The translation initiation factors (eIF) 4B and
eIF2 are phosphoproteins whose phosphorylation state differs between
mature seed and leaves. We examined the isoforms of eIF4B and the
and
subunits of eIF2 during the development and germination of
wheat seed to determine whether the differences in their
phosphorylation state are because of tissue-specific regulation or
occur concomitant with changes in protein synthetic activity during
development. eIF2
underwent phosphorylation through several
intermediate isoforms that correlated with the increase and subsequent
reduction in protein synthetic activity characteristic of seed
development. eIF2
and eIF4B, present as highly phosphorylated
isoforms during early seed development, underwent dephosphorylation
during late development. eIF4B was rapidly phosphorylated within
20 h of germination, whereas eIF2
did not undergo
dephosphorylation until 48-60 h of growth. A third factor, eIF4A, was
predominantly nonphosphorylated throughout most of seed development and
germination. These observations suggest that the phosphorylation state
of eIF2
, eIF2
, and eIF4B is developmentally regulated in a way
that correlates with the changes in protein synthetic activity but that
some differences were also observed.
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INTRODUCTION |
Protein synthesis undergoes dramatic changes during plant growth,
particularly during the development of the seed and its subsequent
germination. From the perspective of protein synthetic activity, the
development of wheat seed can be divided into three stages: early
development (up to 10-12 days after flowering
(DAF)1), mid-development
(approximately 12-30 DAF), and late development (30-45 DAF). During
early development, the endosperm and embryo are surrounded by the
nucellus, a maternal tissue that provides nutrients to the rapidly
growing endosperm (1, 2). The nucellus is the predominant tissue during
early seed development but undergoes a progressive programmed cell
death2 to provide room for
the expanding endosperm tissue. Following 12 DAF, the nucellus is
insignificant and the mass of the seed is dominated by the endosperm
and embryo. The highest level of protein synthetic activity in the seed
occurs between 12 and 30 DAF when the bulk of the seed storage protein
is synthesized in the endosperm and stored to provide the nutrients
required for germination (3-5). This developmental stage is followed
by a rapid drop in protein synthesis when the endosperm undergoes its own programmed cell death and the embryo prepares for quiescence, a
process that continues up to the mature dry stage of the seed by 45 DAF. Despite the reduced protein synthetic activity after 30 DAF, late
embryogenesis abundant (lea) proteins are synthesized, which
are thought to be required for the embryo to survive dessication during
late seed development (reviewed in Ref. 6). At maturity, however, the
seed is virtually metabolically quiescent, and translational activity
ceases despite the continued presence of the translational machinery in
the embryo. Protein synthesis resumes quickly following the initiation
of germination; formation of polysomes and protein synthesis are
detected shortly following the onset of germination (7-10).
Such extreme differences in protein synthetic activity during
development in plants presumably requires considerable control over the
activity of the translational machinery itself. Translational regulation in yeast and animal cells often occurs through changes in
the phosphorylation state of the eukaryotic initiation factors (eIFs)
whose function is to aid 40- and 60-S ribosomal subunit binding to the
mRNA, resulting in the formation of the 80-S ribosome at the
correct start codon. Our previous studies demonstrated that the
phosphorylation state of two initiation factors, eIF2 and eIF4B,
differed substantially in seed versus leaves of wheat (11),
observations suggesting that the activity of these factors may be under
developmental regulation.
Wheat eIF4B, a single polypeptide (59 kDa), is thought to assist eIF4A
and eIF4F in the ATP-dependent unwinding of secondary structure that may be present within a 5'-leader of an mRNA (12, 13). eIF4B is hypophosphorylated in mature wheat embryos, but is
present predominantly as hyperphosphorylated isoforms in leaves (11).
The posttranslational modification of eIF4B is similar in both plant
and animal cells in that the isoforms are divided into an acidic
cluster of four to six isoforms that are phosphorylated forms of a
basic cluster of four isoforms (11, 14). The acidic cluster is
substantially reduced in mature wheat embryos, but predominates in
leaves, and undergoes rapid dephosphorylation in leaves following a
heat shock (11). The alteration in wheat eIF4B phosphorylation
correlates with the repression of translation that occurs
following a thermal stress (15). Further evidence suggesting a
relationship between the phosphorylation state of eIF4B and
translational activity comes from studies in animal cells:
dephosphorylation of mammalian eIF4B occurred following heat
shock (16), serum depletion (17), or mitosis (18). Dephosphorylation of
mammalian eIF4B correlated with the reduction in translation following
these treatments, whereas the phosphorylation of eIF4B that occurs
following insulin treatment correlated with an increase in translation
(19). Moreover, the addition of phosphorylated mammalian eIF4B was
partially able to restore translation in an in vitro
translation lysate prepared from heat-shocked HeLa cells in which the
eIF4B had undergone dephosphorylation (16, 20).
eIF2 is a three-subunit complex in plants as in other eukaryotes and is
responsible for binding the initiator Met-tRNA to the 40-S ribosomal
subunit (21-23). The three subunits of wheat eIF2 are
(42 kDa),
(38 kDa), and
(50 kDa) (24). Phosphorylation of eIF2
occurs
following amino acid starvation in yeast (reviewed in Ref. 25) or viral
infection (reviewed in Ref. 26), heme deprivation, and heat shock in
animal cells (27). Its phosphorylation inhibits the eIF2
-directed
exchange of GTP for GDP, preventing eIF2 from participating in more
than one round of translation initiation (reviewed in Refs. 28-30). In
wheat, eIF2
is also subject to phosphorylation: the factor is
present in a hyperphosphorylated state in embryos of mature seed but is
present in a hypophosphorylated state in leaves (11). However, in
contrast to mammalian eIF2, heat shock, whether of short or long
duration, had little detectable impact on the phosphorylation state of
the
subunit in wheat leaves (11). Mammalian eIF2
is present as a
single major form under normal growth conditions (14), but following
either a heat shock or serum starvation, both a new acidic and basic
species was observed (14, 17). In contrast, approximately four eIF2
isoforms were observed in wheat leaves, the distribution of which did
not change significantly following a heat shock (11).
eIF4A (47 kDa) is an ATP-dependent RNA helicase and an
RNA-dependent ATPase in plants, animals, and yeast (31,
32). It can be found associated with eIF4G or eIFiso4G or as an
independent factor (33, 34). Only a single, dephosphorylated form of
mammalian or yeast eIF4A has been observed; however, in plants and
Drosophila, a phosphorylated isoform has been detected (34,
35). Although dephosphorylated eIF4A normally predominates in leaves
and mature seed, a maximum of 50% of the total available eIF4A can
undergo phosphorylation following hypoxia or heat shock (11, 34).
Our previous observations showing that the phosphorylation state of
several initiation factors is different in seed versus leaves of wheat (11) suggested that this was because of either tissue-specific differences or to differences in translational activity
between mature seed (translationally quiescent) and young leaves
(translationally active). This prompted us to examine whether the
posttranslational modification of these factors may be subject to
developmental control that would correlate with the regulation of
protein synthetic activity known to occur during development. We found
that the phosphorylation state of eIF4B, eIF2
, and eIF2
(and to a
lesser extent, eIF4A) is developmentally regulated during seed
development and germination but that the regulation of each differs
temporally. The differences in the regulation of the phosphorylation state of these initiation factors may affect message selection or
determine translational efficiency during plant development.
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EXPERIMENTAL PROCEDURES |
Antibody Preparation--
eIF4A and eIF2 (31) were purified from
wheat germ (commercially prepared wheat embryos) as described. The
purification of recombinant eIF4B and eIF4A will be described
elsewhere.3 Polyclonal
antibodies to eIF2 and recombinant eIF4B and eIF4A were produced in
rabbits and affinity-purified as described (33, 36). Antibodies raised
against purified PABP were prepared as described previously (37).
Plant Extract Preparation and Two-dimensional Gel
Electrophoresis/Western Blot Analysis--
For the seed development
studies, wheat plants were grown in a greenhouse to minimize any
potential environmental effects and were allowed to self-pollinate.
Whole seeds were collected at various stages of seed development and
frozen in liquid nitrogen. For the germination studies, wheat seed or
seedling tissues were dissected at various stages of germination and
frozen. Total soluble protein extracts were prepared by grinding tissue
in a mortar with liquid nitrogen and then in aqueous buffer (50 mM HEPES, pH 7.5, 120 mM KOAc, 5 mM
MgOAc, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 1 mM EDTA, and 0.5 mM okadaic acid).
The cell debris was pelleted by centrifugation and the protein
concentration determined (38).
Depending on the factor examined, 5-100 µg of protein was loaded on
IEF tube gels (3.6% acrylamide, 9 M urea, 2% ampholytes, 2% Nonidet P-40) and run at 400 V for 4.5 h followed by 0.5 h at 500 V. The protein was then resolved in the second dimension using
standard SDS-PAGE and the protein transferred to 0.22 µm nitrocellulose membrane by electroblotting. Following transfer, the
nitrocellulose membranes were blocked in 5% milk, 0.01% thimerosal in
TPBS (0.1% Tween 20, 13.7 mM NaCl, 0.27 mM
KCl, 1 mM Na2HPO4, and 0.14 mM KH2PO4) followed by incubation
with primary antibodies diluted 1:2000 for eIF4A and 1:1000 for eIF4B,
eIF2
, and eIF2
in TPBS with 1% milk for 1.5 h. The blots
were then washed twice with TPBS and incubated with goat anti-rabbit
horseradish peroxidase-conjugated antibodies (Southern Biotechnology
Associates, Inc.) diluted to 1:10,000 for 1 h. The blots were
washed twice with TPBS, and the signal detected was typically between 1 and 15 min using chemiluminescence (Amersham Pharmacia Biotech). The
range of ampholytes used is indicated in the legend to each figure. The
pH range of the IEF gel following isoelectric focusing was determined
from the measurements of 5 mm sections of a control gel soaked in 1 ml
of 15 mM NaCl.
 |
RESULTS |
eIF4B Undergoes Dephosphorylation Late in Seed Development and Is
Phosphorylated Early during Germination--
To examine whether the
phosphorylation state of eIF4B changes during seed development, wheat
seed were collected at developmental stages following pollination.
Soluble protein extracts prepared from whole seeds were resolved on 2D
gels and analyzed using antibodies raised against recombinant eIF4B
following Western blotting. The early development of wheat seed is
characterized by a small endosperm and embryo and a large nucellus. In
the early developmental time points (4-12 DAF), intermediate acidic
isoforms of eIF4B were present with a second set of lower molecular
weight isoforms also present (Fig. 1).
This apparent degradation of eIF4B correlated with the programmed cell
death of nucellar tissue that occurs at this stage in the development
of the seed (T. Young and D. Gallie, manuscript in preparation). This
observation is supported by the presence of only the full-length acidic
isoforms of eIF4B at the next developmental stage (16 DAF) by which
point the nucellus had virtually disappeared. Also by 16 DAF, the
distribution of eIF4B isoforms had shifted to highly phosphorylated
(acidic) species and remained unchanged through 30 DAF which
corresponds to the period in which the bulk of protein synthesis takes
place (3-5). However, at 30 DAF, the basic cluster of eIF4B isoforms
appeared for the first time, representing a redistribution of the
isoforms away from phosphorylated to dephosphorylated species, a
process that continued through late seed development. By 45 DAF, the
phosphorylated species had disappeared altogether, leaving only the
basic cluster. The eIF4B isoforms were identical in the endosperm and
embryo at this stage (data not shown), despite the fact that the embryo is viable whereas the endosperm has undergone programmed cell death (T. Young and D. Gallie, manuscript in preparation), data suggesting that
dephosphorylation of eIF4B during development is not a consequence of
cell death.

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Fig. 1.
eIF4B undergoes dephosphorylation during late
seed development in wheat. 60-100 µg of soluble protein extract
from developing wheat seed taken at the indicated days after flowering
(DAF) was resolved in the first dimension using IEF with
75%, pH 5-8, and 25%, pH 3-10, ampholytes and on an 8% SDS-PAGE
gel for the second dimension. eIF4B degradation products can be seen in
the first three time points. The acidic and basic clusters of the eIF4B
isoforms are each indicated with a bracket above the
top panel.
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We next examined whether the distribution of eIF4B isoforms was
regulated during germination. Imbibing seed were collected at time
points and the embryo dissected. The endosperm was not examined as this
is a dead tissue in germinating seed. Upon emergence of the shoot and
roots at later stages of germination, these tissues were also collected
and the samples were analyzed as described above. eIF4B was present as
predominantly hypophosphorylated isoforms in seed before imbibition and
up to 10 h of germination (Fig. 2).
By 20 h, a shift to more phosphorylated isoforms was observed that
was completed by 48 h of germination when the shoot had first emerged. eIF4B present in roots of 48 h germinated seed was also present as highly phosphorylated isoforms (Fig. 2). The observed shift
of eIF4B from hypophosphorylated to hyperphosphorylated species during
germination correlates with the resumption of protein synthesis in
wheat (7-10).

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Fig. 2.
eIF4B undergoes progressive phosphorylation
during wheat germination. 10-30 µg of soluble protein extract
from wheat germ, embryos excised from germinating seed (0, 1, 5, 10, and 20 h after imbibition), or from leaves and roots (48 h) was
resolved in the first dimension using IEF with 75%, pH 5-8, and 25%,
pH 3-10, ampholytes and on an 8% SDS-PAGE gel for the second
dimension. Recombinant eIF4B, purified following its expression in
Escherichia coli, was included in the analysis. The eIF4B
acidic and basic clusters are each indicated with
brackets.
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eIF4A Is Phosphorylated at the Nucellus-containing Stage of the
Young Seed but Not during Subsequent Seed Development or during
Germination--
The same extracts were used to determine whether the
phosphorylation state of eIF4A changes during seed development and
germination. Dephosphorylated eIF4A largely predominates in wheat
leaves or maize roots under normal conditions but undergoes
phosphorylation to a maximum 1:1 ratio following a severe heat shock or
hypoxic treatment (11, 34). During early seed development when the nucellus predominates and undergoes programmed cell death (4-12 DAF),
the two isoforms of eIF4A were present in approximately equimolar
amounts (Fig. 3). When this maternal
tissue had disappeared by 16 DAF, the dephosphorylated form of eIF4A
once again predominated and the distribution of the isoforms remained
little altered during the remaining development of the seed. This
observation suggests that the phosphorylated form of eIF4A is present
in the nucellus and may be targeted for inactivation or degradation
during programmed cell death. During germination, only the
dephosphorylated isoform was observed in the embryo (data not shown)
and up through 2 day-old shoots (Fig. 4).
However, the presence of both eIF4A isoforms was observed in 2 day-old
roots (Fig. 4).

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Fig. 3.
Two-dimensional gel electrophoresis/Western
analysis of the change in phosphorylation state of eIF4A during seed
development. 5 µg of soluble protein extract from developing
wheat seed taken at the indicated days after flowering (DAF)
was resolved in the first dimension using IEF with 50%, pH 5-8, and
50%, pH 2.5-5, ampholytes and on a 10% SDS-PAGE gel for the second
dimension. The eIF4A phosphorylated isoform is indicated with a
downward pointing arrow.
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Fig. 4.
eIF4A is differentially phosphorylated in
shoot and root tissue of wheat seedlings. 5 µg of soluble
protein extract from leaves and roots (48 h) was resolved in the first
dimension using IEF with 50%, pH 5-8, and 50%, pH 2.5-5, ampholytes
and on a 10% SDS-PAGE gel for the second dimension. The eIF4A
phosphorylated isoform is indicated with a downward pointing
arrow.
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eIF2
Undergoes Progressive Phosphorylation during Seed
Development and Is Dephosphorylated during Germination--
In the
embryo of mature seed, the
subunit of eIF2 is present in its most
phosphorylated state (pI of 5.4-5.6) whereas in leaves it is
completely dephosphorylated (pI of 6.2-6.4) (11). This large
difference in pI is in striking contrast to that observed in animal
cells in which the
subunit is subject to a single phosphorylation
event at serine-51 (reviewed in Ref. 39) resulting in a small shift in
the pI of the subunit from 5.4 to 5.6 (14, 40). This suggests that the
subunit of eIF2 in plants may be multiply phosphorylated. The
number of
subunit isoforms also appears to differ between plants
and animals. Whereas a single major mammalian eIF2
species is
observed under normal growth conditions (14), at least four eIF2
isoforms were observed in wheat leaves (11). We examined, therefore,
whether multiple isoforms of the
and
subunits were present in
wheat seed and whether their distribution changed during seed
development. As in leaves, approximately four major species were
observed for the
subunit during early seed development (Fig.
5), although the distribution in seed
included more acidic isoforms not detected in leaves. The two most
basic species present at 4-12 DAF (Fig. 5) correspond to the middle
two, and most dominant, species in leaves (11). With further
development of the seed, a progressive redistribution toward the basic
isoforms was observed such that by 35 DAF, only a single basic isoform
predominated. This eIF2
basic isoform serves as a convenient
reference point (see arrows, Fig. 5) for the changes in the eIF2
subunit isoforms described below. In contrast, small amounts of some of
the phosphorylated eIF2
isoforms observed during mid-development
remain present in mature embryos (see 0 h, Fig. 7 and (11)),
suggesting tissue-specific (endosperm versus embryo)
regulation of its phosphorylation state during late
development.

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Fig. 5.
eIF2 undergoes conversion from multiple
phosphorylated isoforms to a single hypophosphorylated species during
seed development. 30 µg of soluble protein extract from
developing wheat seed taken at the indicated days after flowering (DAF)
was resolved in the first dimension using IEF with 75%, pH 5-8, and
25%, pH 3-10, ampholytes and on a 12% SDS-PAGE gel for the second
dimension. Affinity-purified, anti- subunit antibodies were used to
probe for the subunit. The and subunits of eIF2 are
indicated to the left of the gels. The large
arrow in each panel points to the most abundant
subunit isoform.
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At the earliest stages of seed development examined (4-12 DAF),
multiple isoforms of eIF2
were detected (Fig.
6) but mostly were basic to intermediate
acidic species and those at 12 DAF corresponded to those
dephosphorylated species previously observed in young wheat leaves
(11). By 16 DAF, a shift to slightly more acidic species was observed
(Fig. 6). The most basic of the two
isoforms observed at 16 DAF
corresponded to the most acidic of the isoforms present at 12 DAF. The
distribution of eIF2
isoforms remained unchanged up to 20 DAF. By 25 DAF, the isoforms that had been present at 16-20 DAF had disappeared
and a new set of 3-4 highly acidic isoforms (pI range from 5.4 to 5.9)
appeared, the most acidic of which (pI 5.4) corresponded to the fully
phosphorylated
subunit isoform that was observed in mature wheat
embryos (11). At 30 DAF, there was an increasing shift of the
subunit to the most acidic isoform and by 40 DAF, only the most
phosphorylated isoform remained. At 40 DAF, a second, smaller molecular
weight form of the
subunit was observed in addition to the
full-length form. This was also observed with eIF2 purified from mature
wheat embryos (11) and may be because of degradation of the
subunit during late seed development. At 45 DAF, the
subunit was no longer
detectable despite the fact that the
subunit was still present,
data suggesting that the
subunit is degraded during late endosperm
development. In contrast, eIF2
remains present in mature embryos
(see 0 h, Fig. 7 and (11)). These
data demonstrate that the eIF2
subunit is present in a
hypophosphorylated state during early seed development but undergoes a
progressive shift to a highly phosphorylated state that begins at 16 DAF, is completed by 35 DAF, and is followed by degradation
specifically in the endosperm at 45 DAF but not completely in mature
embryos.

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Fig. 6.
eIF2 undergoes progressive phosphorylation
during seed development. Affinity-purified, anti- subunit
antibodies were used to reprobe the membrane from Fig. 5 so that subunits would also be evident to serve as points of reference. The and subunits of eIF2 are indicated to the left of the
gels. The subunit isoforms are indicated by
arrows.
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Fig. 7.
eIF2 begins conversion to its
dephosphorylated state only during late germination. 100 µg of
soluble protein extract from embryos excised from germinating wheat
seed (0, 1, 10, and 20 h after imbibition) or from leaves and
roots (48 h) was resolved in the first dimension using IEF with 75%,
pH 5-8, and 25%, pH 3-10, ampholytes and on a 12% SDS-PAGE gel for
the second dimension. Affinity-purified, anti- subunit antibodies
were used to probe for the subunit (left column of
panels), and the membranes were reprobed with anti- subunit
antibodies (right column of panels). The subunit
isoforms are indicated by arrows.
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Upon imbibition, the
subunit was present solely in its highly
phosphorylated form in the embryo (Fig. 7). No change in eIF2 phosphorylation in the embryo was observed between 10 and 20 h of
germination, the point at which eIF4B was observed to undergo phosphorylation (see Fig. 3). Between 48 and 60 h of growth (see Figs. 7 and 8, respectively), the basic
isoforms of the
subunit are detectable in the emerging shoot.
However, substantial levels of the most phosphorylated isoforms
remained without the appearance of the intermediate species, suggesting
that the two extreme forms of the
subunit temporally coexist during
this stage of germination. This differs substantially from the
observations made during seed development where the conversion of the
subunit from the hypophosphorylated to hyperphosphorylated state
occurred en masse and the two extreme forms of the
subunit were temporally separate. Only by 4 days of growth had the
subunit been completely converted to the basic isoforms (Fig. 8).
Similar results were observed in the roots of 4 day-old seedlings where
the
subunit was present solely in its dephosphorylated form.
Germination in the light promoted the conversion of eIF2
to its
dephosphorylated state as dark-germinated seedlings contained nearly
equimolar amounts of phosphorylated and dephosphorylated
subunit
(Fig. 9). These data show that eIF2
subunit undergoes a shift from being present in a highly phosphorylated
state during early germination to a hypophosphorylated state upon
emergence of the shoot that occurs over a period of several days.

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Fig. 8.
eIF2 undergoes complete dephosphorylation
only during late germination. 100 µg of soluble protein extract
from 1-day-old embryos, shoots of 2-, 2.5-, 3-, and 4-day-old
germinated wheat, or roots of 4-day-old seedlings was resolved in the
first dimension using IEF with 75%, pH 5-8, and 25%, pH 3-10,
ampholytes and on a 12% SDS-PAGE gel for the second dimension. The
membranes were then probed for the and subunits. The subunit isoforms are indicated by arrows.
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Fig. 9.
Dephosphorylation of eIF2 during
germination is promoted by growth in light. 100 µg of soluble
protein extract from 3-day-old shoots germinated in light or in
complete darkness was resolved in the first dimension using IEF with
75%, pH 5-8, and 25%, pH 3-10, ampholytes and on a 12% SDS-PAGE
gel for the second dimension. The membranes were then probed for the
and subunits. The subunit isoforms are indicated by
arrows.
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 |
DISCUSSION |
In this study, we observed that eIF4B was present in a
hyperphosphorylated state during the period of seed development in which protein synthesis is most active (12-30 DAF) and underwent gradual dephosphorylation (35-45 DAF) during the late stage of development concomitant with the decline in protein synthetic activity
that precedes the metabolically quiescent state of the mature seed.
eIF4B remained in a hypophosphorylated state up to 10 h following
imbibition but was rapidly converted to its hyperphosphorylated state
between 20 and 48 h of germination.
In contrast, phosphorylation of eIF2
occurs earlier during seed
development than does the dephosphorylation of eIF4B, and eIF2
undergoes dephosphorylation much later during germination than does
eIF4B. Interestingly, conversion of eIF2
to a hyperphosphorylated state during seed development occurred in a concerted manner, suggesting either that its phosphorylation occurs in a distributive fashion or that phosphorylation of each site is under temporal control
during development. During germination, however, dephosphorylation of
eIF2
occurred without the significant appearance of the intermediate isoforms, even though both the hyperphosphorylated and
hypophosphorylated isoforms were present simultaneously. This suggests
that either eIF2
dephosphorylation occurs in a processive manner or
that its phosphorylation is irreversible, and dephosphorylated eIF2
is generated only through new synthesis.
The presence of multiple eIF2
phosphorylation sites in plants
appears to differ from the single site present at serine-51 in
mammalian eIF2
(41, 42). As in mammalian cells, eIF2
activity in
yeast is also regulated through phosphorylation at Ser-51, however,
three additional sites close to the C terminus are constitutively
phosphorylated in vitro and in vivo by casein kinase II (43) and remain so following heat shock, nitrogen starvation,
or growth in poor carbon sources (44). Mutations at these sites do not
reduce growth, but can exhibit synthetic growth defects in combination
with mutations affecting the activity of eIF2B (the factor required for
GTP/GDP exchange in eIF2) or GCN2 (the kinase responsible
for phosphorylating eIF2
), suggesting that their phosphorylation is
necessary for optimal activity of the factor (43). Although human
eIF2
does not contain consensus sites for and is not phosphorylated
by casein kinase II, Artemia eIF2
contains four such
sites and is phosphorylated by the enzyme in vitro (43, 45),
suggesting considerable variation in the number of phosphorylation
sites of eIF2
from various species.
None of the phosphorylation sites in plant eIF2
can be considered as
constitutively phosphorylated since the most hypophosphorylated species
dominates in leaves and during early seed development. How
phosphorylation at each site of wheat eIF2
affects its activity remains to be determined. However, as changes in eIF2
phosphorylation correlate with the substantial changes in protein
synthetic activity during seed development, they do suggest a possible
role for phosphorylation as a means to regulate eIF2
activity in
plants. Phosphorylation at some sites may serve to promote translation
initiation, whereas phosphorylation of other sites may serve to inhibit
protein synthesis or alter those mRNAs selected for translation. In
this respect, it should be noted that phosphorylation at Ser-51 in
eIF2
inhibits factor activity in mammalian cells (reviewed in
Ref. 39), whereas it affects message selection in yeast
(e.g. GCN4) through alterations in its
re-initiation activity (46, 47).
eIF2
is required for ribosomal start site selection (48), and
phosphorylation of mammalian eIF2
stimulates eIF2 activity (49).
eIF2
is multiply phosphorylated in wheat leaves (11) as it is during
early seed development, where approximately 4-5 isoforms were
observed. During subsequent seed development however, eIF2
underwent
progressive dephosphorylation until a single basic species remained at
45 DAF. The presence of phosphorylated eIF2
correlated with the high
rate of protein synthetic activity characterizing the mid-development
of the seed whereas dephosphorylation of eIF2
during late
development correlated with a corresponding low level of protein
synthesis, observations suggesting that phosphorylation of eIF2
may
play a role in regulating translational activity.
In contrast to the dynamic changes in phosphorylation of eIF4B,
eIF2
, and eIF2
, eIF4A remained little changed during seed development or germination. The only exception to this was during the
earliest stage of seed development when the seed is dominated by the
nucellus, a maternal tissue that undergoes programmed cell death.
During this developmental stage, 50% of the total eIF4A in the seed
was phosphorylated, similar to the observations made in wheat leaves
following a heat shock (11) or in maize roots following hypoxia (34).
Therefore, phosphorylation of eIF4A appears to occur in response to
stress and as part of some types of programmed cell death. During this
same early developmental period, eIF4B was subject to degradation and
eIF2
was present as phosphorylated intermediates, which may reflect
the fate of these factors in the nucellus and other tissues as they
undergo rapid programmed cell death.
Our observations suggest that the phosphorylation state of eIF4B,
eIF2
, eIF2
, and even eIF4A is developmentally regulated but that
the regulation of each exhibits some unique aspects. These differences
may play a role in the changing translational environment of the
developing seed and during acquisition of photosynthetic competence
following germination as the complement of mRNAs can change
dramatically from one developmental stage to another. Whether the
translational requirements of mRNAs expressed only during specific
developmental stages differs is unknown. However, changes in the
phosphorylation status of the protein synthetic machinery may be a
means by which necessary changes in the translational environment are
achieved during plant development.
We thank the Department of Botany and Plant
Sciences, University of California, Riverside for the generous use of
their greenhouse facilities.