subjected to SDS-PAGE (10%
acrylamide) according to the method of Laemmli (1970). After electrophoresis, proteins were electroblotted to nitrocellulose membranes (N-8017 from Sigma) at 0.8 mA/cm2 for
2 h using an electrophoretic transfer kit (Novablot, LKB, Bromma,
Sweden). Membranes were blocked in TBS (20 mm Tris-HCl, pH
7.5, and 150 mm NaCl) containing 5% (w/v) powdered milk,
and PEPC bands were immunochemically labeled by overnight incubation of
the membranes at 4°C in 20 mL of TBS and affinity-purified polyclonal
sorghum C4 PEPC IgG (27 µg of protein). Control
experiments were carried out by incubating the membranes under the same
conditions with a preimmune serum. No bands were detected in these
controls. Subsequent detection was performed by a peroxidase assay
(affinity-purified goat anti-rabbit IgG horseradish peroxidase
conjugate from Bio-Rad).
Wheat PEPC cDNA Cloning and Northern Analysis
A cDNA library constructed in  gt10 with
poly(A+) RNA from roots of 7-d-old wheat
seedlings was screened with 32P-labeled CP28 cDNA
encoding a sorghum C3-type PEPC (Lepiniec et al.,
1991). The library (approximately 40,000 plaque-forming units) was
plated, transferred to Hybond-N filters (Amersham), and hybridized at
60°C according to manufacturer's instructions. Four positive plaques
were selected and purified, and the size of their cDNA inserts was
analyzed. None of them contained a full-length cDNA; the longest one
(1.3 kb) was subcloned in pBluescript SK+
(Stratagene) and sequenced in both chains.
For northern analysis, total RNA samples were extracted from liquid
N2-frozen tissue, fractionated on
agarose-formaldehyde gels, and blotted onto Hybond-N filters according
to the manufacturer's instructions. Hybridization was performed as
described by Sambrook et al. (1989) with
32P-labeled probe (the wheat PEPC cDNA clone
described above). After hybridization, filters were washed at 60°C in
washing solution (0.5× SSC and 0.1% SDS). Signal intensities of the
autoradiographs were quantitated with a densitometer (Millipore).
Variations in RNA loading were corrected for by normalizing each value
to the level of the corresponding radish 18S rRNA sequence (Grellet et al., 1989).
Immunohistochemistry
Grains harvested at different stages of development and
germinating grains at the indicated times after imbibition were fixed in 4% paraformaldehyde, dehydrated in a graded series of aqueous ethanol solution, and embedded in Paraplast Plus (Sigma) as described by Cejudo et al. (1992). Sections (10 µm thick) were cut in a microtome (model RM2025, Leica) and placed on
poly-l-Lys-coated microscope slides. After deparaffinizing
in xylol and rehydrating in decreasing concentrations of ethanol,
sections were blocked for 30 min in TBS buffer containing 1% (w/v)
BSA. Then, 100 µL of the same solution containing affinity-purified
anti-PEPC antibodies (1 µg of protein) or preimmune serum was placed
on the samples and incubated overnight at 4°C. Unbound primary
antibodies were removed by three 10-min washes in TBS. Tissue sections
were then incubated with alkaline phosphatase-conjugated goat
anti-rabbit IgG for 2 h at 37°C. The reaction of alkaline
phosphatase was developed with nitroblue tetrazolium and
5-bromo-4-chloro-3-indolyl-phosphate.
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RESULTS |
PEPC in Developing Wheat Grains
To study the amount of PEPC and its polymorphism during wheat
grain development and germination, a polyclonal sorghum
C4 PEPC IgG was used that effectively
cross-reacted with PEPC from wheat grains, as shown by the
immunoinhibition of PEPC activity (Fig. 1; see also Osuna et al., 1996). No
inhibitory effect was observed in the presence of the preimmune serum.
Extracts prepared from whole grains harvested at different stages of
development showed PEPC activity (Fig.
2A). Activity increased at early stages
of development to reach a maximum 15 DPA and then progressively
decreased as grains approached maturity (up to 31 DPA).
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| Figure 1.
Immunotitration of wheat grain PEPC. Aliquots (75 µL) of an extract (2.7 mg protein/mL) from the dissected aleurone
layer of germinating grains were incubated with increasing amounts of sorghum C4-PEPC IgG ( ) or preimmune serum ( ). PEPC
activity remaining in the supernatants was measured as indicated in
``Materials and Methods''. One hundred percent activity corresponds
to 56 milliunits/mg protein.
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| Figure 2.
Analysis of PEPC during wheat grain development.
A, Time course of PEPC activity during wheat grain development.
Activity was assayed in crude extracts from grains at different stages of development. Results are means ± se of three
independent determinations. mU, Milliunits; and prot, protein. B,
Western-blot analysis of PEPC in developing grains. Crude extracts (75 µg of protein) from grains harvested at different stages of
development were resolved in SDS-PAGE, transferred onto nitrocellulose,
and probed with polyclonal PEPC IgGs (27 µg of protein/20 mL of
incubation medium). Molecular masses of standard proteins are indicated
on the left. C, Northern-blot analysis. Total RNA samples (10 µg)
extracted from grains at the indicated DPA were fractionated in 1%
agarose/formaldehyde gels, transferred onto Hybond-N filters, and
hybridized to 32P-labeled PEPC cDNA. As a control of the
amount of RNA loaded per lane, filters were stripped of radioactivity
and rehybridized with 32P-labeled 18S rRNA.
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When extracts of developing wheat grains were analyzed by western blot
using the polyclonal sorghum C4 PEPC IgG as a
probe (Fig. 2B), the antibodies detected two main polypeptides with molecular masses of 103 and 108 kD during the first part of the development process (5-15 DPA). During the second half of the development process (15-31 DPA), the 108-kD polypeptide progressively disappeared to become hardly detectable in grains 31 DPA. In these samples (15-31 DPA), three additional polypeptides of molecular mass
ranging between 75 and 85 kD were recognized by the anti-PEPC antibody.
These bands are very specific to these developmental stages, since they
were not observed at the early stages (up to 9 DPA). These bands may
correspond to PEPC-degradation products, although this possibility was
not tested further.
To carry out studies on PEPC expression in wheat grains, a partial PEPC
cDNA clone was isolated from a cDNA library made with poly(A+) RNA from roots of wheat seedlings (see
``Materials and Methods''), which encoded the C-terminal region (331 amino acids) of wheat PEPC. The deduced amino acid sequence presented a
high identity (more than 70%) to the C-terminal region of PEPCs from
different sources, including tobacco, alfalfa, sorghum, and maize
(Zea mays). Using the wheat partial cDNA as probe, we
studied the accumulation of PEPC mRNA during grain development by
northern-blot hybridization (Fig. 2C). When membranes were washed under
low-stringency conditions (60°C, 0.5× SSC, and 0.1% SDS), a single
band corresponding to an mRNA of approximately 3.4 kb was detected
throughout the process of grain development. However, wheat grain
maturation was accompanied by a decline in PEPC mRNA (Fig. 2C), in
concert with reductions in PEPC activity and in the 108-kD subunit
(Fig. 2, A and B).
PEPC in Germinating Wheat Grains
As mentioned above, mature wheat grains contain almost exclusively
a single type of PEPC subunit with a molecular mass of 103 kD (Fig.
3, lanes 0). We analyzed by western
blot the evolution of PEPC content and polymorphism in the different
parts of germinating grains. In dissected scutellum the 103-kD
polypeptide was detected throughout the 4 d after imbibition (Fig.
3A). The 108-kD polypeptide, barely detectable in mature grains, was
found to increase during the 1st d of imbibition and then did not show
any significant variation following grain germination. It should be
mentioned that the higher amount of the 103-kD polypeptide observed at
d 4 (Fig. 3A) is not significant and is most probably due to variation of the detection reaction.
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| Figure 3.
Western-blot analysis of PEPC in germinating wheat
grains. Wheat grains were allowed to germinate for the indicated times and scutella (A) or aleurone layers and starchy endosperm (B) were
carefully dissected. Crude extracts from scutella (70 µL, containing
140 µg of protein), aleurone layer (30 µL, containing 159 µg of
protein), and starchy endosperm (30 µL, containing 160 µg of
protein) were resolved in SDS-PAGE, transferred onto nitrocellulose, and probed with polyclonal PEPC IgGs (27 µg of protein/20 mL of incubation medium). 0, Dry grains; O/N, 14 h after imbibition; and
1 to 4, days after imbibition. al, Aleurone layers; and se, starchy
endosperm.
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The polymorphism of PEPC was also analyzed in the endosperm (the
aleurone layer and starchy endosperm) of germinating grains (Fig. 3B).
In mature grains the endosperm contained exclusively the 103-kD subunit
(Fig. 3B). After the 2nd d of imbibition the aleurone layer could be
easily dissected and therefore it was possible to analyze separately
the PEPC pattern in the aleurone layer and the remaining starchy
endosperm. In the aleurone layer the most abundant 103-kD polypeptide
was constantly present up to 4 d after imbibition, whereas the
108-kD band was already detected 2 d after imbibition (Fig. 3B).
In the starchy endosperm the level of PEPC enzyme decreased so that
4 d after imbibition it was no longer detectable in western blots
(Fig. 3B), suggesting that PEPC, like other enzymes and storage
proteins that accumulated in the starchy endosperm during grain
development, was hydrolyzed during grain germination to serve as a
nutrient for seedling growth.
PEPC activity was also analyzed in dissected tissues of germinating
grains (Table I). In the scutellum
PEPC activity increased after 1 d of imbibition and then remained
invariable. In the aleurone layer a progressive increase in PEPC
activity was observed (Table I), whereas in the starchy endosperm
activity was almost undetectable 2 to 3 d after imbibition (not
shown), in agreement with the disappearance of PEPC polypeptides
described above (Fig. 3B). Therefore, the overall increase of PEPC
activity observed in the scutellum and the aleurone layer of
germinating grains is concomitant with the appearance of the 108-kD
subunit, whereas the 103-kD subunit shows no significant variation.
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Table I.
PEPC activity in dissected scutellum and aleurone
layer of wheat germinating grains
Values are means ± se of at least three replicate
experiments.
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Northern-blot analysis revealed that PEPC mRNA in the scutellum of
germinating grains was sharply and strongly enhanced by the 1st d of
imbibition and then quickly declined (Fig.
4A). A similar pattern was observed in
the aleurone layer (Fig. 4B).
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| Figure 4.
Northern-blot analysis of PEPC in germinating
wheat grains. Grains were allowed to germinate, and at the indicated
times scutellum (A) and aleurone layers (B; de-embryonated grains) were
dissected. Total RNA (10 µg) extracted from dissected tissues was
fractionated in 1% agarose/formaldehyde gels, transferred onto
Hybond-N filters, and hybridized to 32P-labeled PEPC cDNA.
mRNA levels were quantified and normalized with the corresponding rRNA
sample. The results were plotted as a percentage of the maximum level
of PEPC mRNA. 0, Dry grains; O/N, 14 h after imbibition; and 1 to
4, days after imbibition.
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Immunolocalization of PEPC in Developing and Germinating Wheat
Grains
The distribution and possible variations of PEPC in wheat grain
tissues were studied by immunolocalization. At the early stages of
development (5 DPA), PEPC was abundant in the nucellus, the multinucleate syncytium, and the vascular tissue over the crease area
of the grain, whereas no signal was detected in the pericarp (Fig.
5A). As grain development proceeds (10 DPA), the endosperm is formed by the cellularization of the syncytium.
At this stage, PEPC was mostly present in the endosperm and the
vascular tissue, as was shown in transverse sections of the grain (Fig.
5B). The differentiation of the aleurone cells was already initiated
and was immunodecorated, as observed in longitudinal sections (Fig. 5C). At 16 DPA the aleurone cells, which show a high mitotic activity, were fully differentiated and contained a high level of PEPC (Fig. 5D). Remarkably, well-differentiated phloem and xylem
in the vascular tissue of the crease area were immunodecorated by the
PEPC antibody (Fig. 5D). A similar section probed with preimmune
antiserum showed no signal (Fig. 5E). At 22 DPA the pattern of PEPC
localization in the grain was similar to that of the preceding stage,
PEPC being very abundant in the aleurone layer (Fig. 5F) and also
detected in developing embryo (Fig. 5G). No significant differences
were observed at 31 DPA, when the maturation process was almost
complete (results not shown).
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| Figure 5.
(Figure continues on facing page.)
Immunological localization of PEPC in developing wheat grains.
Immunolocalizations were carried out as described in ``Materials and Methods''. Sections of grains (10 µm thick) 5 DPA (A), 10 DPA (B and
C), 16 DPA (D and F), and 22 DPA (G) were probed with polyclonal PEPC
IgGs (1 µg of protein per slide). A control section from 16-DPA
grains was probed with preimmune serum (E). al, Aleurone layer; ch,
chalaza; cv, central vacuole; em, embryo, n, nucellus; p, pericarp; ph, phloem; s, syncytium; se, starchy endosperm; vt, vascular tissue; and
x, xylem. Magnification ×50.
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In a second set of experiments, the localization of PEPC was studied in
germinating grains. Early after imbibition (14 h), a wide distribution
of the enzyme was observed in the embryo (Fig. 6A) in the epicotyl, radicle, scutellum,
and aleurone layer. No signal above background was detected in sections
probed with the preimmune antiserum (Fig. 6B). Following germination
(24 h after imbibition) several differences were noted in the pattern
of PEPC localization in the scutellum and the part of the starchy
endosperm facing it (Fig. 6, compare C and D). On a magnified image it
can be seen that as germination proceeded (24 h after imbibition) the
signal increased considerably in the scutellar epithelium, a fact that
may reflect the changes in the 108-kD PEPC seen in western blots (Fig.
3A). However, the PEPC signal started to decrease in the part of the
starchy endosperm facing the scutellar epithelium (Fig. 6D), most
probably because of the effect of secreted proteases from the scutellum
and the aleurone layer to the starchy endosperm, in agreement with the
previously described disappearance of PEPC polypeptides observed in the
starchy endosperm of germinating grains (Fig. 3B).
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| Figure 6.
Immunological localization of PEPC in germinating
wheat grains. Grains were soaked for 14 h (A-C) or 24 h (D).
Sections (10 µm) were probed with polyclonal PEPC IgGs (1 µg per
slide; A, C, and D) or preimmune serum (B). al, Aleurone layer; cr,
coleorhiza; ep, epicotyl; ept, epithelium; sc, scutellum; se, starchy
endosperm; and r, radicle. Magnifications: A and B, ×50; and C and D,
×100.
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DISCUSSION |
The results presented in this paper establish the presence and
wide distribution of PEPC in developing and germinating wheat grains.
They confirm and extend a previous study of the regulatory phosphorylation of the enzyme in this organ (Osuna et al., 1996). The
study of PEPC polymorphism in wheat grains was carried out with
polyclonal antibodies raised against a C4
(sorghum) enzyme, which effectively cross-reacts with wheat PEPC,
indicating a high similarity between these enzymes.
Several lines of evidence show that wheat PEPC is very homologous to
PEPCs from other sources, including C4 plants.
The high degree of homology was confirmed by the sequence comparisons
of the partial cDNA clone from wheat, which shows more than 70%
identity at the amino acid level in the C-terminal region to PEPC from different sources, including both C3 and
C4 plants. Although no sequence of the N-terminal
domain of wheat PEPC is yet available, we have shown (Osuna et al.,
1996) that the wheat 103-kD enzyme undergoes a true phosphorylation
that is prevented by an antibody raised against a 21-mer synthetic
peptide representing the phosphorylation site of the sorghum enzyme at
the N-terminal region (Pacquit et al., 1995). This result suggests that
the high degree of identity may also be extended to the N-terminal
domain of plant PEPCs.
The polyclonal sorghum C4 PEPC IgG used in this
study recognized two polypeptides in developing and germinating wheat
grains with molecular masses of 103 and 108 kD. A similar PEPC
polymorphism has been described in castor bean (Ricinus
communis) seeds using anti-PEPC antibody raised against maize
C4 PEPC (Sangwan et al., 1992). In developing
wheat grains, the 108-kD subunit progressively disappears after 15 DPA;
therefore, most of the PEPC in mature grains has the 103-kD subunit.
The disappearance of the 108-kD subunit coincides with the detection of
three lower-molecular-mass polypeptides (ranging from 75-85 kD) in
western blots, which were very efficiently immunodecorated by the PEPC
antibodies (Fig. 2B). The possibility that these bands correspond to
unspecific detection may be ruled out, since the antibody shows a high
specificity in the detection of wheat PEPC polypeptides either at the
early stages of grain development or in germinating grains. Because no
PEPC subunit from any source has been described with a molecular mass
as low as 75 to 85 kD, we conclude that these bands probably correspond
to PEPC-degradation products. The strong intensity of these bands
could have been due to the fact that buried epitopes on the entire PEPC
are accessible to corresponding antibodies on these PEPC fragments.
Provided this assumption is valid, the result suggests that the 108-kD
subunit is degraded during the second half of the development process.
One attractive explanation for these collective results could be that
the upper band corresponds to ubiquitinated enzyme prior to its
degradation via the ubiquitin pathway, as has been reported in leaves
of broad bean (Schulz et al., 1993). However, using commercially
available anti-ubiquitin antibodies, we could not detect any
recognition of the PEPC molecule (data not shown). Another possibility
is that the 103-kD PEPC is the stable, housekeeping enzyme of the
grain, whereas the 108-kD form is inducible in nature. This view is
supported by the transient accumulation of mRNA in the scutellum and
the aleurone layer of germinating grains (see below).
These findings underscore the fact that the 103-kD PEPC subunit is very
stable throughout the entire process of development and germination of
wheat grains. In addition, they suggest that the newly synthesized mRNA
encodes the inducible 108-kD subunit in the scutellum and the aleurone
layer of germinating grains, but more information about the PEPC gene
family in wheat is needed before a firm conclusion can be drawn. Since
both PEPC subunits are found together in different tissues of the
grain, the question is raised whether the tetrameric enzyme has a
chimeric nature.
Such a hypothesis has been proposed by Sangwan et al. (1992) and
Podestá and Plaxton (1994a) in the case of PEPC in germinating but not developing broad bean seeds. In the wheat grain, it might be
that the quaternary structure of PEPC changes from mixed 103- and
108-kD subunits during early development and following germination to
mainly 103-kD subunits when maturation is being reached in the mature
grain and early after imbibition. Such a chimeric enzyme with modified
subunit composition could have specific regulatory and functional
properties as required by its cytosolic environment and developmental
stage of the grain. This interpretation awaits further work before a
conclusion is drawn.
The immunolocalization experiments (Figs. 5 and 6) showed that PEPC is
localized in tissues with high metabolic activity in developing and
germinating grains; it is present in developing endosperm (5-10 DPA)
when cellularization is taking place (Bosnes et al., 1992). The high
PEPC content of the aleurone layer during differentiation also
coincides with mitotic activity in these cells, as is the case in the
developing embryo. The presence of PEPC in the aleurone layer of
developing grains may also be related to the accumulation of malate in
the starchy endosperm, as has been described by Macnicol and Jacobsen
(1992) for late stages of barley grain development. A remarkable result
described in this study is the presence of high amounts of PEPC in the
vascular tissue of developing grains. The role played by PEPC in this
tissue is not clear yet; one possible explanation is that it produces dicarboxylic acids, which act as counterions for the translocation of
cations (such as K+) to the starchy endosperm.
Following imbibition, the enzyme is abundant in most tissues of
the grain. This localization pattern probably reflects the crucial role
of PEPC activity in maintaining the C content of the grain by
refixation of the CO2 evolved by actively
respiring tissues. This activity may generate C skeletons to help
supply the demand of amino acid biosynthesis in these tissues. This is particularly important for the scutellum and the aleurone layer of
germinating grains, since both tissues are very active in the synthesis
and secretion of hydrolytic enzymes that mobilize the storage compounds
of the starchy endosperm during germination (Cejudo et al., 1995;
Domínguez and Cejudo, 1995). It is also important for the
developing starchy endosperm, the PEPC of which is associated with
protein bodies during protein filling of the grain (Araus et al.,
1993).
Especially significant is the high and transient accumulation of PEPC
mRNA in the scutellum and the concurrent increase in the 108-kD PEPC
subunit after the first 24 h of imbibition (Fig. 4). The
immunological study reveals a parallel PEPC enrichment in the scutellar
epithelium at this time after imbibition. Collectively, these results
lend support to the hypothesis that the corresponding PEPC gene is
stimulated to produce PEPC mRNA encoding the 108-kD PEPC, whereas the
103-kD PEPC subunit would already be present to form the housekeeping
enzyme. A specific physiological task for the inducible 108-kD PEPC
could be linked to the fact that in germinating grains the scutellar
epithelium is deeply committed to the synthesis and secretion of
hydrolytic enzymes and the transport of nutrients (such as sugars and
amino acids) from the starchy endosperm (Drozdowicz and Jones, 1995).
The expression pattern of PEPC and  -amylase-1
(-Amy1) genes in wheat grains have several features in
common. Both mRNAs accumulate transiently in the scutellum of
germinating grains, showing a maximum 1 d after imbibition (Cejudo
et al., 1995). In addition, both genes are predominantly expressed in
the scutellar epithelium. It has been shown that -Amy1
expression is down-regulated by the availability of sugars in the
scutellum of rice grains (Yu et al., 1991; Karrer and Rodriguez, 1992;
Chen et al., 1994; Thomas and Rodriguez, 1994). Furthermore, PEPC
expression is metabolically repressed by sugars in maize mesophyll
protoplasts (Sheen, 1990). A similar repression might be taking place
in the scutellum of germinating wheat grains due to sugars that are
produced in the starchy endosperm by the action of -amylase activity
and taken up by the scutellum. The data presented in this study suggest a coordination of PEPC and -Amy1 expression in
embryos of germinating wheat grains. The possibility that PEPC
expression is under metabolic control in wheat grains is an open
question. In this regard, it is worth mentioning the presence of common
elements in the promoter region of PEPC and -Amy1
genes (Karrer and Rodriguez, 1992). Whether or not these elements
are relevant in the control of PEPC expression in response to
sugars remains to be determined.
| |
FOOTNOTES |
1
This work was supported by grant no. PB92-0675
from Dirección General de Investigación Científica
y Técnica, Ministerio de Educación y Ciencia, and grant no.
CVI 0118 from Junta de Andalucía, Spain.
*
Corresponding author; e-mail fjcejudo{at}cica.es; fax
34-5-446-0065.
Received September 17, 1997;
accepted December 31, 1997.
The nucleotide sequence data reported in this paper appear in the EMBL,
GenBank, and DDBJ nucleotide sequence databases under accession number
Y15897.
| |
ABBREVIATIONS |
Abbreviations:
DPA, days postanthesis.
PEPC, PEP carboxylase.
| |
ACKNOWLEDGMENTS |
Thanks are due to Dr. C. Hartman and E. Bismuth for the generous
gift of the wheat PEPC probe. The photography work of M.J. Cubas is
deeply appreciated.
| |
LITERATURE CITED |
Araus JL,
Bort J,
Brown RH,
Bassett CL,
Cortadellas
(1993)
Immunocytochemical localization of phosphoenolpyruvate carboxylase and photosynthesis gas-exchange characteristics in ears of Triticum durum Desf.
Planta
191:
507-514
Blanke MM,
Lenz F
(1989)
Fruit photosynthesis.
Plant Cell Environ
12:
31-46
[CrossRef]
Bosnes M,
Weideman F,
Olsen O-A
(1992)
Endosperm differentiation in barley wild-type and sex mutants.
Plant J
2:
661-674
Bradford MM
(1976)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254
[CrossRef][ISI][Medline]
Cejudo FJ,
Cubo MT,
Baulcombe DC
(1995)
Amy1 expression during wheat seed germination.
Plant Sci
106:
207-213
[CrossRef]
Cejudo FJ,
Murphy G,
Chinoy C,
Baulcombe DC
(1992)
A gibberellin-regulated gene from wheat with sequence homology to cathepsin B of mammalian cells.
Plant J
2:
937-948
[ISI][Medline]
Chen M-H,
Liu L-F,
Chen Y-R,
Wu H-K,
Yu S-M
(1994)
Expression of -amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient.
Plant J
6:
625-636
[CrossRef][ISI][Medline]
Chollet R,
Vidal J,
Oleary MH
(1996)
Phosphoenolpyruvate carboxylase: a ubiquitous, highly regulated enzyme in plants.
Annu Rev Plant Physiol Plant Mol Biol
47:
273-298
[CrossRef][ISI]
Domínguez F,
Cejudo FJ
(1995)
Pattern of endoproteolysis following wheat grain germination.
Physiol Plant
95:
253-259
[CrossRef]
Drozdowicz YM,
Jones RL
(1995)
Hormonal regulation of organic and phosphoric acid release by barley aleurone layers and scutella.
Plant Physiol
108:
769-776
[Abstract]
Duff SMG,
Chollet R
(1995)
In vivo regulation of wheat-leaf phosphoenolpyruvate carboxylase by reversible phosphorylation.
Plant Physiol
107:
775-782
[Abstract]
Duffus CM,
Rosie R
(1973)
Some enzyme activities associated with the chlorophyll containing layers of the immature barley pericarp.
Planta
114:
219-226
Grellet F,
Delcasso-Tremousaygue D,
Delseny M
(1989)
Isolation and characterization of unusual repeated sequence from the ribosomal intergenic spacer of the crucifer Sisimbrium irio.
Plant Mol Biol
12:
695-706
[CrossRef]
Hamabata A,
García-Maya M,
Romero T,
Bernal-Lugo I
(1988)
Kinetics of the acidification capacity of aleurone layer and its effect upon solubilization of reserve substances from starchy endosperm of wheat.
Plant Physiol
86:
643-644
[Abstract/Free Full Text]
Huppe HC,
Turpin DH
(1994)
Integration of carbon and nitrogen metabolism in plant and algal cells.
Annu Rev Plant Physiol Plant Mol Biol
45:
577-607
[ISI]
Karrer EE,
Rodriguez RL
(1992)
Metabolic regulation of -amylase and sucrose synthase genes in planta.
Plant J
2:
517-523
[ISI][Medline]
Khayat E,
Dumbroff EB,
Glick BR
(1991)
The synthesis of phosphoenolpyruvate carboxylase in imbibing sorghum seeds.
Biochem Cell Biol
69:
141-145
[Medline]
Laemmli UK
(1970)
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685
[CrossRef][Medline]
Latzko E,
Kelly GJ
(1983)
The many-faceted function of phosphoenolpyruvate carboxylase in C3 plants.
Physiol Veg
21:
805-815
Lepiniec L,
Santi S,
Keryer E,
Amiet V,
Vidal J,
Gadal P,
Crétin C
(1991)
Complete nucleotide sequence of one member of the Sorghum phosphoenolpyruvate carboxylase gene family.
Plant Mol Biol
17:
1077-1079
[Medline]
Lepiniec L,
Vidal J,
Chollet R,
Gadal P,
Cretin C
(1994)
Phosphoenolpyruvate carboxylase: structure, regulation and evolution.
Plant Sci
99:
111-124
[CrossRef]
Macnicol PK,
Jacobsen JV
(1992)
Endosperm acidification and related metabolic changes in the developing barley grain.
Plant Physiol
98:
1098-1104
[Abstract/Free Full Text]
Mikola J,
Virtanen M
(1980)
Secretion of l-malic acid by barley aleurone layers (abstract no. 783).
Plant Physiol
65:
S-142
Osuna L,
González MC,
Cejudo FJ,
Vidal J,
Echevarría C
(1996)
In vivo and in vitro phosphorylation of the phosphoenolpyruvate carboxylase from wheat seeds during germination.
Plant Physiol
111:
551-558
[Abstract]
Pacquit V,
Giglioli N,
Crétin C,
Pierre JN,
Vidal J,
Echevarria C
(1995)
Regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase from Sorghum: an immunological study using specific anti-phosphorylation site antibodies.
Photosynth Res
43:
283-288
[CrossRef]
Podestá FE,
Plaxton WC
(1994a)
Regulation of cytosolic carbon metabolism in germinating Ricinus communis cotyledons. I. Developmental profiles for the activity, concentration, and molecular structure of the pyrophosphate- and ATP-dependent phosphofructokinases, phosphoenolpyruvate carboxylase and pyruvate kinase.
Planta
194:
374-380
Podestá FE,
Plaxton WC
(1994b)
Regulation of cytosolic carbon metabolism in germinating Ricinus communis cotyledons. II. Properties of phosphoenolpyruvate carboxylase and cytosolic pyruvate kinase associated with the regulation of glycolysis and nitrogen assimilation.
Planta
194:
381-387
Sambrook J,
Fritsch EF,
Maniatis T
(1989)
Molecular Cloning. A Laboratory Manual, Ed 2.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Sangwan RS,
Singh N,
Plaxton WC
(1992)
Phosphoenolpyruvate carboxylase activity and concentration in the endosperm of developing and germinating castor oil seeds.
Plant Physiol
99:
445-449
[Abstract/Free Full Text]
Schulz M,
Klockenbring T,
Hunte C,
Schnabl H
(1993)
Involvement of ubiquitin in phosphoenolpyruvate carboxylase degradation.
Bot Acta
106:
143-145
Sheen J
(1990)
Metabolic repression of transcription in higher plants.
Plant Cell
2:
1027-1038
[Abstract/Free Full Text]
Sugiharto B,
Sugiyama T
(1992)
Effects of nitrate and ammonium on gene expression of phosphoenolpyruvate carboxylase and nitrogen metabolism in maize leaf tissue during recovery from nitrogen stress.
Plant Physiol
98:
1403-1408
[Abstract/Free Full Text]
Sugimoto T,
Kawasaki T,
Kato T,
Whittier RF,
Shibata D,
Kawamura Y
(1992)
cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean.
Plant Mol Biol
20:
743-747
[Medline]
Suzuki I,
Crétin C,
Omata T,
Sugiyama T
(1994)
Transcriptional and posttranscriptional regulation of nitrogen-responding expression of phosphoenolpyruvate carboxylase gene in maize.
Plant Physiol
105:
1223-1229
[Abstract]
Thomas BR,
Rodriguez RL
(1994)
Metabolite signals regulate gene expression and source/sink relations in cereal seedlings.
Plant Physiol
106:
1235-1239
[ISI][Medline]
Van Quy L,
Champigny ML
(1992)
Nitrate enhances the kinase activity for phosphorylation of phosphoenolpyruvate carboxylase and sucrose phosphate synthase proteins in wheat leaves.
Plant Physiol
99:
344-347
[Abstract/Free Full Text]
Van Quy L,
Foyer C,
Champigny ML
(1991)
Effect of light and nitrate on wheat leaf phosphoenolpyruvate carboxylase activity. Evidence for covalent modulation of C3 enzyme.
Plant Physiol
97:
1476-1482
[Abstract/Free Full Text]
Vazquez-Tello A,
Whittier RF,
Kawasaki T,
Sugimoto T,
Kawamura Y,
Shibata D
(1993)
Sequence of a soybean (Glycine max L.) phosphoenolpyruvate carboxylase cDNA.
Plant Physiol
103:
1025-1026
[CrossRef][Medline]
Vidal J,
Chollet R
(1997)
Regulatory phosphorylation of C4 PEP carboxylase.
Trends Plant Sci
2:
203-241
[CrossRef]
Yu S-M,
Kuo Y-H,
Sheu G,
Sheu Y-J,
Liu L-F
(1991)
Metabolic derepression of -amylase gene expression in suspension-cultured cells of rice.
J Biol Chem
266:
21131-21137
[Abstract/Free Full Text]
Zhang X-Q, Bin L, Chollet R (1995) In vivo regulatory
phosphorylation of soybean nodule phosphoenolpyruvate carboxylase. Plant Physiol 108:
1561-1568
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Top
Abstract
Introduction